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Tracer Technique Main
Tracer Technique Main
BIOSYNTHETIC PATHWAYS
Biosynthesis of Primary Metabolites
Living plants are solar-powered biochemical and
biosynthetic laboratory which manufactures both
primary and secondary metabolites from air, water,
minerals and sunlight.
The primary metabolites like sugars, amino acids & fatty acids
that are needed for general growth & physiological development of
plant which distributed in nature & also utilized as food by man.
~ requires energy
glucose to glycogen
Introduction
• Tracer technique
• Use of isolated organ
• Grafting methods
• Use of mutant strain
• Geiger–Muller counter.
• Liquid Scintillation counter.
• Gas ionization chamber.
• Bernstein–Bellentine counter.
• Mass spectroscopy.
• NMR electrode meter.
• Autoradiography.
• Radio paper chromatography.
Methods
1. PRECURSOR PRODUCT SEQUENCE:
• In this technique, the presumed precursor of the constituent under
investigation on a labelled form is fed into the plant and after a
suitable time the constituent is isolated, purified and radioactivity is
determined.
Disadvantage:
• The radioactivity of isolated compound alone is not usually
sufficient evidence that the particular compound fed is direct
precursor, because substance may enter the general metabolic
pathway and from there may become randomly distributed through
a whole range of product.
Application:
• Stopping of hordenine production in barley seedling after 15–20
days of germination.
• Restricted synthesis of hyoscine, distinct from hyoscyamine in
Datura stramonium.
• This method is applied to the biogenesis of morphine & ergot
alkaloids.
2. DOUBLE & MULTIPLE LABELLING:-
• This method give the evidence for nature of
biochemical incorporation of precursor arises double & triple
labelling.
• In this method specifically labelled precursor and their
subsequent degradation of recover product are more
employed.
• Application: -
• This method is extensively applied to study the biogenesis of plant
secondary metabolite.
• Used for study of morphine alkaloid. E.g. Leete, use Doubly
labelled lysine used to determine which hydrogen of lysine
molecule was involved in formation of piperidine ring of
anabasine in Nicotina glauca.
2. Competitive feeding experiments can be of value
in determining which of two possible intermediates is
normally used by the plant. In its simplest form,
without taking into account a number of other factors,
competitive feeding could distinguish whether B or B
´ was the normal intermediate in the formation of C
from A.
Inactive B and B´ are fed with labelled A to separate
groups of plants and a control is performed by feeding
labelled A only to another group. If the incorporation
of radioactivity into C is inhibited in the plants
receiving B, but is unaffected in the group receiving B
´, then we may conclude that the pathway from A to
C probably proceeds via B.
COMPETITIVE FEEDING:
• If incorporation is obtained it is necessary to consider whether this infact, the
normal route of synthesis in plant not the subsidiary pathway.
• Competitive feeding can distinguish whether B & B’ is normal intermediate
in the formation of C from A.
Application: -
• This method is used for elucidation of biogenesis of Tropane alkaloids.
• Biosynthesis of hemlock alkaloids (coniine, conhydrine etc) e.g. biosynthesis
of alkaloids of Conium maculactum (hemlock) using 14C labelled
compounds.
3. SEQUENTIAL ANALYSIS:
• The principle of this method of investigation is to grow
Chlorella in atmosphere of 14CO2 & then analyze the at
given time interval to obtain the sequence in which various
correlated compound become labelled.
Application:-
• 14CO2 & sequential analysis has been very
successfully used in elucidation of carbon in
photosynthesis.
• Determination of sequential formation of opium,
hemlock and tobacco alkaloids.
• Exposure as less as 5 min. 14CO2, is used in
detecting biosynthetic sequence as-
• Piperitone --------- (-) Menthone ---------- (-) Menthol in
Mentha piperita.
AUTORADIOGRAPHY
An autoradiograph is an image on an x-ray film or
nuclear emulsion produced by the pattern of decay
emissions (e.g., beta particles or gamma rays) from a
distribution of a radioactive substance. Alternatively,
the autoradiograph is also available as a digital image
(digital autoradiography)
High sensitivity.
Applicable to all living organism.
Wide ranges of isotopes are available.
More reliable, easily administration &
isolation procedure.
Gives accurate result, if proper metabolic time &
technique applied.
Isolated Organs, Tissues and Cells
• The cultivation of isolated organs and tissues of
plants eliminates interference from other parts of the
plant which may produce secondary changes in the
metabolites. It can be used for feeding experiments in
conjunction with labelled compounds and is also
useful for the determination of the site of synthesis of
particular compounds.
• Isolated shoots of plants, when placed in a suitable
solution or in water, will usually remain turgid for
some days and during this time presumably have a
normal metabolism; soon, however, a pathological
metabolism commences.
• The technique can be refined by aseptically
connecting the cut end of the shoot to a reservoir
of suitable sterile nutrient, when the shoots will
remain normal for much longer periods. Such
shoots often develop roots at the cut ends—a
factor which could invalidate the results of an
experiment.
• Isolated leaves can be similarly maintained. Rooted
leaves have been used in studies on Nicotiana and
Datura. By this method a large quantity of root is
obtained with a relatively small amount of aerial
parts. It has the advantage that the nutrient solution
requires no sugar, as sufficient starch is synthesized
in the leaf and consequently bacterial and fungal
growth in the nutrient solution is minimized.
Grafts