Wuolah Free BIOCHEMISTRY NOTES

BIOCHEMISTRY-NOTES.
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Carlota_Trigo
Bioquímica
2º Grado en Ingeniería Biomédica
Escuela Politécnica Superior

Universidad Carlos III de Madrid
Reservados todos los derechos.

No se permite la explotación económica ni la transformación de esta obra. Queda permitida la impresión en su totalidad.
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Carlota Trigo (100383134)
BIOMEDICAL ENGINEERING 2018/19.
BIOCHEMISTRY

1. ENZYMES
DEFINITION
Enzymes are biological catalysts. The catalyze efficiently and selectively chemical
reactions without altering equilibrium.
The highest rate discovered is 7.2*10^27 but the usual is that they work at 10^7.
Their name comes from the substrate they attach to + ase
CLASIFICATION
● REACTION CATALYZED
○ Oxydoreductase: Transfer of electrons
○ Transferase: Group transfer
○ Hydrolase: Hydrolysis
○ Lyase: Addition and removal of groups to destroy/form double bonds

○ Isomerase:
○ Ligase: They form bonds by coupling to an ATP cleveage
Kinases are transferases because they take a phosphate from ATP to a molecule
As of today, the IUPAC has established a number code for each enzyme, each number
referring to a specific classification of the enzyme (Hexokynase 2.7.1.1)
Proteases are hydrolases.
● COMPLEXITY
○ Simple: Only contains proteins.
○ Complex or holoenzymes:
■ Apoenzyme: polypeptide (protein)
■ Coenzyme or prosthetic groups: (extra group)
- Cofactor that is a small organic group, often derived from
metals. Very often, enzymes are not active until a cofactor
binds to them. Enzymes that require a metal cofactors are
known a metalloenzymes.
- Most of them derive from vitamins. (Diet is important!).
- Coenzyme - loosely. PG - tightly.
- Coenzymes act as transporters of chemical groups from
one reactant to another.
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● SUBSTRATE SPECIFICITY: They are highly specific for the kind of reaction
they catalyze. There are enzymes that are very specific to the substrate they
catalyze and other that are not so specific (they can use several similar
substrates)
FUNCTION & HOW THEY WORK
Enzymes allow reaction to carry on by acting as catalysts. They accelerate processes
that would only occur at high temperatures.
Enzymes present the so called active site, which is highly structured. It has amino acid
residues with substituent groups that bind the substrate and catalyze the
transformation.
The substrate attaches to the active site and then the reactions carries on.
In order to go from reactant to products you need to surpass an energetic barrier
(transition state). Enzymes reduce that barrier in living systems for the reactions to
take place.

Enzyme rate enhancement
When a substrate binds to the active site what typically happens is:
1. Entropy reduction: Bimolecular reaction, unimolecular reaction or constraint of.
They need to hit into each other in the proper orientation. The entropy of the
system opposes the reaction.
2. Desolvation: most common, every enzyme is always surrounded by a water or
a buffer solution. Water tends to surround the enzyme - substrate complex,
which makes it more thermodynamically favorable.
3. Acid-base and covalent catalysis: acid base reactions promote the movement
of the electron in the S/E complex. Some aa acting as nucleophilics may
participate in covalent catalysis. Nucleophilic attack.
4. Induced fit in Hexokinase: Assumes that the enzyme does not have the same
structure as the substrate. The enzymes participating have lower Km as som
of the energy must be used to change the enzyme.
5. 5. structures to orient groups better.

PROPERTIES
All enzymes are affected by temperature and pH and they all have an optimum
temperature and pH at which they work better
ENZYME KINETICS
Chemical processes occur with a speed rate proportional to the concentration of the
participant species until the saturation of the enzyme occurs.
Chemical kinetic equations:

Everything happens very quick until the max velocity is reached when the enzyme
becomes saturated. Km is the half of the max velocity (in the graph).
The main equation you need to know when you talk about enzymes and about doing
exercises related is the Michaelis Menten equation. It is applied when the stationary is
reached, which means, Rate of ES formation = rate of ES breakdown.

The same enzyme working on a different substrate gives you different graphs. The v
max will be the same but the km will change. The lower the km the higher the efficiency
or specificity of the enzyme. If you can measure the km you can now how well an
enzyme works. When v= vmax/2, km=[S]
Turn over number or catalytic constant kcat indicates the number of products that can
be produced in a catalytic process. They typically go from 0. something to a hundred.
There is also an equation. The highest k cat found is 40*10^6

The kcat values for most enzymes have not been experimentally measured thereby
there are some doubts in the relevance of kcat under in vivo conditions.
Enzyme activity unit: During a reaction, the units of velocity are [P]/time. From that we
derive the units for enzyme activity which are [P]/time/protein(mg)
Lineweaver-Burke plot.
It is derived from the Michaelis Menten equation.
Enzyme inhibition
Plots:
- Competitive: Enzyme only and competes with the substrate. Increases the
concentration of substrate ‘stops’ the inhibition.
- Non competitive: Enzyme and transitional state. Increasing the concentration
of substrate will not overcome the inhibition.
- Uncompetitive: Transitional state only. Increasing the concentration of
substrate favors the inhibition.

- Allosteric Inhibition: VERY IMPORTANT WAY. Whenever you have a chain
of different enzymes catalyzing different reactions, you usually have a product
of the pathway that acts as an inhibitor. El propio producto actúa como inhibidor.
Sigmoid curve.
You can do both enhance and inhibit enzymes.

ENZYMES and BIOENGINEERING
They modified Rubisco, the main enzyme doing photosynthesis. This way they
improved the absorption of CO2.
rhTG1 s not soluble because is very hydrophobic so they thought of covering them
with a hydrophobic thing to favor absorption.
The last article explains an experiment in which by the modification of an enzyme, they
are able to correct the architecture of some of the cells in charge of cicatrizing.

2. INTRODUCTORY CONCEPTS
Charges in proteins vary depending on the pH of the surroundings. Histones for
instance, are positively charged in a physiological medium (pH=7).
DISSOCIATION OF WEAK ACIDS AND WEAK BASES
The pH of pure water is 7. Water is a molecule that is very slightly dissociated. WE

may consider water as an extremely weak base or acid which dissociates into proton
and Oh- groups. Why is the pH of water 7?

The pH is measured through a logarithmic scale in which the lower the number is, the
more acidic the pH is.
Acid {1-7} Neutral {7} Basic {7 - 14}
Normal engineering depends a lot on temperature, but bioengineering depends also
on the pH because enzymes have both an optimal pH and temperature.
This graph shows how
Pepsin works better at pH 2
than it does at any other pH
but at pH 6.5 trypsin works
better.
Each enzyme has a pH in
which they work better.

Dissociation of strong acids: Strong acids tend to dissociate completely and appear
ionized in water. There won’t be an equilibrium but a dissociation.
Dissociation in weak acids: The dissociation won’t be complete and an equilibrium will
be stablished. The equilibrium constant is given by [H+][A-]/[HA]. Stronger acids have
bigger Ka as the concentration of protons is bigger.

If you take the logarithm of the equilibrium constant, you obtain a value called pKa. It
is possible to measure the pKa of an acid
by performing a titration curve.
You put the acid in a water solution at a
given concentration and you start adding
a base like NaOH in small ammounts
until oyu reach the exact same amount
of the weak acid that is in solution. While
you are adding this equivalents, the pH
is starting to increase as when adding
the base you are removing protons (H +

OH → H2O ). When you are half in equivalents, you reach a number at which pH =
pKa. From that you reach a time in which there won’t be a big change in the pH; this
is considered the Buffer Area of this molecule. It is a pH range that resists to change
when you add the titrating base. This buffering area is about (pKa - 1,pKa + 1).
HENDERSON - HASSELBALCH EQUATION
The Henderson - Hasselbalch equation

provides the pH in terms of the pKa and
the amount of acid dissociated over the
undissociated.
We can think of weak acids as proton
suppliers within certain pH range.
Weak acids help us keep the pH of the
solution in which they are, relatively
constant.

BUFFERS, BIOLOGICAL/PHYSIOLOGICAL EXAMPLES
Buffers are composed of a weak acid/base and its conjugate that cause a solution to
resist changes in pH when an acid or base are added.
The buffering capacity is greater when pH=pKa.
To measure the effectiveness of a buffer we need to take into account the pH of the
solution and the concentration of the buffer (the more, the better).
Why are weak acids/bases bufers? IT is becaus they don’t fully dissociate. The HA
can neutralize OH- and the A- neutralized H+. The rapidly bind to protons and that
keeps the pH unchanging.
Examples:
- Carbon dioxide - bicarbonate: Blood pH
- Phosphate system: cytosolic pH
- Lungs and kidneys: Lungs increase or reduce O2. Filters more or less
bicarbonate depending on the needs.

IONIZATION OF AMINO ACIDS
The amino and carboxyl group along with R groups in aa acts as weak acids and
bases. When an aa lacking an ionizable R groups
is dissolved in water, it presents the form of a
zwitterion: a neutral molecule with both positive
and negative charges.
An additional amino or carboxyl group will be
responsible for the changes in the protein.
Titration of amino acids: The group with lower
pKa will be the first on to be titrate. In the end, the carboxylic group will become

dissociated but we still have the amine group. Why COOH dissociates first? The amino
group begins to attract electrons towards itself making the carboxyl group unstable.
After the Isoelectric point is reached, the removal of protons from the amino group
begins.
From the titration curves of amino acids we get clear that aa have 2 regions of buffering
power. But also,know that it depends on the amino acid.
The titration curves also vary depending on the amino acids and mainly on the
presence/absence of an ionizable R group.
Extra:
We can consider both groups in the amino acid as weak acids. Their dissociation gives
you the charge.
Nonionic + Zwitterionic forms = net charge is 0
ISOELECTRIC POINT
The isoelectric point is the pH at which the net charge is 0. At this point the amino acid
is mostly present as a zwitterion.
QUICK REVIEW OF ORGANIC CHEMISTRY AND MAIN REACTIONS
The charge and weight of the protein influences electrophoresis, a technique used to

analyze proteins.
When the protein is mostly positive charged, it will migrate to the positive side. (pH >
pKa)
the opposite will happen if it is negatively charged. (pH < pKa)
Biochemical reactions allow for the maintenance of homeostasis and for metabolism
to occur.

Groups:
Carbon oxidation states:
Reduced: Alkane → Alcohol → ALdehyde → Carboxylic acid → Carbon dioxide :Oxidized
Reactions:
- REDOX: Redox reactions are the ones in which electrons are exchanged.
Nucleophiles: O2-, S2-, C2-, uncharged amine, imidazole, OH-
Electrophiles: Carbon in a carbonyl, imine, phosphorus in a phosphate, H+
- Hydrolysis: A molecule is broken due to the consumption of a water molecule.
Nucleophilic attack.

3. PROTEIN ANALYSIS
WHY DO WE ANALYZE PROTEINS
Proteins play crucial roles in all biological processes: folding proteins into 3D
structures.
Pharmaceutical industries are interested in knowing all data about proteins (purity,
composition, structure, function…)
Allows to understand the final changes at DNA/RNA level resulting in possible
malfunction or disease.
ISOLATION OF SPECIFIC CELL COMPARTMENTS / ORGANELLES
The cell is composed of different subcompartments and from that organization there
are different types of proteins: nuclear, cytosolic, plasma membrane, mitochondria,

endoplasmic…
1. Homogenization: First step always. Consists of breaking cells and tissues, so
that the proteins can be solubilized. You will need to take decisions depending
on the buffer you are using. En las imágenes: Arriba Derecha - Muestra
pequeña. Abajo derecha - muestra más grande.
2. Separation of components: Next step is to use and ultra high speed
centrifugation to separate the different components from the cell. Differential
centrifugation (speed changes) works under high vacuum conditions. Inside
you have different types of rotors which you need to use one or the other
depending on the tissue.

EXTRACTION OF PROTEINS
Efficient extraction of protein and peptides in a biologically alive form.

During this process, inactivation of proteolytic enzymes are required so you don’t
cause the degradation of the proteins contained in the tissue.
Homogenize in physiological buffer containing proteolytic enzymes inhibitors (EDTA,
Pestatin) and chaotropic agents (to keep the protein soluble).
Protein solubilization:
1. Osmotic lysis: placing cells in hypotonic solution/distilled water. H2O gets in the
cell through osmosis and the cell ends up exploding.
2. Mechanical: crushing / grinding cells, sonication
3. Detergent: used if protein is in the lipid membrane.
Conditions:
- Stable and adequate pH: buffer.
- Temperature: 0-4C (any molecule)
- Proteases inhibited
- Store under inert gas, frozen -80 to -196 (DNA)
At this point we have a crude extract/lysate/homogenate
To measure the total concentration we need to measure the absorbance.
You use a spectrophotometer to measure the absorbance or transmittance of color.
Beer Lambert Law: A=Ebc → Absorbance is proportional to a
constant*width*concentration. Desde aquí despejas lo que necesitas.
Nanometer by nanometer you will have a registry of what is the absorbance of what
you put in your sample.
280 nm Protein / 260nm DNA. The bigger the ratio between those absorbances the
better, as you have more concentration of protein REVISAR APUNTES LAB DEL AÑO
PASADO.
PURIFICATION OF THE PROTEIN
They can be purified according to their differential properties that allow us to employ
different techniques to get proteins purified.
- Solubility:Differential Precipitations.
- Molecular size: gel filtration chromatography

- Molecular charge: Ion exchange chromatography
- Hydrophobicity: Reversed phase chromatography
- Biological activity: Affinity chromatography.
You can combine them as you wish.
If you have a solution with protein A (120,000 kDA); B (charge 2+) and an antibody
you can separate them doing first a Gel filtration chromatography, then Ion exchange
chromatography and finally you could do a gel filtration again.
Differential precipitation
The effect of salt on different proteins may differ. Certain proteins precipitate from
solutions under conditions in which others remain soluble.
Salting out: When the salt concentration is increased, some of the water molecules
are attracted by salt ions which decreases the number of molecules that are able to
interact with the charged protein and therefore the protein precipitates.
Salting in: Allows for a neutralization of the charges in the protein to occur and
therefore increases the solubility as it is more stable.
When (NH4)2SO4 o PEG are added to a protein solution, a precipitate forms and it
can be separated from the solution after precipitation. The more concentration of the
added, the more precipitate forms.

Dialysis: after a salting out step, you have a high salt concentration that can be
disruptive to subsequent chromatographic steps. The salt can be removed through
dyalisis: tubing with tubes that have pores with specific diameters allowing molecules
of different sizes to go through.
Chromatography: process that separates molecules
involving differential retention of the components
contained in a mobile phase while they move along the
stationary phase. Usually done after a precipitation.
Gel Filtration Chromatography

Separates protein according to their molecular size
(Dalton).
The solution is inserted to the top of a specialized column
which consists of different pores. These pores allow

smaller proteins to go through but larger molecules wont.
Smaller proteins elute later. The first things you will collect
will be the bigger things.
Bigger things go faster down the column because they are
too big to get into the spheres. Smaller ones have to get in and out of the sphere, so
it takes them a longer time.
Ion exchange chromatography

The beads of column have specific charges result of a molecule that is attached to
these beads. They can be + charged attaching them to DEAE or - charged
(carboxymethyl). The beads depend on the proteins you want to purify. If the protein
is negatively charged, you need to use positive beads and vice versa.
Imagine a negatively charged protein and positively charged beads.The protein will
bind to the beads and it can be released by increasing the concentration of NaCl. The
Na+ ion will compete and bind to the beads in the column instead of the protein.
PRoteins that are highly positively charged will emerge first.
Reversed Phase HPLC (High Performance Liquid Chromatography)

Purifies according to their hydrophobicity.

On the surface of the beads you now have aliphatic chains of carbons (C4, C6, C8…);
the longer the chain, the stronger the retention of the proteins.
The hydrophobic groups will attach more to the beads and they will elute later in time.
But proteins that are characterize by having a lower hydrophobicity will emerge first.
The hydrophobic remain in stationary phase until the organic modifier concentration
rises and desorbs polypeptides.
Affinity chromatography

Takes advantage to the high affinity of some proteins to specific chemical groups or
molecules.
Concanavalin A is a CH binding protein. It can be purified by passing through a column
that has beads with glucose attached.
Antibodies have the advantages that they are highly specific and well known → easy
to produce and to use in the beads for this kind of chromatography.

Gel electrophoresis: Method that allows to separate
and counts molecules based on their molecular weight.
An agarose gels with different size pores is placed and
an electric current is applied. The molecules (al
negatively charged -- SDS) run from negative to
positive going through the pores. YOu usually do this
with a marker from which you know the size of the
molecules that compose
it, that way you can compare and know the sizes of

everything that is running. This technique relies on the
combination of the isoelectric point and the molecular
weight.
1 - An ampholyte solution is incorporated into a gel and a pH gradient is achieved. The
protein solution is then added and after electricity is applied, the proteins are separated
based on their pI (each proteins stops at their pI).
2 - You make the molecules run through the gel as explained before. This allows for
the separation according to the molecular size of the components.
IDENTIFICATION OF THE PROTEINS (WESTERN BLOT / MASS

SPECTROMETRY)
Western Blot:
A gel is placed between filter paper and soaked in transfer buffer; you squeeze it and
then apply the voltage. Proteins migrate and attach a piece of PVDF. After that, they
can be identified by using a specific antibody against the protein you are looking for.
Proteomics: First use 2d gel and the add the proteins, the proteins need to be
transformed into peptides. in a single sample all the peptides from all the proteins from
the whole repertorio of proteins can be identified using mass spectrometry
Other: First tryptic digestion, then peptide analysis with mass spectometry.
STRUCTURAL CHARACTERIZATION OF THE PROTEIN.
Determination of molecular mass by mass spectrometry (MALDI-TOF): There is

another technique in which you put the protein on top of a solid phase and that solid
phase is covered with a matrix that can absorb the energy of a laser. The proteins
become volatile and later on they become ions so you are able to apply voltage and
force them to go into a high vacuum channel which allows to detect them. With the
time they spend trying to get to the scanner you are able to identify the molecular mass
of each protein.
Electrospray ionization (ESI): You break the proteins into peptides (trypsin)and then
separate them and ionize and measure the mass of each fragment so you are able to
identify them using bioinformatics.

4. PROTEIN ANALYSIS II - PTM
PROTEIN COMPLEXITY
DNA - genome; mRNA - transcriptome; protein - proteome

From genome to transcriptome the modifications are done by alternative splicing.
From transcriptem to proteame, the modifications are called posttranslational
modification.
Alternative splicing
IT is the different combinations that can be done with the exons. Different enzymes
indicate what exon goes after the other. Therefore you can combine different
sequences to form different proteins.
The results may be non functional proteins, proteins with different functions or proteins
with the same function but located at different points in the cell or human body.
The same piece of gene can be controlled by different promoter.
Example A - Each promoter send the same signal.
Example B - The strongest promoter send the signal. The only difference is a small
portion of the N terminal.

Example C - Depending on which promoter acts, the protein will be different.
Post translational modifications
Modification Charge dependent modification
Acetylation Loss of amino positive charge
Alkylation Alteration of amino positive group
Phosphorylation Gain of negative charge
Carboxylation Gain of negative charge
Proteolytic processing Truncation leads to change of pI

TYPES AND FUNCTIONS OF DIFFERENT PTM
On these On these Enzymes Mechanism Mass

aa groups Added
Phosphoryl Serine ** PO3(2-) Kinases add Addition of a 80

ation (OH) and phosphate (1P + 3O)
Threonine phosphatases group.
** (OH) remove.
Tyrosine
Acetylation Lys (-N) NH Acetylases Addition of 42

Arg (-N) (KATs) acetyl
Deacetylases groups
(DKATs)
On histones it
is histone

acetylasesand
histone
deacetyases.
Methylation Arg NH Methylases Addition of 15

Lys Demethylase methyl
groups onto
proteins.
You can
add 1 -

mono; 2 - di;
3 - tri)
Glycosylatio Asn Nitrogen ... Hydroxyl on Depends on

n Ser, Tyr Oxygen the sugar the sugar.
reacts with Glucose:
nitrogen 180
from a
Nitrogen
form a
compound
releasing a
water
molecule.
Hydroxyl on
the sugar
reacts with
the other
molecule
forming the
bond and
releasing a
water
molecule
A water
molecule is
released.
Ubiquitinati Lys (N)(K) Ubiquitin

on added to a
protein and
the protein
is the
signaled for
destruction
by the
proteasome
.

ATP - stores energy. AT-P-P-P (adenosine triphosphate). When ATP is converted into
ADP energy is released from the high energy bond.
Sumoylation: addition of sumo 1. ITs function is to be a target to subnuclear structures
and it’s in charge of proper nuclear location.
Electro carriers
The molecules involved are molecules obtain from redox reactions. NAD+/NADH;
NADP+/NADPH; FAD+/FADH2.
The chemical bond energy can be transformed into electrical energy and then into
kinetic energy in the ETC and during oxidative phosphorylation.
PROTEIN DEGRADATION
Ubiquitination
First, ubiquitin becomes activates through the ubiquitin activating enzyme (ATP
required). LAter, ubiquitin is transferred from E1 to E2 (ubiquitin conjugating). Finaly
an isopeptide bond is created between a lysine and the C terminal glycine of ubiquitin
(Enzyme E3 ubiquitin protein ligase.
1 - E1 ubiquitin activating 2 - E2 ubiquitin conjugating 3 - E3 ubiquitin protein ligases

The proteasome works as an enzyme with catalytic sites on the inside. The protein is
forced into the proteasome and the ubiquitin molecules are released. Then the protein
is linearized and degraded.
Once you have four ubiquitin molecules, the protein can be degraded. Each E1 binds
an extra ubiquitin molecule until 4 or more are reached.
- Mono ubiquitination: histone regulation
- Multi ubiquitination: endocytosis
- Polyubiquitination: proteasomal degradation and DNA repair.
The shape of the modification can also lead to different finals.

5.BIOENERGETICS AND GLYCOLYSIS
ENERGY CONVERSIONS AND METABOLISM
The ultimate source of energy is the sun.

The energy comes from process called fusion. it is the fusion of the nuclei of the
Hydrogen that turns into Helium (4 atoms of hydrogen become 1 atom of Helium). The
thing is 4 atoms of H = 4,032 daltons and 1 atom of He = 4,003 daltons. There is some
mass lost = 0,029 daltons. This mass is the one used to calculate the energy obtained
from the sun: 2,7*10^^2 J/mol (A LOT).
The energy from the sunlight + H2O +CO2 from the atmosphere is transformed into
organic molecules in a process called photosynthesis.
Once we have this organic molecules we oxidize them in a very controlled process (to
maximize the energy obtained). This process occurs in many small steps that are
catalyzed by enzymes.
Oxidation is not simply the addition of oxygen molecules. It is the removal of electrons
of an atom, but in general, it implies the addition of an oxygen (NOT ALWAYS). When
oxidation occurs, there is a reduction happening at the same time (Redox reactions).
When the carbon atom is very reduced, Hydrogen surrounds it. When carbon is
oxidized, oxygen surrounds it.
The oxidation of glucose produces 4kcal while fatty acids provide 9kcal. This is
because in fatty acids there are more carbon atoms that can be oxidized.

FREE ENERGY FROM ATP AND OTHER HIGH ENERGY COMPOUNDS
The energy that comes from the oxidation of this bond comes in the form of adenosine
triphosphate: ATP. But it is not the only energetic molecules, it is like if it was a dollar
but there where other molecules that were euros or pounds.
The energy that is stored in ATP is released by hydrolysis (breaking high energy bond
with water) and you can couple this energy with reactions that have a positive delta G.
The most energetic molecules is phosphoenolpyruvate.
Catabolism: polymers → monomers (energy release)
Anabolism: monomers → polymers (energy input required)
GLYCOLYSIS AND FERMENTATION
The storage of glucose doesn’t bother osmotic equilibrium as it ha a high molecular

weight.
Glycolysis is a partial oxidation of sugar molecules.
Glycolysis is the transformation of glucose into pyruvate.
Glycolysis is the partial oxidation of glucose into pyruvate by a series of enzyme
catalyzed reactions during which some of the energy released is stored as ATP or
NADH.
It occurs in the cytoplasm of the cells (which means that it can also occur in
prokaryotes!!).
The pyruvate then goes to the mitochondria and the full oxidation occurs there.

Glucose (6C) → Pyruvate (3C) + Pyruvate (3C)
But glycolysis is not the only pathway glucose may follow:
- Extracellular matrix and cell wall polysaccharides → synthesis of structural
polymers.
- Glycogen, starch, glucose → storage
- Pyruvate → oxidation via glycolysis
- Ribose 5-phosphate → oxidation via pentose phosphate pathway.
It is important to remember the shape/s of glucose:

The cycle is between carbon 1 and 5.

Normally we will see the cyclic form.
The difference between fructose and glucose is that in carbon 2 in glucose we have a
ketone instead of an aldehyde.
Key concepts in glycolysis:
- It literally means sugar splitting
- 10 enzyme catalyzed reactions

- 1 molecule glucose (6C)--> 2 Pyruvates (3C)
- A small amount of ATP is produced.
- Produced reductor power in the form of NADH which can be used to make more
ATP in the mitochondria
- The enzymes are located in the cytosol
- No oxygen is required (anaerobic)
There are 3 reactions but we only need to know by heart 1, 3 and 10. The three of
them are irreversible, once they happen you can’t return.
Phases in glucose:
- Energy investment phase
Glucose getting into the cell by some transporters in the membrane (that depend on
which cell it is).
Step 1: Phosphorylation in carbon 6 by a family of enzymes called hexokinases
(transferase). To add a phosphate in carbon 6, it first requires an input of energy from
ATP. This is a favorable process because of the hydrolysis of ATP.

Step 2: Isomerization of glucose 6 phosphate into fructose 6 phosphate (ring is now
formed between carbon 2 and 5). Somehow this allows carbon 1 to be additionally
phosphorylated in next step.
Step 3: Catalyzed by the phosphofructokinase 1(another transferase). It
phosphorylates fructose 6 phosphate and it becomes fructose 1,6 - bisphosphate. As
in the first step, it also requires an input of ATP. Energetic and irreversible steps.

Phosphofructokinase 1 (PFK-1) is an enzyme that is extremely regulated by different
molecules:
- Protein level: post translational modification
- RNA level: action of insulin
- Activity: action of fructose 2,6 - diphosphate (F26BP → allosteric regulator and
activator of the enzymes) .

The PFK1 kinetics change dramatically in the presence of Fructose 2,6
Bisphosphate. The enzyme doesn’t work in a standard Michaelis Menten
function but as an allosteric molecule. In low levels of substrate there is no
sensitivity of it until it reached very high levels.
- Cleavage phase
Step 4: Fructose 1,6 bisphosphate is converted by aldolase into 1
dihydroxyacetone phosphate and 1 glyceraldehyde 3 phosphate.
Step 5: Dihydroxyacetone phosphate is converted into glyceraldehyde 3
phosphate by triose phosphate isomerase.
- Energy liberation phase
Step 6: Glyceraldehyde 3 phosphate (2 molecules), there is an oxidation G3P
that requires NAD+. NAD+ reduces and forms NADH and the aldehyde groups
transforms into COOH.
Then, the product is phosphorylated by an inorganic phosphate to form 1,3
Bisphosphoglycerate. But this phosphorylation occurs by an ester. (Ester
arriba, eter abajo)
Step 7: ATP formation by substrate level phosphorylation. Transfer of a

phosphate from 1,3 bisphosphoglycerate done by phosphoglycerate kinase.
(1 molecule ATP/molecule * 2 molecule = 2ATP)
Summary of 6 & 7:
NADH production in
6 and ATP
formation in 7.
Step 8: 3-phosphoglycerate becomes 2 phosphoglycerate by
phosphoglycerate mutase.
Step 9: 2 phosphoglycerate becomes phosphoenolpyruvate by the action of
enolase (1 water molecule is required)
Step 10: Phosphoenolpyruvate is converted into pyruvate by the enzyme
pyruvate kinase. This process involves the dephosphorylation of
phosphoenolpyruvate and the phosphorylation of ADP.
(1 molecule ATP/molecule * 2 molecule = 2ATP)

GLUCOSE + 2 ADP + 2 Pi + 2 NAD+ → 2 PyruvIc Acid + 2 ATP + 2 NADH + 2H+
Fates of pyruvates:
- Acetyl CoA and further oxidation in Respiration
- Lactate (Fermentation)
- Acetaldehyde → Ethanol (Fermentation)
There are two kinds of fermentatoin:

- Lactic: The product is lactate. Eukaryotic and Prokaryotic. Muscles.

Lactobacillus produces lactate out of the pyruvate.
Ketone group in the pyruvate is reduced into an alcohol group in lactate (Lactate
dehydrogenase). In this step the NAD+ is regenerated and it can be reused.
If do not completely oxidize glucose in our muscles, it will undergo fermentation
and lactate will form → AGUJETAS
- Alcoholic: The product is ethanol.
Decarboxylation of pyruvate (Pyruvate decarboxylase): Pyruvate loses its
carboxyl group and 2 CO2 molecules are produced.
Acetaldehyde (2 molecules) is converted by alcohol dehydrogenase into
ethanol (2 molecules).
Humans cannot do alcoholic fermentation because we lack pyruvate
decarboxylase.
If nature made us have pyruvate decarboxylase it would also give us a way to
detoxify so we don’t generate ethanol under ethanolic conditions.

Muscle activity
Example:
When running for 30 minutes, your body does glycolysis with the glycogen stored.
When sprinting, your body uses creatine phosphate to obtain energy.
But if oxygen is present, you will deplete completely de glycogen and you will start
using fatty acids.
ATP is not used by our muscles, they use different source of energy depending on the
duration of the activity.
- LEss than 30 seconds: phosphocreatin
- More than 30 seconds: glycogen
- Extended time: oxygen from blood → oxidative phosphorylation
- Long time: Fatty acids via beta oxidatoin
WARBURG EFFECT
It is fermentation under aerobic conditions. Instead of undergoing cellular respiration,

pyruvate undergoes lactic fermentation.
Differentiated tissues in the presence of oxygen use it to do oxidative phosphorylation.
However, when oxygen is limiting, cels car redirect pyruvate towards the generation
of lactate. However, the production of ATP is minimal.
It was observed that cancer cells tend to accumulate lactate due to this process,
regardless of whether oxygen is present.

6. CELLULAR RESPIRATION. KREBS CYCLE
AND OXIDATIVE PHOSPHORILATION
REVIEW OF GLYCOLYSIS

INTRODUCTION
Krebs cycle is essential in the metabolism of the cell since it is a crossroad between
anabolism and catabolism. It consists of the oxidation of the remaining carbons in
pyruvate which leads to the production of CO2 and NADH and FADH”. This last two
will be used later in the Electron Transport Chain.
The main enzyme involved in ATP production during this two processes is ATPase,
located in the inner mitochondrial membrane.
The brain can also work with ketone bodies. Diabetes I patients may form ketone
bodies when they do not control the blood sugar. THis bodies are good for the brain
but very bad in blood.
The rest of our body can work with Lipids but the brain can only work with glucose.
Glycogen - polymer of glucose
Starch - polymer of glucose in plants

Fat - many lipids all together. The red things in the picture are fat deposits in the
cells.
We have 2 main sources of energy: glucose and fat. But in high starvation state, our
proteins can also be used as energy source.
the main energy source in a healthy person is fat. Glucose is also good but it is of a
very fast use while fat lasts longer. In the case of starvation, as a last resource for
your body, you can also use proteins.
Sugars and fats are absorbed by the cell and transformed into acetyl CoA
Pyruvate from glycolysis travels to the mitochondria and becomes Acetyl CoA by the
action of pyruvate dehydrogenase complex. This process is also known as oxidative
decarboxylation
It is a three step process:
1. Decarboxylation of pyruvate
2. Reduction of NAD+ to obtain

3. Addition of Coenzyme A
The process of transforming pyruvate into Acetyl CoA is not only irreversible but also
highly controlled because depending on the feeding situation of the user it needs to
be stopped or enhanced.
Once the ACoA is formed, the production of CO2 or the use of ACoA to produce lipids.
If there is a lot of food intake, or a lot of energy; ACoA transforms into lipids. The other
way around if you are starving, lipids will be reversed transformed into ACoA which
will be used in cellular respiration.
To produce ACoA from fats:

1. Degradation of fat. We can have three fatty acids in a molecule called
triacylglycerol. This molecule is hydrolyzed and transformed into fatty acids and
glycerol. This step is catalyzed by a lipase. Fatty acids go into the bloodstream
and are absorbed by the cells in need.
2. Formation of ACoA: These cells oxidize the fatty
acid, releasing ATP, and producing CO2. Acyl CoA
synthetase with ATP and CoA you synthesize what
is called as Fatty acetyl CoA that goes into the next

state.
The process is known as beta oxidation. The fatty
acids will be degraded two by two carbons, producing a molecules of ACoA
from each loop the cycle takes.
IMPORTANT → N carbons yield to N/2 acetyl coenzymes A.
KREBS CYCLE (TRICARBOXYLIC ACID CYCLE - TCA)
It is the source of reduction power (NADH and FADH2) that will be transformed into
energy.
It is the source of CO” from oxidative decarboxylation of TCA
It is also the crossroad between anabolism and catabolism.

The enzyme that catalyzes the conversion from Oxaloacetate to Citrate is Citrate
synthase.
Oxaloacetate Citrate synthase Citrate
Citrate Isocitrate
Isocitrate alfa ketoglutarate 1 NADH

1 CO2
alfa ketoglutarate succinyl CoA 1 NADH

1 CO2
Succinyl CoA Succinate 1GTP
Succinate Malate 1 FADH2
Malate Fumarate
Fumarate Oxaloacetate 1 NADH

The net result of the citric acid cycle is three NADH, one GTP, one FADH2 and 2
molecules of CO2
The KC is a clearly catabolic cycle. But some of the molecules involved are
intermediate molecules in the synthesis of other important biochemistry molecules
which are aa, purines, pyrimidines, fatty acids… This are called anaplerotic reactions.
OXIDATIVE PHOSPHORYLATION
Introduction
Its main step is the Electron Transfer Chain: oxidation of NADH and FADH2
Produces and electrochemical gradient
Production of ATP from the gradient.

ETC occurs in between the membranes of the mitochondria.
ELECTRON TRANSPORT CHAIN
The electron transport chain is a set of proteins that have metals and that are the

responsible of the movement of the redox reactions. This protein complexes are
ubiquinone, cytochromes a, b and c, other heme containing proteins and iron sulfur
proteins.
The transfer of electrons goes in the direction of the picture, from lower to higher
potential. The acceptor of electrons is oxygen and will become water.

The four complexes where electrons move are:
1. NADH dehydrogenase
2. Succinate dehydrogenase
3. Ubiquinone cytochrome c oxidoreductase
4. Cytochrome oxidase.
Electrons don’t always have to enter in the first complex, they may enter through the
second, as if the ones that come from the conversion of succinate to fumarate.
I transfers to III. H+ pumped

II transfers to III.
III transfers to IV. H+ pumped
IV transfers to O2. H+ pumped
Electrochemical gradient
As electrons fall into lower potentials due to the ETC, protons sre pumped to the
intermembrane space.
Transport of an elecron yields energy used for:
- Chemical gradient: pumping of protons
- Potential difference: + charges outside - charges inside
Energy in the electrochemical gradient: E=-RT(pHo-pHi)
Electromotive force is dissipated by ATP synthase.
Formation of ATP
The flowbak of electrons through the ATP synthase casuses that ADP and Pi bind
together forming ATP.

Some respiratory poisons are CN- and CO.
Higher concentration of protons yield to a lower pH.

The translation of the gradient of protons is not a biochemical reaction but more like a
mechanical mean.
Some of the experiments that lead to the DISCOVERY THAT THE PROTON
GRADIENT WAS RESPONSIBLE OF THE SYNTHESIS OF ATP.

If you have a mitochondria isolated from
cells you can do this experiment which is
just changing the pH in the
compartments of the mitochondria. You
need a buffer with a higher pH, ADP,
Phosphate and some tricks for the
mitochondria.
YOu have to copartments with protons at
the same concentration, and then you
lower the concentration in one of them

and you block the movement of
potassium. You get that the mitochondria
has different pH in the matrix and the
inner membrane.
EXPLICAR EXPERIMENTO
Coupling of electron transport chain to ATP synthesis EXAMEN!!!
RESPIRATION POISONS
The two graphs are coupled, one follows the other and vice versa.
You need a source of ATP
and Pi and a source of
Electrons (succinate in this
case)
At the beginning when oyu
don’t have electrons, the
graph stays flat. When you
add e- the ETC begins to
work and the production of
ATP is increased due to the
proton gradient. When you
add an ETC blocker, no more
oxygen is consumed bc electrons won’t reach it and when this happens, the proton
gradient will be dissipated and no more oxygen will be consumed.
Uncoupling drug
There are two possibilities, to block the ETC (ATP synthesis will be blocked) or you
can block the ATP synthase.

If you add venturicidin or oligomycin, which are ATP synthase blockers, the proton
gradient starts to accumulate like a balloon. The oxygen is still being consumed (at a
lower rate) but the ATP is not being synthesize.
How can I differentiate between a block in
the ETC or inthe synthase? By using drugs
known as uncoupling drugs. What they do is
to make a hole in the inner membrane and
all the protons go through the whole and
they don’t go through the ATP synthase, this
way they dissipate the proton gradient.
When this happens, you trick the
mitochondria so the two lines in the graph
are not coupled anymore, and the balloon
effect disappears. Since there is a new
proton source, the ETC is now unblocked,
oxygen will receive more e- and therefore its
consumption will increase while the ATP
won’t be synthesized.
ESTO CAE EN EL EXAMEN.

There are proteins that are real that are also unblockers like the one present in the
brown fat. It has special mitochondria that are natural uncouplers and the protons
instead of producing ATP produce heat passing through the channel.

7. BIOSYNTHETIC AND DEGRADATION
PATHWAYS
CARBOHYDRATES
- Gluconeogenesis
Transforms pyruvate into glucose.
Many steps are reversible but 1, 3, 10 are not. (Just as in glycolysis bc it is the inverse).
1. Conversion of Pyruvate to PEP: Pyruvate is translocated in the mitochondria
and transformed into oxaloacetate and this is reduced to malate which is
transported to the cytosol and reoxidized. Then PFP carboxykinase produced
PEP.

2. & 3. Many catalyzed by phosphatases. Fructose 1,6 bisphosphate promotes
the irreversible hydrolysis of C1 phosphate and Glucose 6 phosphatase which
recovers glucose from glucose 6 Phosphate.

PYR-[pyruvate carboxylase] → OXALOACETATE -[malate dehydrogenase] →
MALATE -[phosphoenolpyruvate carboxykinase] → PEP -... → F16BP -[F16
bisphosphatase]→ F6P → GLUCOSE 6 PHOSPHATE -[glucose 6 phosphatase] →
GLUCOSE
- Glycogen synthesis and hydrolysis

Glycogen is a compact glucose storage molecule. It is like short time fuel. IT will be
used before fats.
Synthesis: Molecules of glucose are added to form a long homopolysaccharide. Large
amount of hexose units can be stored without altering osmotic concentrations. The
enzyme involved is glycogen synthase. To start the reaction you need 4 molecules
already bonded together so Glycogen synthase can start adding residue from UDP
Glucose to the non reducing end of the chain forming a new 1→ 4 linkage.
Glycogen branching enzyme (Carbon 1-6). It makes little branches making the
molecule more thermodynamically stable.
Hydrolysis: Glycogen phosphorylase separates a glucose at the non reducing end via
phosphorylation. This yields to glucose 1P which is later on transformed into G6P by
phosphoglucomutase. This final molecule can undergo glycolysis.
Glucose (n+1) --[Glycogen phosphorylase + Pi]-> Glucose (n) + Glucose 1 Phosphate.
Glucose 1 phosphate --[Phosphoglucomutase]-> Glucose 6 phosphate→ Glycolysis

This two processes are highly regulated
by cell signaling. Insulin promotes the
incorporation of glucose via Glut4 and the
activity of hexokinase and glycogen
synthesis. It also phosphorylates the
glycogen synthase kinase, preventing it
form inactivating GS and promotes the
activity of PP1.
Glycogenesis → Insuline
Glycogenolysis → Glucagon and epinephrine.
Summary

Gluconeogenesis → Reversal steps of glycolysis: 10, 3 and 1. Enzymes (1,2,3,4 )
Glycogen synthesis enzyme (UDP-glucose) 1-4 links

Glycogen branching enzyme (1-6 links)
Glycogen Breakdown: Glycogen phosphorylase (GLucose 1 P ) and glycogenesis.
Pentose Phosphate pathway
Source of NADPH during process. It is part of the nucleotide formation process.
Glucose 6 phosphate is transformed into ribose 5 phosphate (used in nucleotides)
Deficiency of Glucose 6 phosphate in red blood cell is related with malaria .
TRIGLYCERIDES
Fat biosynthesis:
IT IS NOT THE REVERSE OF BETA-OXIDATION.

1. Synthesis of fatty acids
ACoA + CO2 -[Acetyl CoA Carboxylase]→
MAlonyl-CoA (real precursor) Consumes ATP.
1 enzyme (Fatty acid synthase) → 4 Steps to
row chain.
In each step two carbons are added (NADPH

releases CO2).
2. Synthesis of triglycerides
Glycerol 3 phosphate + 3 fatty acid -[acyl transferase] → Triglyceride
Acyl transferase sterifies alcohol groups.
AMINOACIDS
Non essential aa are produced by the body: Alanine, asparagine, aspartate, glutamate
and serine.

Essential aminoa acids need to be incorporated from food: Histidine, Isoleucine,
Leucine….
Urea cycle: Takes places in the mitochondria of the liver cells.

Mitochondria:
Precise aminoacid:
Amino group of the ingested aa is exchanged.
- WIth ketoglutarate: Which is converted into glutamate that releases ammonia.
- By the enzyme aminotransferase the now ketone group goes to the ingested
amino acid that is now alfa-Ketoacid.

Exam question: What happens when OTC is not working: Ammonia will accumulate
and it will be toxic to the liver.
8. CELL SIGNALING
INTRODUCTION
Transmission → Reception → Decodification → Response
Some of the signals to which cells respond: light (antigens), mechanical
(glycoproteins), neurotransmitters, nutrients…
THEY USUALLY HAVE A SPECIFIC RECEPTOR.
The signal finds a receptor (99% of the times is a protein). This protein changes its
conformation and triggers a cascade of events that end up in an action (cell growth,
movement, hormone secretion…)
This signal transduction patterns are combined. A combo of signals will have an effect
or modulation of this effect (not only one signal).
Usually the signals are at low concentration and the receptors bind to them with high

affinity.
RECEPTORS CLASIFICATION
Location
- Membrane: For hydrophilic signals. They need not a carrier.
- Nuclear: For hydrophobic signal molecules. They need a carrier protein.
SIGNALLING PROCESSES
How signals may act on a target cell depends on the distance between the signalling
cell and the target cell.
- Contact dependent: During development and immunity. Antigen presenting cell
and lymphocyte for example. Cells that are very close to each other and the
signal molecule attaches to the receptor that is in the nearby target cell. General
signalling way but the synapsis affects only to neurons. Synapsis is a kind of
contact dependent signalling that works only in neurons.
- Paracrine: The signalling molecule is close to the target cell (but not in contact!)
The signalling molecules travels to find a receptor but not very far away. The
message travels through a fluid that surrounds the cell (interstitial fluid), in
general; but it may also travel through blood but is not common. Skin: fibroblasts

in the epidermis send signals to the keratinocytes also in epidermis and vice
versa. This also happens in tumor cells
- Autocrine: Typical of cells that are transformed, tumorgenics. The signalling
molecules is produced and received by the same cell. For example tumor cells
produce their own GF and also produce their receptors.
- Endocrine: The message and the target are far away and the message travels
through blood (both conditions must be fulfilled). For example an endocrine cell
produces a hormone that travels through the bloodstream until the target cell is
reached.
- Synaptic: It is a double signal: The synapsis requires that the neuron is
activated in some way in order to release the neurotransmitter. Specific of
neuron - neuron and neuron - muscle. Neuron is excited → Neurotransmitter
released → Neurotransmitter finds receptor. The distance in the synapse is so

small that it can be considered as contact dependent.
PROPERTIES OF SIGNALLING MOLECULES
- Specificity: The signal molecules fits the binding site on its complementary
receptor, other signals don’t fit on the receptor.
- Speed of the effect of the signal ( not the velocity at which the signalling
molecule reaches the receptor).
- Fast: Adrenaline reaches the receptor in the muscle and the degradation
of glycogen begins. VERY QUICK.
- Slow: The ones involved in cell growth, they depend on the transcription
and translation processes before the effect happens; this takes hours or
even days. There are some exceptions, some genes may actually be
activated very quickly.
- Amplifications: When enzyme activated enzymes, the number of affected
molecules increases geometrically in an enzyme cascade.
- Integrations: Cells are not receiving one single message, they receive several.
In some cases they need to integrate or differentiate from the signals. Picture.
You need A+B+C to make the cell survive. When it receives additional singals:

A+B+C+D+E the cell survives and divides. But if it receives A+B+C+F+G the
cell differentiates. However if the cell does not receive any signal it will die.
Cross talk: YOu have two signals A and B which transduced through different
transduction mechanisms but in a given moment they affect the same effector
molecules that transduces the signal downstream.
TYPES OF RECEPTORS
We have different kinds of receptors because they use different transduction signaling
pathways. The receptor itself has some primary activities which are different.
There is a signal → transduction → effect on DNA most of the cases this will be the
activation of some genes and new proteins will be synthesized.
Receptor tyrosine kinases

This receptors are associated to growth factors, they are the receptors that will receive
the molecules that produce the growth of some cell. Paracrine signal. The receptor
has an endogenous enzyme activity, the receptor becomes an activated kinase.
Family of receptor tyrosine kinases: EGF R (stalin colin) epidermal growth factor
receptor; NGF R (rita levi montalcini) neurotrophic growth factor receptor. Buscar a los
investigadores en internet, the discovered how the neural tissue grow. MIRAR
DIAPOSITIVA, FALTAN NOMBRES
There are other factors like insulin, vascular endothelial growth factor, fibroblast
growth factor. (the thing in front of the R is the molecule that is being received).
Tabla de diferentes cosas que son recibidas a traves the RTKs.
Most of them stimulate proliferation of different kinds of cells.
Characteristics:
- Enzyme coupled receptors: The receptor crossing the plasma membrane has
associated an enzymatic activity. This activity is started when the ligand binds
to the receptor. In normal conditions they don’t do anything; but when they bind
to the signalling molecule, it produces a dimerization: the two molecules of the
receptor get together, and this leads to the activation of the enzymatic activity
of the receptor. The enzymatic activity is dormant while no signal attaches to
the receptor. It phosphorylates the opposite chain of the receptor. The receptor
on the left becomes activated and finds the substrate on the other chain. Across

phosphorylation occurs and they are recognized by the first transducers of the
cascade.
The receptor gets phosphorylated, the two chains are dimerized and
phosphorylated. Y (tyr) gets
phosphorylated. These phospho
tyrosines can trigger to response
MAPK pathway and PI3K - AKT
pathway.
and These are the pathways that led to effect, mostly cell growth.
The receptor gets phosphorylated by the attachment of the signal. Some other

molecules recognize this phosphorylated tyrosines.
Pathways:
- MAPK pathway: First a docking protein recognizes the phosphotyrosine and
attaches to it. The key molecule is the Ras molecule (it is a G protein (ras proto
oncogene); it is a typical signalling molecule; in the normal conditions it is in the GDP
form but after the beginning of the signal phosphorylation and docking of the molecule,
Ras transforms into GTP form, it becomes activated and binds a series of kinases
which activate the next in the cascade. So the cascade of the signaling pathway is
called MAPK SP. Raf = MAPKKK. Mek = MAPKK. Erc = MAPK. The molecules that is
up in the pathway is upstream of the following. Then MAPK will go to the nucleus and
activate whatever pathway. In order of this molecule to be activated it needs to be
phosphorylate.The phosphorylation of this molecules is produced by Erc, but this
molecule to be phosphorylated needs to be phosphorylated too, and that is produced
by the Ras. Ras phosphorylates Mek which phosphorylates Erc. But for this to happen,
Ras has to go from GDP to GTP.
Imagen. Ver video.
Dimerizacion → cross phosphorylation → Docking → SOS → Ras → Raf (Ras
phosphorylated) → Mek → Erc → nucleus.
- PI3K AKT pathway: PDGF receptor is activated and many tyrosines are
phosphorylated. Depending on which is phosphorylated, a different transduction
pathway will be started. IF the PI 3 kinase (regulatory subunit) is attached, the pathway
begins.
The PI· kinase signaling pathway starts with the cross phosphorylated receptor in
tyrosine, and an enzyme (a kinase) PI 3 kinase binds to the phosphorylated tyrosine.
This kinase does not phosphorylate a protein, it phosphorylates a membrane
phospholipid (phosphatidylinositol - PI(4,5)P2). and now we have a PI(3,4,5)P3. This
molecules will be the docking and will find other molecules and will bind to it and will
continue a kinase cascade.
PI(4,5)P2-[PI3kinase] → PI(3,4,5)P3 when this happens, PDK 1 and Akt attach to the
PI(3,4,5)P3 and it activates PDK1 phosphorylates akt. One t is phosphorylated, it
detaches from the membrane and phosphorylates some other molecules.
PDK1 se añade a la membrana. Akt se añade a la membrana. Se encuentran. PDK 1

fosforila Akt. Akt se quita de la membrana y se va a fosforilar.
For example, when insulin binds to the receptor, it starts the PI(3,4,5)P3 cascade to
activate glycogen synthase and synthesize glycogen.

Akt phosphorylates GSK3 (glycogen synthase kinase) and it becomes inactive. when
this molecule is inactive the glycogen synthase cannot be phosphorylated by GSK3
(because normally GSK3 phosphorylates glycogen synthase and it gets activated by
phosphorylation.) so it will work perfectly synthesizing glycogen. Si GSK3 se fosforila,
no puede fosforilar glycogen synthase. It is the inhibition of the inhibitor.
Laboratory example:
This is a western blot, bueno 3, from using three different antibodies. 1 anti
tubulin, 1 anti Akt, 1 anti phospho akt.
When insulin is absent, there is more Akt and no Aktp. When insulin is present there
is a little bit less of AKt and little bit more of AKtp.
Phosphorylated Akt is only present when insulin is present.
This means that akt is phosphorylated when insulin is added.
How would you recognize if the PI3KT AKT pathway is active? check if when adding
insulin, pakt appears.
Insulin may act as a growth factor. One case is when women get pregnant and they
don’t control the glucose metabolism-> pregnancy induced diabetes. Los hijos suelen
ser gordos al nacer y la madre gorda pa siempre.

This happens when insulin follows the MAPk pathway (only when there is a lt of
insulin).
After the baby is born the mother goes back to normal conditions, this is something
that has to be controlled.
This happens because insulin gets crazy and starts to function as a growth factor.
Receptor cytokine
Molecules involved in the immune system. They are receptors mainly for growth factrs.
Comprobar.
JAK/STATs Signaling Pathway
Jaks are intracellular non receptor tyrosine kinases.
STATs transcription factors.
There are different signaling proteins that are received by different receptor associated
to a kind of JAKs and STATs from the family. what changes is the receptor bu also the

receptors engage different combos of JAKs and STATs. Anyhow, most of them
stimulate the growth of different things (mostly immune system, but no only).
Pathway:
Interferon binds receptor → dimerization of the receptor: this doesn’t trigger the
activation of the pathway but engages the next step. → autophosphorylation of JAK
and phosphorylation of the receptor: once the JAK is phosphorylated, the receptor
becomes phosphorylated and therefore activated. Now instead of engaging the
MAPKs, it ngages the STATs. → Binding to receptor and phosphorylation of STATs
by JAKs→ Nuclear translocation of STATs→ Binding to interferon response element →
Expression of genes

There are no covalent bonds between the receptor and the JAKs.

The cytokine first binds and that induces the cross link ofthe adjacent receptors. Then
JAKs phosphorylate each other on tyrosines. The activated JAKS phosphorylate
receptors on ttyrosines. THis make for a docking place for STATs, and they are
phosphorylated by JAKs. When this phosphorylation occurs, a conformational change
in the STAT occurs and they are released from their phosphate group. The bind to
each other (dimerize) via their SH2 domain. This dimer is the effector molecule
because it migrates insie the nucleus and it’s a transcription factor itself.
When the JAKS get phosphorylated, the receptors becom etyrosine kinase.
DIFFERENCE WITH MAPK: MAPK are not transcription factors, they enter the
nucleus and phosphorylate transcription factors. STATs are transcription factors
themselves.
Exercise from an exam:
Interferon gamma (IFNγ) activates peritoneal macrophages, resulting
inenhanced bacteria killing and protection of mice against lethal doses of
Listeria monocytogenes infection through enhanced production of a protein
called Listericyn. STAT1 mediates IFNgamma signalling. Considering this
information and using the diagram in the figure give the appropriate names to
the signalling/component molecules and explain the events that take place in 1,
2, 3 and 4.

Red triangle: Interferon
gamma
2. Interferon gamma receptor
(which is also a cytokine
receptor but that is too
general)
3. STAT
4. STAT 1 dimer

5. Listerycin gene is the gene
transcribed
Recapitulating: Receptor tyrosine kinases: HAve endogenous kinase activity ad the

autophosphorylation of the receptor engages either MAPK pathway or PI3K pathway.
Cytokine receptors: Do not have enzymatic activity. The associated molecules to the
receptor is the JAK. There is no phosphorylation unless the JAK auto phosphorylates
and activates the receptor.
EPO - famous protein for doping. The normal activity of EPO is going through the
STAT pathway and one of the target genes is the globin → leading to more hemoglobin.
In some cases EPO may become a growth factor and instead of regulating the amount
of hemoglobin in blood, they may engage in a cross activation of the MAPK pathway
and this could also be like a growth factor for some tissues (hematopoietic).
G protein coupled receptors
This pathway involves not the typical cascade of phosphorylation but also an enhancer
that ys the cAMP:
The first messenger is the signalling molecule but after this happens there is a second
message that amplifies more the signal.
G protein coupled receptor doesn’t have any enzymatic activity. IT is associated to the
receptor and when the receptor binds to the signalling molecules, it trigger the
activation of the alfa subunits of the associated proteins which are bounded to GDP
and when the receptor binds to the signal, it becomes to GTP which is functional and
activated in this form.
There are many GDP or GTP binding
proteins and some are associated to G
protein coupled receptor and otehrs are
part of the RAS family.
there is always a GDP/GTP exchange
factor or GEF.
- RAS family: SOS.
- Receptor coupled: Receptor.
There also modulators that goes the

opposite way: GTP/GDP.
Exchange factors: te lo ponen ellos de su
propia mano.
Modulators: Son GTPasas.
The guanine exchange facto rfor this
GTP binding protein is the receptor.
The receptor activates by binding of
th eligan and triggers the exchange
of GDP for GTP and then onces this
alfa subunit is activated it produces
some conformational changes in the
beta and gamma subunits.
Alfa subunit: G protein → activated
and exchanges GDP and GTP.
When this happens the alfa subunit
dissociates and binds to AC
(adenylyl cyclase) which transforms

ATP into cAMP and this becomes a second messenger which activates an intracellular
kinase: protein kinase (PKA)
GPCR → cAMP → Activity PKA.
And this will phosphorylate other things.

AMPLIFICATION: Epinephrine is one of the things that this kind of receptors receive.
Epinephrine binds to the receptor and it produces 20 molecules of cAMP. Each
molecule will activate 10 molecules of PKA. each PKA activates 100 molecules of
PKB. PKB activates 100 molecules of glycogen phosphorylase. Each GP will produce
10000 glucose molecules.
Signal → cAMP → PKA → PKB → Glycogen phosphorylase
Three phosphorylations that lead to the activation of glycogen phosphorylase.

TGF Receptors

Cascade of phosphorylation.
RTK - direct tyrosine phosphorylation
CR - indirect tyrosine phosphorylation
TGF - serine threonine phosphorylation
This receptors are oposie to the cytokine or receptor tyrosine kinase. when they are
activated in general they are blocking or decreasing proliferation of a given tissue.
They are anti proliferative receptors.
cAMP is also a serine threonine kinase.
The TGF beta (ligand) binds to the receptor and the type II TGF beta receptor
phosphorylates the opposite chain of the receptor. The type I receptor recruits and
phosphorylated SMAD 2 and 3, and dimerizes them; which dissociates from the
receptor and oligomerizes with SMAD4.
Type II activates type I → activates SMADS 1 2 5 or 9 → SMAD released from the
receptors and dimerizes with other SMAD forming the heterodimer that are inhibitors
of transcription.
Muscled cow: the TGF beta is not working. It normally inhibits proliferation. When the
receptor has a mutation instead of inhibiting proliferation it enhances it.

The ligand is not TGF beta but myostatin (same family though). Myostatin blocks the
proliferation of the muscle. If the inhibitor for myostatin doesn’t work, this proliferation
won’t be inhibited and the muscle will grow without control.

9. CANCER
CANCER BASICS
Cancer cells break that basic rules of cell behavior.

If a mutation affects a cell and makes it grow more vigorously than others, that cell
may become the parent of a non-controlled colony of mutant cells.
Cancer is an abnormal growth of cells that tend to proliferate in an uncontrolled way.
Cancer cells have two main characteristics: the proliferate at a rate faster than
allowed on normal cell growth and the invade and colonize territories normally

reserved for other cells.
The process cn occur in any tissue as well as in blood.
Cancer starts in a tumor and they can evolve into a metastasis (when it spreads via
blood or lymphatic system).
A tumor is considered benign unless it invades other cells. Cancer is only when the
tumor is malignant, that means, it colonizes and invades spaces reserved for other
cells. Another characteristic for a tumor to be malignant is its capability to spread
through the lymphatic system or blood.
In the early stages when the tumor is small, it can be extracted through surgical
procedure
Tackling cancer development is fundamental to improve life quality in developed
countries. The target for most therapies is the early stages of cancer, when it can be
handled genetically.
An adenoma is a benign tumor of a glandular structure or of glandular origin.
A carcinoma is a malignant tumor of epithelial origin.
This image shows the progression from adenoma to carcinoma.
Most cancers derive from a single mutant cell. They can be traced to its origin in an
specific organ where a cell experiences a mutation that loows it to outgrow its
neighbors.
Tumors arise as clones of this transformed cell. This clones usually diverge giving
rise to different clones through a secondary or tertiary genetic alteration.
Clones and subclones may originate in progenitor cancer cells. All of which
constitute a spectrum of cells with different genetic alterations and states of
differentiation. The heterogeneity contributes to differences in clinical behavior and
responses to treatment of tumors of the same diagnostic type.
CANCER CAUSES
If a single cell can give rise to a tumor that means that the mutation is heritable.
Epigenetic regulation plays an important role in the development of cancer but we
know that cancers development s mainly due to DNA mutations (directly on the
sequence). This alteration are usually somatic but some germ line mutations can
predispose a person to heritable or familial cancer.

However, a single genetic change is not sufficient to give rise to a malignant tumor.
Most evidences point to a multistep process of sequential alterations.
Tumor progression= rounds of random inherited mutations + natural selection.
Cancer arises from a cell population (descendants of an aberrant cell) that evolve
from bad to worse through a sequence of mutations. In each round of the cycle, one
of the cells will also acquire a mutations that gives a selective advantage, making its
offspring proliferate and becoming the dominant clone.
Some mutations can be induced due to the exposure to some agents. Occupational
exposure (por tu trabajo – limpiadores de chimeneas) usually relates to specific
cancer types. Some chemical agents are both mutagenic and carcinogenic like
benzopyrines. Ionizing radiation can also produce damage at DNA level leading to
cancer.
Two fundamental steps:
2. Initiation: Due to the exposition to a carcinogenic agent or due to random

mutations, a cell suffers a
mutations that allows for
proliferation (advantage).
Repeated exposure to
initiators can cause
cancer but that is not
what normally happens.
3. Promotion: Promoter
agents are not
carcinogenic nor they
produce mutations in
cells but they facilitate the
proliferation of the tumor
by releasing the
restraints. Many closely
in time promotion steps
can produce cancer. If
the promotion occurs widely spaced in time from the mutation it can giver rise
to cancer too, but it’s not common. During promotion, further mutations may
occur.

ONCOGENES
Proto oncogenes are the genes that encode dominant acting proteins that control
cell proliferation apoptosis or both. When one of this genes suffers a mutation, it
becomes an oncogene and it stops regulating the process that it is supposed to
regulate, giving rise to proliferation. Normally cells are regulated by homeostasis,
when homeostasis is disturbed, the cell begins to malfunction.

This genes are activated by structural alterations resulting from a mutation or gene
fusion, by juxtaposition to enhancer elements or by amplifications.
Translocations an mutations can occur as initiation evens or during tumor
progression, but amplification often occur during progression/promotion.
A point mutation or deletion can produce a hyperactive protein made in normal
amount but when the mutation is in the regulatory sequence, this can lead to
overproduction of the protein.
Gene amplification has a similar effect, overexpressing a protein that works normally.
Chromosomal rearrangement as in the case of Philadelphia chromosome also plays
a role: translocation can make a gene be regulated by a sequence that is not its
regulatory sequence; overexpressing it or forming a hyperactive fusion protein.
The products of oncogenes can be classified:
- Transcription factors
- Chromatin remodelers
- Growth factors
- Growth factor receptors
- Signal transducers
- Apoptosis regulators.
One of the most important cases is those that act on signaling pathways such as
Ras. Mutant Ras can’t hydrolize GTP making it ermanently active and activating the
MAPK pathway.
Many oncogenes can alter the regulation of the cell cycle. Cell cycle control is based
on a connected series of switches. Adult or starving cells are usually in G0 but they
reenter the cycle when they receive growth factors. The key controllers of the cycle
are the CdKs. In a resting cell the retinoblastoma protein binds to the regulatory
proteins preventing the synthesis of proteins required fro proliferation.
The activation of a mitogen leads to a signal transduction pathway in which Ras

relays the signal to the MAPK. The activated transcription factors promote the
synthesis of gene regulatory proteins such as Myc that lead to the expression of
cyclins and the activation of CdKs.
The kinases phosphorylate the Rb protein, inactivating it and allowing other
transcription factors to become active. This in turn produces DNA synthesis and a
positive feedback loop by promoting phosphorylation.
TUMOR SUPRESOR GENES
Tumor suppressor genes are those that with a loss of function can contribute to cancer.
The function of both alleles of the critical gene must be lost to drive a cell towards
cancer. A single mutation is not sufficient.
These genes are normally expressed, and their function is to prevent an uncontrolled
proliferation. When the cell is signaled to over proliferate. (mutation, telomere
shortening etc) they become active and induce the cell
arrest, senescence or apoptosis. When they cannot
perform properly, mutations tend to accumulate, and
the cells start proliferating at a faster rate, which may
lead to cancer.
Oncogenes and tumor suppressor genes usually act in

an alternating sequence.

Example: P16 is a tumor suppressor which inhibits the formation of the cyclin D-CdK4
complex, which inhibits Rb by phosphorylation. This in turn will inhibit E2F that will
transcribe the genes that regulate the entry in S phase. Thus a gain of function of an
oncogene or a loss of function of a tumor suppressor gene will alter the sequence of
inhibition leading to the last step.
In a non-proliferative cell, p16 binds to the CdKs and prevent cyclin binding, thus
maintaining Rb active. In proliferative cells with a mutation in P16, the protein is unable
to prevent the binding so Rb becomes phosphorylated and the transcription regulators
are released.
P53 is one of the main tumor suppressor genes that becomes activated either by
DNA damage or oncogenes. When there is DNA damage, the ATM/ATR kinases
become active and they activate the chk1/chk2 kinases. These last ones

phosphorylate p53 preventing the binding of mdm2 (which usually binds to p53 for
ubiquitination. Non-degraded p53 regulate the expression of proteins such as P16 or
P21 locking the CdKs and inhibiting proliferation. When oncogenes are overactive
such as My, a protein called Arf becomes active and sequesters the mdm2 releasing
p53 and leading to the same result.

Other tumor suppressor gene is the PTEN which undoes the action of PI3K.
Some viruses contain genes that can easily lead to cancer. Such is the case o the
papilloma virus. E7 captures Rb while E6 inactivates P53, allowing the
hyperproliferation.

ENVIRONMENT – ANGIOGENESIS
Once a tumor begins to grow and to undergo metastasis, the nutrient supply is smaller
because the tumor is very big. Since the tumor can only exist if there is enough oxygen
and nutrients, new vessels to transport it are required.
Angiogenesis is the production of new bloods vessels from preexisting ones. Tumors
can continuously grow if there is enough oxygen and nutrients, so new blood vessels
are needed.
- Vasculogenesis is the formation of blood vessels by differentiation from progenitors. It
creates the primary network of vascular endothelial cells that will become major blood
vessels. This happens when you get hurt too. Vasculogeneiss is used when it is

necessary. THE RULER.
- Angiogenesis: Sprouting of cells from preexistent endothelial cells of the vessel wall.
Angiogenesis happens when it is related to cancer cells.
Under low oxygen conditions, HIF (hypoxia inducing factor) starts to work. IT is I
charge of sensing that we have a low concentration of oxygen. Tis protein binds to
specific portions of the cell, it is a transcription factor. It activates some proteins that
will try to solve the situation of low oxygen: VEGF (Vascular Edothelial Growth Factor)
.
Tumor angiogenesis starts with cancerous tumor cells releasing molecles that send
signals to surrounding normal host tissue. This signaling activates certain genes in the
host tissue that make proteins encourage growth of new blood vessels. When there is
high oxygen some hydrolases are able to add hydroxyl groups to HIF. This
modification allows the binding of VHL and the ubiquitynation. When there is not
sufficient oxygen, the PTM does not occur, HIF is active and activates the transcription
of the signals such as VEGF.
Avastin is a drug that is injected in the blood of people that suffers any kind of tumor
and it blocks VGEF, which is fundamental in angiogenesis. When VGEF is received,
they start sprouting, providing the tumor with a vascular system. A high expression of
VGEF with a mutant Ras will probably cause a tumor. The opposite however is not
true.

Any drug that finishes in -mab means monoclonal antibody. It is specific, so it has to
be specifically modified to specifically bind VGEF.
- Glioblastoma, cancer in the brain. It is really difficult to treat because you have a
physical frontier.
- Carcinoma – epitelial cells that cover the inside and outside surfaces in our body.
Depending on where they begin they will have different names
o adenocarcinoma – epithelial cells that produce fluid sor mucus;
o basal cell carcinoma – begins in outer layer of skin;
o squamous cells carcinoma – right beneath the outter Surface of the skin, also
stomach, intesitnes, lungs,, bladder…
o transitional cell carcinoma – transitional epithelium: ureters, part of the kidneys.
- Sarcoma: bone and soft tissues. OSteosarcoma is the most common cáncer in bones.
- Leukemia: blood forming tissue of the bone marrow.
- Lymphoma: lymphocytes. They affect cells of the immune system.
- Multiple myeloma: plasma cells
- Melanoma: cells that become melanocytes.
- Brain ans spinal cords

10. CLINICAL BIOCHEMISTRY
INTRODUCTION
It involves the study of different molecules and markers of health and pathology to
perform a diagnostic or profiling process. It involves studying molecules present in
specimens and determine their concentrations to see if they are at abnormal ranges.
Biomarkers:
- Electrolytes
- Acid base homeostasis related products
- Glucose
- Lipids
- Proteins
- Hormones
- Degradation product/ metabolites

- Toxics
- Microorganisms
Most of these parameters are regulated by the endocrine system.
In the last decades the methods used in clinical biochem have improved,
nowadays many things are done by complex machines.
SPECIMEN TYPES
There are different specimen types:

- Blood:
- Serum: fluid from blood after blood cells and clot is removed. Doesn’t
have any proteins because they were in the clot.
- Plasma: fluid from blood when centrifuged at low speed in a vial with
anticoagulants (EDTA chelator of calcium, for example.) Plasma has
the proteins.
- Erythrocytes: red blood cells
- Leukocytes: white blood cells

- Urine
- Feces
- Less common: saliva, semen…
- Other
- Non Invasive Sligthly Invasive Highly Invasive
Blood Spinal fluid

Urine
Inner organ

The physicians dream is to be able to know many things from urine, since it is non-
invasive. However, that is not possible therefor what they usually do is blood
analysis.
BLOOD
Blood functions
- Respiratory
- Transport of O2 from lungs to tissues
- Transport of CO2 from tissues to lungs
- Nutrition: Transport of food from gut to tissues
- Excretory: Transport of water form tissues to kidneys
- Regulatory: Regulates water content of tissues, the water is exchanged
through the vessels walls to tissues.
- Regulates body temperature
- Protective: antibodies, antitoxins
- Acid – base balance: 7.35 – 7.45 pH through carbonate buffer.
- Coagulation

Blood is composed of:
- Cells
- Plasma
- Water (90%)
- Plasma solutes (10%)
- Inorganic components: NaCl, bicarbonate, phosphate, salts.
- Organic metabolites and waste
- Plasma proteins: serum albumin, VLDL, LDL, HDL,
immunoglobins (antibodies), fibrinogen, prothrombin (blood
clotting), specialized transport proteins such as transferrin
Blood – urea, glucose and gases
Plasma and Serum – enzymes, metabolites and electrolytes.
Urine – glucose, protein, bile salts, pigments and blood steroids.
Cerebrospinal fluid – Nervous system
EVOLUTION OF CLINICAL CHEMISTRY LABS
New devices are making giant steps in the laboratories: from the blood sugar
analyzer to the lab on a chip.
BIOCHEMICALMARKERS OF HEALTH AND PATHOLOGY
We can measure:
- Electrolytes
- Acid base homeostasis
- Glucose: The normal range of
glucose is between 60-60
mg/100ml. Getting out of that
range may have secondary effects
such as sweating, trembling,
lethargy, convulsions or coma.
- Lipids
- Proteins
- Hormones
- Degradation products /

metabolites
- Toxics
- Microorganisms
CLINICAL BIOCHEMISTRY PANELS

Basic Metabolic Panel
Also known as Chem 7. Information about fluid and electrolyte status, kidney
function, blood sugar levels and response to various medications and other medical
therapies.
Electrolytes, Na+, K+, Cl-, CO2, Glucose, Creatinine, BUN
Comprehensive Metabolic Panel
Extension of BMP. It includes 6 of its tests (not CO2).
Checks kidney and liver function and electrolyte level.
Specific tests:
- Albumin
- Alkaline phosphatase
- Aspartate aminotransferase
- Alanine aminotransferase
- Bilirubin: indicator of degradation of erythrocytes.
- Phosphorus

- Cholesterol
- Triglycerides: indicator of metabolism of fat.
A non-expected concentration of the aminotransferases may indicate a not so good
metabolism of amino acids.
Specific panels
To study the function of a specific organ. Depending on the organ, the assays vary.
Liver function:
The liver produces and excretes bile (a yellowish liquid) required for emulsifying fats.
Some of the bile drains directly into the duodenum, and some is stored in the
gallbladder. The liver produces coagulation factors I (fibrinogen), II (prothrombin), V,
VII, IX, X and XI, as well as protein C, protein S and antithrombin.
It also produces Insulin like growth factor 1 (IGF-1). Hormone that plays an important
role in childhood growth and continues to have anabolic effects in adults. Leo Messi
hormone, they provided it to Messi because he was too small. FCB payed the

injection of this to Messi and he was very lucky because he did not develop cancer.
Liver diseases: hepatitis, alcohol damage, cirrhosis, cancer,drug damage. Many

diseases that affect the liver make you yellowish (jaundice=ictericia). It is caused by
high levels of bilirubin. Bilirubin is produced from the breakup of the hemoglobin of
dead red blood cells., it is normally removed by the liver and excreted in the bile.
Also many pediatric diseases like biliary atresia or progressive familial intrahepatic
cholestasis.
Albumin (low) Cirrhosis (low in nephritic syndrome)
Ala Transaminase (high) Damage (e.g Hepatitis)
Asp Transaminase (high) Damage (e.g Hepatitis)
Alkaline Phosphatase (high) Obstruction
Bilirrubin (high) Hepatitis, Obstruction, Anemia,

Hemorrhage
Gamma glutamil transpeptidase Chronic alcohol toxicity

Lactate dehydrogenase (high) Damage
Coagulation (delayed) High damage
Amino transferase, glut DH à higado
BMP: ELECTROLYTES
The major electrolytes are sodium, potassium, chlorine, phosphate, calcium (not in
Chem 7), magnesium and bicarbonate.
You obtain them from what you eat/drink.
They affect most biochemical reactions including muscle, bone or brain.
Their levels can be low or high, the cause for that is water intake, vomiting,
diarrhea…
Problems most often happen with levels of sodium, potassium and calcium.

Sodium
Most abundant extracellular cation.
Main contributor to plasma osmolality/volume.
Na/K ATPase: pumps Na out and K in the cells. Without this active transport pump,
cells would fill with Na and the osmotic pressure would rupture the cells. It is also
important to transport any big molecule across the membrane (glucose).
Range: 135-145.
It is regulated through hormone mechanism by: plasma osmolality, water
intake/output, anti-diuretic hormone (ADH or vasopressin)à thirst mechanism,
together with aldosterone.

Potassium
Main intracellular cation. Together with sodium it helps maintain the osmotic
pressure.
In blood only 2% is in plasma will red blood cells have a high intracellular
concentration.
It is fundamental in neuromuscular activity. Sodium potassium flux generates the
electrical potential that aids with the conduction of nerve impulses. This potential is
the responsible for muscle contractions and regulates the heartbeat. High potassium
promotes muscular excitability (arrhytmias) while low potassium reduces it

(paralysis).
Calcium
About 99% is associated with bone tissue while only 1% is in plasma. Of this
percent, 40% is bound to proteins, 5-15% is found in other compounds and the rest
is the only hat has a physiological function.
It is a critical component in cardiac function, being responsible for the coordinate
contraction of actin and myosin. Hypercalcemia can have very different results
depending on the level of calcium, ranging from asymptomatic behavior to cardiac
arrest or coma. Hypocalcemia is usually related to tetany, dementia and arrhythmia.
Regulation of calcium levels is performed by the endocrine system. Decreases
plasma ionize Ca stimulates the release of PTH. This hormone increases renal
reabsorption of calcium and stimulate Vitamin D synthesis. This increases
gastrointestinal absorption of Calcium and release of calcium from the bone. On the
other hand, calcitonin has the opposite function of PTH.
PTH also increases plasma magnesium and decrease plasma phosphate.
Chloride
Main extracellular anion. Moves passively with Na+ or against HCO3- to maintain
neutral electrical charge in the extracellular fluid. Cl- usually follows Na+ and
therefore if one is abnormal so is the other.
Cl- is reabsorbed in the renal proximal tubules along with sodium. Deficiencies on
either one limit the reabsorption of the other. The reference range for chlorine is
between 100 and 110 meq/l.
Bicarbonate
It is the second most abundant anion after chlorine. Essential in acid base
homeostasis.
The total amount of CO2 in plasma is divided in CO2, H2CO3 and HCO3-.
The anionic form of HCO3- accounts for 90% of total plasma.
The system CO2-HCO·- represents the most important plasma buffer, required for
acid base regulation.
Reference range: 20-30 meq/l
Creatinine
Healthy kidneys filter creatinine and the waste products leave your body in the form
of urine. If there is an increased level in blood, it means that your kidneys don’t work
properly.
The BMP also includes: glucose, creatinine and BUN (blood, urea, nitrogen). This
last one informs about how well your kidneys and liver are working.

ACID BASE HOMEOSTASIS
Buffer keeping homeostasis
- Cells/tissue: Phosphate buffer
- Blood: Carbonate
Carbonate buffer is regulated thanks to the intervention of lungs to exchange the gas
the kidney that exchange blood and filters things.
Kidney mainly regulates the concentration of protons

What Water Ca, Mg Electrolytes
Hypothalamus Thyroid Adipose tissue

Gland
Pituitary Parathyroid Adrenals
Pancreas
Kidneys
Ovaries
Testes
Neurons release hypothalamic

factors into arterial blood which is
received by the pituitary.

The posterior pituitary receives
neuron conditioned signals to
release vasopressin or oxytocin.
The anterior pituitary receives the
signal in the media, but it is not
conditioned to the neurons. It
releases anterior pituitary
hormones such as tropins.
ADH – Antidiuretic hormone

Water deprivation -> High plasma osmolality àHypothalamic osmoreceptor
stimulation à Increased ADH release à Increased plasma ADH
à Thirst and water intake à Renal water retention à Antidiuresis
Water intake à Low plasma osmolality à Hypothalamic osmoreceptor block à ADH

block à decreased plasma -ADH à Renal water excretion à Large water diuresis

Electrophoretic proteinogram: Electrophoresis of proteins from blood, to analyze their
concentrations. It is a normal electrophoresis, the bigger the band, the higher the
concentration of the protein.
The liver is a fantastic organ regulating almost anything.
An increase in gamma globins may be due to any kind of disease or infection that
causes oyu body to porduce antibodies, isnce in fact, gamma globulins are
antibodies. Autoimmune diseases is when you body reacts against you own body.

11. DIABETES AND OBESITY
All methods to see the level of glucose in blood involves an invasion of the body. The
future of bioengineering is to design something to measure metabolic stuff without
invasion.
The normal range of glucose in blood is between 60 to 90 mg/100mL. Over a
100mg/mL, it is called prediabetes or directly diabetes. Through your genetic
conditions or your feeding habits you may get diabetes. It is usually related to obesity
but that is only one type of diabetes.

Around 79 million US adults between 20 and older have prediabetes.
Prediabetes is a medical condition where blood glucose is higher than normal but not
high enough to be diagnosed as diabetes.
Most people have prediabetes before they develop type two diabetes.
Type I diabetes: No insulin. The cause is immunological.
Type II diabetes: A lot of insulin but it doesn’t work. The cause is your alimentation
habit.
Diagnostic criteria.
Glycated Fasting plasma 2 Hour oral glucose
hemoglobin glucose test challenge
Normal N//A <100mg/dl <140mg/dl
Pre diabetes 5.7-6.4% 100-125 mg/dl 140-199 mg/dl
Diabetes >6.5% >126mg/dl >200 mg/dl
What glucose challenge does is to expose the patient to high levels of glucose and
measuring its concentration after a period of time.
The pancreas is the main organ involved in diabetes. It has many functions:
- Exocrine: producing enzymes for food digestion
- Endocrine: clusters of endocrine cells, called islets of Langerhans. The cells
responsible for the secretion of insulin are beta cells in the pancreas. The islets
contain alfa, beta and delta d cells, each of them secretes a specific peptide
hormone. (alfa – glucagon, beta – insulin, d -somatostatin)
Diabetic have always had to control the amount of glucose they have and the insulin
required, but nowadays the devices can secrete that by themselves when they see
that you have low levels of glucose.
Beta cells specialized in the
secretion of insulin. When
glucose appears, it enters the cell
and triggers glycolysis, Krebs and
oxidative phosphorylation. When
this happens, it triggers a
potassium channel. Potassium is
then depolarized, and it triggers a
current of calcium that gets in the
cell and it acts on the secretory

machinery that produces insulin.
Insulin is a hormone that wants
the cell to have lots of energy. It
wants the cell to synthesize macromolecules and it does it by controlling different
pathways.
When you eat a big meal, plasma glucose increases because it is absorbed. Beta cells
detect that increase and secrete insulin. This induces an increase in the glucose
uptake of adipocytes and muscle. It also may produce an effect in the glucose (many
times it is doing gluconeogenesis which is stopped after secretion of insulin). This
makes glucose level in plasma get to normal levels.
Biochemistry level:
Muscle: Uptake of glucose and utilization, glycogen synthesis, net amino acid uptake,
net protein synthesis.
Adipocytes: glucose uptake and utilization
Liver: glucose uptake, net glycogen synthesis, net triglyceride synthesis, no ketone
synthesis.
We start generating adipocytes through malonyl. CoA.
Sometimes muscle cells may use adipocytes to obtain energy.

Well-fed state: insulin works.

Glucose comes from food, travels to the blood vessels, plasma feels that it has
increased and glucose is transformed into glycogen in the liver so the glycogen
synthase will be activated.
Effects of insulin in blood glucose:

There is no increase in Glut 4
but there is a translocation when
AKT is activated, that is the
trigger for glucose to get inside
the cell.
This signaling pathway ending in
glycogen synthesis is very
important.
When glut 4 is in the plasma
membrane, glucose can get in.
Early fasting state.

There is a drop in glucose level so instead of insulin, glucagon is secreted.

The brain only uses in normal conditions glucose. This glucose is produced by
degradation of glycogen or by gluconeogenesis. Gluconeogenesis comes from
pyruvate which may come also from deamination of aminoacids.
The energy that we need for our muscles comes from fatty acids and this goes to be
used for Krebs cycle.
Fuel metabolism in the liver during prolonged fating or in uncontrolled diabetes mellitus
(insulin doesn’t work).

1 Protein degradation yields glucogenic amino acids
2 Urea is sexported to the kidney and excreted in urine
3 Citric acid cycle intermediates are diverted to gluconeogenesis
4 Glucose is exported to the brain via the bloodstream
5 Fatty acids are oxidized as fuel, producing ACoA.
6 Lack of oxaloacetate prevents ACoA entry into the citric acid cycle; ACoA
accumulates.
7 ACoA accumulations favor ketone body synthesis
8 Ketone bodies are exported via the bloodstream to the brain, which uses them as
fuel.
Ketone bodies in very big amounts are toxic for the body.
The production of oxaloacetate in gluconeogenesis.
Glucose is not the only fuel for the brain but the majority, ketone bodies also fuel the
body.
Fasting: need glucose. Call glucagon.
OBESITY
People with type II diabetes first become obese and then resistant to insulin.
There are few families in the world that has a mutation in the leptin gene. The mutation
in that gene helped us understand a lot of things about the feeding patterns.
Obese mice do not have leptin. Leptin controls the food intake at the hypothalamus
level. The brain tells the animal to eat or not to eat.
Leptin stimulates the hormones that tell you not to eat. ‘Eat less, metabolize more’.

Leptin is an adipokine that when reaching the brain acts on the receptors in the
hypothalamus to retrain appetite. Leptin was the first identified as the product of a
gene designated OB in laboratory mice. The leptin receptors are expressed primarily
in the regions of the brain known to regulate feeding behavior. Leptin carries the
message that fat reserves are sufficient, and it promotes a reduction in fuel intake
and increased expenditure of energy.
Leptin in the anorexigenic neurons acts through the JAKSTAT signaling pathway. As
a consequence of that, the effectors are the following:
- Adipose tissue
- Leptin
- Hypothalamus
- Adrenergic neuron
- Peripheral tissues (adipose, muscle, etc.)
Leptin also stimulates the sympathetic nervous system, increasing blood Reservados todos los derechos.
pressure, heart rate, and thermogenesis by uncoupling. Recall that

thermogenin forms a channel in the inner mitochondrial membrane that allows
protons to reenter the mitochondrial matrix without passing through the ATP
synthase complex. This permits continual oxidation of fuel (fatty acids in an
adipocyte) without ATP synthesis.
Two types of neurons in the arctuate nucleus (place where leptin works)
control fuel intake and metabolism.

- Orexigenic: neurons stimulate eating by producing and releasing
neuropeptide Y whose concentration presumably underlies the obesity of
these mice, who eat voraciously. They are also called appetite stimulating
hormones
- Anorexigenic: they produce alfa melanocyte stimulating hormone. When
weight loss decreases the mass of lipid tissue, leptin levels in blood decrease.
This are called appetite suppressing neurons.
There is a connection between leptin and insulin which is probably the base of
obesity.
Type I diabetes no insulin
Obesity or mutation related OBOB- no leptin.
There may be some cross signaling between the leptin and the insulin

receptor in the brain.

THE END

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BIOCHEMISTRY-NOTES.

2º Grado en Ingeniería Biomédica

Escuela Politécnica Superior

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FUNCTION & HOW THEY WORK

Enzymes allow reaction to carry on by acting as catalysts. They accelerate processes

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DISSOCIATION OF WEAK ACIDS AND WEAK BASES

The pH of pure water is 7. Water is a molecule that is very slightly dissociated. WE

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You put the acid in a water solution at a

given concentration and you start adding

until oyu reach the exact same amount

of the weak acid that is in solution. While

you are adding this equivalents, the pH

is starting to increase as when adding

the base you are removing protons (H +

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HENDERSON - HASSELBALCH EQUATION

The Henderson - Hasselbalch equation

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Reduced: Alkane → Alcohol → ALdehyde → Carboxylic acid → Carbon dioxide :Oxidized

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WHY DO WE ANALYZE PROTEINS

ISOLATION OF SPECIFIC CELL COMPARTMENTS / ORGANELLES

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Efficient extraction of protein and peptides in a biologically alive form.

constant*width*concentration. Desde aquí despejas lo que necesitas.

PURIFICATION OF THE PROTEIN

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Gel Filtration Chromatography

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Ion exchange chromatography

Reversed Phase HPLC (High Performance Liquid Chromatography)

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to produce and to use in the beads for this kind of chromatography.

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IDENTIFICATION OF THE PROTEINS (WESTERN BLOT / MASS

Determination of molecular mass by mass spectrometry (MALDI-TOF): There is

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