417

Biochimica et Biophysica Acta, 538 ( 1 9 7 8 ) 4 1 7 - - 4 2 5

© E l s e v i e r ] N o r t h - H o l l a n d B i o m e d i c a l Press

BBA 213414

ENZ~VIATIC D E G R A D A T I O N OF SUCCINYL-COENZYME A BY RAT

L I V E R HOMOGENATES

T A K E Y O S H I M I N A G A , M A N O H A R L. S H A R M A , E R N E S T K U N * and W A L T E R N.
PIPER *

Department of Pharmacology and the Cardiovascular Research Institute, University of

California, San Francisco, Calif. 94143 (U.S.A.)
( R e c e i v e d J u n e 1 3 t h , 197.7)

Summary

When a dilute suspension of the mitochondrial fraction of rat liver homo-

genates was incubated with chemically synthesized succinyl-CoA, a product
was rapidly formed which was retained at pH 3.9 on D o w e x 50 (H+). Although
its acid-base properties were indistinguishable from those of 5-aminolevulinic
acid, the p r o d u c t did n o t form a pyrrole with acetylacetone, nor was its enzy-
matic formation dependent on added glycine. The enzyme which cleaved suc-
cinyl-CoA to the 5-aminolevulinic acid-like product was inhibited b y phenyl-
methyl sulfonylfluoride. The first substance formed by the peptidase was the
unstable thioester of succinic acid and cysteamine which underwent rearrange-
ment to the more stable N-succinyl cysteamine above pH 4.0.
It is apparent that the assay of 5-aminolevulinic acid synthetase (EC
2.3.1.37) b y the ion-exchange m e t h o d of Ebert et al. (Ebert, P.S., Tschudy,
D.P., Choudhry, J.N. and Chirigos, M.A. (1970) Biochim. Biophys. Acta 208,
236--250) can yield erroneous results with succinyl-coenzyme A as substrate,
especially when incubations are carried o u t for less than 25 min.

Introduction

It is generally recognized that the enzymatic formation of 5-aminolevulinic

acid from succinyl-CoA and glycine is an important rate-limiting step in heme
biosynthesis [ 1 ]. Subcellular localization of the enzyme 5-aminolevulinic acid
synthetase (EC 2.3.1.37) appears to be predominantly mitochondrial [2,3].
Because of its low concentration in tissues of normal adult animals and its sus-

* To whom reprint requests should be addressed.

Abbreviations: DTNB, 5,5'-dithiobis(2-nitrobenzoic aeid)~ HEPES, 4-(2-hydroxyethyl)-l-pipera-
zineethanesulfonic acid.
418

ceptibility to induction by a large variety of drugs [4--8], analyses of 6-aminoo

tevalinic acid synthetase content of tissues provide a sensitive test for drug-
induced derepression of chromatin template activity for R N A synthesis, a
problem which led us to study assay procedures for 8-aminolevalinic acid syn-,
thetase in some detail. The colorimetric m e t h o d for assay of 5=aminolevutinic
acid synthetase is based on the condensation of ~-aminolevu!inic acid with ace-
tytacetone to form a pyrrole, which is then detected colorkmetricalty by its
subsequent reaction with Ehrlich's reagent [ 9 , ! 0 ] . This m e t h o d is relatively
insensitive, and the radiochemical assay developed by Ebert et alo [ t l ]
appeared to offer significant advantages. The procedure of Ebert et alo con-
sists of radiochemical detection of 8-aminolevulinic acid, which is selectively
adsorbed on D o w e x 50(H ÷} ion-exchange resin at pH 3.9 and subsequently
eluted by a strong base. Unreacted suecinyt-CoA and glycine should not be
b o u n d to the resin under these conditions; thus the m e t h o d promises a simple
procedure for separating substrates from the product. Other laboratories have
reported results with Ebert's m e t h o d for the assay of 8-amino!ewalinic acid syn-
thetase [12--16] under conditions where succinyl-CoA was generated enzyo
matically from its precursors, although it was stated [11] that chemically syn-
thesized succinyl-CoA was also suitable as a directly added substrate for the
assay of 8-aminolevulinic acid synthetase in crude tissue preparations° Since it
is possible that drugs can indirectly influence apparent 8-aminolevu!inic acid
synthetase activity of tissue preparations by primarily acting on the succinyl=
CoA-generating system [17], we decided to evaluate the use of chemically syn-
thesized ~4C-Iabelled succinyl-CoA. It was observed that a substance which was
n o t 5-aminolevulinic acid but behaved exactly like ~-aminolevulinic acid on
D o w e x 50(H+) Was rapidly formed from succinyl-CoA by liver homogenates or
isolated mitochondria. The present report deals with the identification of this
p r o d u c t of succinyl-CoA degradation.

Materials and Methods

Labelled [1,4-14C]succinic anhydride (9.3 Ci/mol) and 4-~4C-labelled

5-aminolevulinic acid (25.4 Ci/mol) were obtained from New England Nuciear~
Boston, Mass. [2,3-14C]Succinate ( ! 9 Ci/mol) was purchased from ICN, irvine,
Calif., and CoA, glycine, pyridoxal 5'-phosphate, ~-mercaptoethanol, EDTA,
phenylmethyl sulfonylfluoride and 5,5'-dithiobis(2-nitrobenzoic acid} (DTNB)
from Sigma, St. Louis, Mo. The resin AG 50W-X8 H* (identical with Dowex 50°
200--400 mesh) was a preparation of Bio-Rad Labs, Richmond, Calif.
Tissue preparations. Male Sprague-Dawley rats (180 200 g b o d y weight)
were fasted 24 h before killing. Livers were quickly removed, and 10% homo-
genates (w/v) prepared in a medium composed of 70 mM sucrose, 220 mM
mannitol, 2 mM HEPES and 2 mM Tris • HC1, pH 7.2 [18] or in 10 mM Tris/
acetate buffer (pH 7.4) both containing 1% Triton X-100. Both media gave the
same results. Lysosome- and microsome-free mitochondria were prepared by a
method developed in this laboratory [18,19]. Protein was assayed either by a
biuret method [20] or by the method of Lowry et al. [21].
Synthesis of [14C]succinyl-CoA. [14C]Succinic anhydride (3.1 rag) and CoA
(3! mg) were dissolved in 3 ml of cold water, and the pH adjusted to 7.3 with
 419

5% NaHCO3 (aqueous) as described b y Simon and Shemin [22]. The formation

of succinyl-CoA was monitored by recording the absorbance at 232 nm. When
the reaction reached 92% of completion (in 15 min) it was terminated by
adjusting the pH to 1.0 with 1 M HC1 and the p r o d u c t was immediately
lyophilized. The p r o d u c t was then dissolved in 28 ml of cold water and the pH
adjusted to 1.0 with HC1. The solution of [1,4-14C]succinyl-CoA (28 pmol;
1 . 4 4 . 1 0 7 cpm/pmol) was divided into 120-td aliquots, and stored frozen
(--15°C) until used.
Conversions o f A G 50W-X8 I-I+ to the Na ÷ or NH~4 form. The resin (about
300 g) was washed three times in succession with 1 1 aliquots of 1 M HC1, suffi-
cient distilled water to remove Cl-, 1 1 of 1 M NaOH (or NH4OH) and again
with excess water until the pH of the eluate was 6--7. The resin was stored at
this pH. Before assay, the pH of the resin suspension was adjusted to 3.9 with
either 1 M sodium or ammonium acetate buffer of pH 3.9.
Radiochemical assay o f the 5-aminolevulinic acid-like enzymatic product.
Enzymatic formation of products from 14C-labelled succinyl-CoA (0.1 Ci/100
M) was determined in a reaction mixture (100 pl) which contained liver homo-
genate (200 t~g protein), 100 mM Tris, HC1, pH 7.4, 100 pM pyridoxal 5'-phos-
phate, and 10 mM EDTA, in the presence and absence of 100 mM glycine. Incu-
bations were performed at 30°C for various lengths of time and the reaction
terminated by 1.2 ml 6% trichloroacetic acid. After centrifugation, 1 ml of the
supernatant was adjusted to pH 3.9 with 2 ml of I M sodium (or ammonium)
acetate buffer of pH 3.9. Products were separated by ion-exchange chromatog-
raphy as follows. Small columns (1 × 2 cm) were filled with resin (Na ÷ or NH~
form adjusted to pH 3.9). After deproteinization the supernatant (at pH 3.9)
was directly applied to the columns. The eluate contained the non-adsorbed
components. The columns were subsequently washed with a 3-ml aliquot of 0.1
M sodium or ammonium acetate buffer (pH 3.9) and with 3 ml of methanol/
acetate (0.1 M) buffer mixture (1 : 1, v/v). Depending on the ionic composition
of the; buffer (Na ÷ or NH~ form) the columns were finally washed with 2 ml of
0.01 M HC1 or acetic acid, respectively. Thereafter, final elution was carried out
with 13 ml 1 M NaOH (or NH4OH) and the radioactivity of the alkaline eluate
was determined by scintillation spectrometry (Packard 3320) with a scintillator
(composed of 4 g 2,5-diphenyloxazole and 50 mg p-bis-[2-(5-phenyloxazoyl)]
benzene per 1 of toluene/Triton X-100 (2 : 1, v/v).
Pyrrole formation. The pH of the samples was adjusted to 4.6 with I M ace-
tate buffer. Acetylacetone (200 td) was added to the samples in glass-stoppered
tubes, which were immersed into a boiling water bath for 20 min. After cool-
ing, the pyrrole was extracted with ethyl acetate that had been saturated with
water. The radioactivity of the ethyl acetate extract was determined.
Determination o f -SH content. The disulfide form of the product after lyo-
phylization was treated with fi-mercaptoethanol (1 ml aqueous solution of the
disulfide +20 pl mercaptoethanol) for 6 h at 25°C. The sample was then lyo-
phylized and the dry material redissolved in 1 ml 5 mM Tris. HC1 (pH 7.2).
The colorimetric assay for -SH was carried o u t with DTNB by the method of
Deakin et al. [23].
420

Results and Discussion

When dilute liver homogenates were incubated with succinyl°CoA, a product

was rapidly formed, which according to the m e t h o d of Ebert et at. [ 1 ! i ,
behaved exactly like ~-aminolevulinic acid on a D o w e x 50 column° The reac=
tion was linear with time only up to 5 6 min (Fig. 1)~ b u t the product rapidly
decayed thereafter. For this reason, proportionality between initial rates and
the amount of liver homogenate was determined in incubations lasting on~y
3 rain. Addition of glycine failed to alter p r o d u c t formation. A direct propor=
tionatity between initial (3 min) rates and protein concentration was obtained
(Fig. 9.).
The tim< course of the reaction was similar when the activities of total
homogenates and l y s o s o m e - a n d microsome-free [18,19] mitochondria were
compared. Product formation was n o t altered by the addition of glycine. There
was no detectable enzymatic activity in the particle-free cytoplasmic fraction
(supernatant after centrifugation at 100 000 X g for I h}. When the activity of
total homogenates and lysosome-free mitochondria were compared on a mg
protein basis, it was f o u n d that they were virtually identical. These seemingly
paradoxical results suggest that a significant portion of enzymatic activity is
probably lysosomal, because these particles were absent in the preparation of
purified mitochondria while total liver homogenates contain disrupted iyso=
somes. The precise subceltular localization of the succinyl-CoA-degrading
enzyme was not investigated further at this time, because the primary purpose
of the present study was to identify the nature of the pathway of the break-
down of succinyl-CoA to a product which by the method of Ebert e~ alo [11]
was indistin~aishable from 5-aminolevulinic acid.
Since the presence of glycine did n o t influence product formation from suc-
cinyl-CoA in both homogenates and mitochondria, it seemed unlikely that the
substance retained on the column at pH 3.9 was 5-aminolevulinic acid. The

700-
#' '7,o\

"~ 3 5 0

36 12 24 48 72

Time of incubation(minutes)
Fig. 1. A p p a r e n t r a t e s o f p r o d u c t f o r m a t i o n f r o m s u c c i n y l - C o A as a f u n c t i o n o f t i m e o f i n c u b a t i o n w i t h
l i v e r h o m o g e n a t e . L i v e r h o m o g e n a t e ( 2 0 0 ~ g p r o t e i n ) w a s i n c u b a t e d in t h e p r e s e n c e o f 1 0 0 m M T t i s o
HC1, p H 7.4, 1 0 0 ~ M p y r i d o x a l p h o s p h a t e ~ 10 m M E D T A a n d 1 0 0 / ~ M [ 1 4 C ] s u c c i n y t - C o A (9.1 ,,zCi) w i t h
1 0 0 m M g l y c i n e (o --e) o r w i t h o u t g l y c i n e (o . . . . . . o). T h e p r o d u c t w a s i s o l a t e d a n d d e t e r m i n e d as
described i n M a t e r i a l s a n d M e t h o d s .
 421

O
J50- j ~je
.c
E
ro

75 "-

-5

/O/°
,~o I I I J
50 I00 200 400
.~g protein
Fig. 2. L i n e a r r e l a t i o n s h i p b e t w e e n r a t e s o f p r o d u c t f o r m a t i o n a n d c o n c e n t r a t i o n o f l i v e r h o m o g e n a t e .
V a r y i n g a m o u n t s o f l i v e r h o m o g e n a t e w e r e i n c u b a t e d in t h e p r e s e n c e o f 1 0 0 m M Tris • HC1, p H 7.4, 1 0 0
p M p y r i d o x a l p h o s p h a t e , 1 0 m M E D T A , 1 0 0 m M g l y c i n e a n d 1 0 0 p M [ 1 4 C ] s u c c i n y l - C o A (0.1 pCi) f o r
3 r a i n at 30°C. ,The p r o d u c t w a s i s o l a t e d a n d d e t e r m i n e d as d e s c r i b e d in Materials a n d M e t h o d s .

estimated endogenous glycine content of the amount of liver homogenates used

was too low to be contributory as the second substrate of ~-aminolevulinic acid
synthetase because the Km for glycine is about 10 mM [24]. When initial velo-
cities were extrapolated to hourly rates, the estimated apparent ~-aminolevu-
linic acid synthetase activity of liver homogenates with succinyl-CoA as sub-
strate was about 30--40 times higher than values obtained by the colorimetric
assay, again indicating that the enzymatic reaction measured with succinyl-CoA
as substrate was not 5-aminolevulinic acid synthetase. The product formed
from succinyl-CoA could not be extracted into ethyl acetate after treatment
with acetylacetone (Table I), providing further evidence that it was not
5-aminolevulinic acid. Furthermore, injection of rats with the well-known

TABLE I
P Y R R O L E F O R M A T I O N F R O M ~ - A M I N O L E V U L I N I C ACID AND ITS ABSENCE IN T H E ENZY-
MATIC P R O D U C T S OF SUCCINYL-CoA OR S U C C I N A T E
2 5 0 # M (1 /~Ci) [ 1 4 C ] s u c c i n a t e w a s i n c u b a t e d in 2 m l v o l u m e s w i t h 10 m g ( p r o t e i n liver h o m o g e n a t e , 50
m M Tris • HC1 b u f f e r ( p H 7.3), 1 0 0 p M p y r i d o x a l p h o s p h a t e , 1 0 m M E D T A a n d 20 m M MgC12 (cf. ref.
1 3 ) in t h e a b s e n c e a n d p r e s e n c e o f 1 0 0 m M glycine. A f t e r i n c u b a t i o n f o r 5 rain (at 3 0 ° C ) t h e r e a c t i o n w a s
t e r m i n a t e d b y 1 m l 15% t r i c h l o r o a c e t i c acid a n d c h r o m a t o g r a p h i c p r o c e d u r e s o n D o w e x 50 w e r e per-
f o r m e d as d e s c r i b e d in Materials a n d M e t h o d s . T h e final e l u a t e s w i t h N H 4 O H f r o m t h e c o l u m n s w e r e l y o -
p h i l i z e d a n d a d j u s t e d to p H 4 . 6 w i t h 1 M s o d i u m a c e t a t e b u f f e r . A e e t y l a c e t o n e ( 2 0 0 p l ) w a s a d d e d a n d
t h e m i x t u r e i m m e r s e d in a b o i l i n g w a t e r b a t h f o r 20 rain. A f t e r c o o l i n g , t h e 8 - a m i n o l e v u l i n i c a c i d - p y r r o l e
w h e n present was extracted f r o m the aqueous phase into ethyl acetate. Authentic 5-amino[ 14]levulinic
acid w a s p r e s e n t in c o n t r o l s . E a c h v a l u e r e p r e s e n t s t h e a v e r a g e o f t h r e e e x p e r i m e n t s .

S o l v e n t :phase 5 - A m i n o [ 14 ] . 14 C-labelled p r o d u c t f r o m :
levulinic acid
[ 14 C] S u e e l n y l - C o A [ 14 C ] S u c c i n a t e -

With 1 0 0 m M Without With 100 m M Without

glycine glycine glycine glycine

Ethyl acetate 93% 13% 8% 12% 4%

Aqueous 7% 87% 92% 88% 96%
422

inducer of 5-aminolevulinic acid synthetase, allylisopropylacetamide, had no

augmenting effect on the measured reaction rates. All these results strongly
indicated that products other than 5-aminolevulinic acid were assayed by the
technique of Ebert et al° [11].
It seemed possible that a peptidase present in liver homogenates could
hydrolyze succinybCoA. A peptidase (or amidase) would be expected to act on
succinyl-CoA at two possible sites~ either at the cysteamine-p~I~tothenyl or the
~-alanyl site. The hydrolytic products formed from peptidase activity at either
site are thioesters containing terminal amino groups, Thus, their ionic proper-
ties would be similar to 5oaminolevuiinic acid°
The mechanism of the enzymatic formation of the product of [14C]succinyio
CoA was partly elucidated by the powerful inhibitory effect of phenylmethyl
sulfonylfluoride, as shown in Fig~ 3o About 9 8 % inhibition was obtained.
Again, even in the presence of this inhibitor, added glycine did not alter the
reaction rate. However, pheny!methyi sulfonyifluoride markedly depressed the
total degradation of succinyl-CoA, indicating that the inhibitor-sensitive enzy.o
matic step is on the main catabolic pathway.
The enzymatic attack of succinyl-CoA by a peptidase can yield two distinct
products. Discrimination between the thioester of succinic acid and cysteamine
and the thioester of alanylcysteamine and succinic acid was carried out as fol~
lows. The alkaline elution product of the D o w e x column obtained from reaco
tion mixtures using succinyl-CoA as substrate was hydroiyzed in 6 M HCI at
120°C for 24 h. Amino acid analyses of the hydrolyzate revealed no ~-alanine
but only NH3, which would be the expected breakdown product of cysteamine,
suggesting that the product retained on Dowex 50 at pH 3.9 might be the thio-
ester of succinic acid and cysteamine. However, the 6°anqinolevulinic acid-like
product eluted with 1 M NaOH (or NH4OH) could not be readsorbed on the
cation-exchange resin at pH 3o99 but was retained on an anion (Dowex 1 Cl-)
exchange resin. These acid-base prope~ies of the product suggested that the
thioester of succinic acid and cysteamine might have undergone a pH-depen-

. o

wO 50

-~
~ I~ IO-~M pheny[mefhytsulfonylfluodde
I~~ ........... ~:
0,5 a.O 2-0

mg protein
Fig. 3. I n h i b i t i o n o f p r o d u c t f o r m a t i o n f r o m s u c c i n y l - C o A b y p h e n y l m e t h y l s u l f o n y l ~ u o r i d e . V a r y i n g
a m o u n t s o f l i v e r h o m o g e n a t e w e r e i n c u b a t e d f o r 3 r a i n i n t h e p r e s e n c e o f i 0 0 m M T r i s • HCI~ p H 7.4, 1 0 0
# M p y r i d o x a ] p h o s p h a t e , 10 m M E D T A a n d 1 0 0 # M [ 1 4 C ] s u c c i n y l - C o A (0.1 # C i ) w i t h 1 0 0 m M glycL~le
(e $ ) o r w i t h o u t g l y c i n e (o . . . . . . o), T h e s a m e e x p e r i m e n t s w e r e p e r f o r m e d in t h e p r e s e n c e o f
phenylmethyl s u l f o n y l f i u o r i d e ( I 0 -4 M) w i t h 1 0 0 m M g l y e i n e (m -~) o r w i t h o u t glycine
(u . . . . . . ~)o
 423

dent rearrangement to a p r o d u c t which had lost its NH~ group and could n o t
be retained on D o w e x 50.
Experimental demonstration of the pH-dependent rearrangement is shown in
Fig. 4. Aliquots of the acidic eluate of the p r o d u c t of enzymatic degradation of
succinyl-CoA (see legend of Fig. 4) were brought to pH values as shown in the
abscissa, then passed through a D o w e x 50 column. The percent retention on
the column was followed b y radiochemical analysis of the effluent. It was
apparent that rearrangement to N-succinyl-cysteamine occurred readily above
pH 4.0. The most probable mechanism, an internal nucleophilic attack resulting
in the acylation of the NH3 group, is illustrated in Fig. 5. The rearrangement
above pH 4.0 to the more stable N-succinyl-cysteamine, which cannot be
retained b y D o w e x 50 at pH 3.9, explains the biphasic time course of product
formation from succinyl-CoA (Fig. 1). Additional characterization of the
product was obtained by analysis of the thiol content. It was found that the
p r o d u c t which was eluted with 2 M HC1 (see legend of Fig. 4) contained no
thiol i~oups. When the alkaline eluate was treated with ~-mercaptoethanol (see
Materials and Methods) and lyophilized, analysis revealed 1 mol of -SH-group
per mol of succinate. Thus, the failure to detect -SH groups in the alkaline
eluate was readily explained by the oxidation to the disulfide, as shown in Fig.
5.
These results demonstrate that the most rapid enzymatic reaction which
occurred when succinyl-CoA was incubated with a dilute liver homogenate or
mitochondria was the cleavage of succinyl-CoA at the cysteamine site, resulting
in the thioester of cysteamine and succinate. A significant portion of this sub-

I00 -
o
I
'~-o- e--~.~.

\
%
~ 50
I
I

I
\
I I I I r I ~"'0~6--o
1 2 3 4 5 6 7 8 9 I0
pH

Fig. 4. p I i - d e p e n d e ' n t b i n d i n g o f t h e e n z y m a t i c a U y f o r m e d p r o d u c t of s u c c i n y l - C o A o n D o w c x 50. I n c u -

b a t i o n o f [ 14 C] s u c c i n y l - C o A f o r 10 rain at 30°C w a s c a r r i e d o u t as d e s c r i b e d in Materials a n d M e t h o d s .
T h e r e a c t i o n (5 m l ) c o n t a i n i n g liver h o m o g e n a t e (10 m g p r o t e i n ) , 1 0 0 m M Tris - HC1, p H 7.4, 1 0 0 # M
p y r i d o x a l p h o s p h a t e , 10 m M E D T A a n d 1 0 0 ~ M [ 1 4 C ] s u c c i n y l - C o A (5 #Ci) w a s t e r m i n a t e d b y 1 m l 30%
t r i c h l o r o a c e t i e acid. A f t e r s e p a r a t i o n of p r e c i p i t a t e d p r o t e i n s b y c e n t r i f u g a t i o n , t h e p H o f t h e s u p e r n a t a n t
w a s adj~mted t o 3.9 a n d a p p l i e d t o a D o w e x 50 resin f N H + f o r m , 1 × 2 0 c m , e q u i l i b r a t e d to p H 3.9). T h e
c o l u m n w a s w a s h e d w i t h 50 m l o f 0.1 M a m m o n i u m a c e t a t e b u f f e r ( p H 3.9), 50 m l o f m e t h a n o l / 0 . 1 M
a m m o n i u m a c e t a t e (2 : 1, v / v , p H 3.9), 25 m l o f 0 . 0 1 M a m m o n i u m a c e t a t e a n d t h e n e l u t e d w i t h 50 m l
2 M HCL P o r t i o n s (1 m l e a c h ) o f t h e 2 M HC1 e l u a t e w e r e a d j u s t e d to d i f f e r e n t p H v a l u e s w i t h N H 4 O H ,
r a n g i n g b e t w e e n p H 1 a n d 10. T h e s e s a m p l e s w e r e m a i n t a i n e d at 25°C f o r 10 h t o insure e q u i l i b r a t i o n .
T h e p H o f all s a m p l e s w a s t h e n r e a d j u s t e d to p H 3.9 a n d t h e y w e r e r e a p p l i e d t o small D o w e x c o l u m n s
(1 X 2 c m ) e q u i l i b r a t e d t o p H 3.9. T h e a m o u n t s o f r a d i o a c t i v e m a t e r i e l r e t a i n e d o n t h e c o l u m n s w e r e
determi~Led b y e l u t i o n w i t h 1 M NI-I4 O H a n d b y r a d i o c h e m i c a l a n a l y s e s o f eluates.
424

~"o-
s o-
Vi °
c1"--. O-
II CH2
CN 2 CH2 ,
] pH 4 4 . 5 I oxidation i I
CH2 - - ~ CH 2 ~ ~ CH a
I (SN[) ] ' [
. ~ G --~-~0 C~---O C~O
"-~ / /
H2N S HN SH NN
| i
CH2-- CH2 CH 2"--- CH2 t
L 42
I E Z

Z. Succlny~-cysteamine thioester
]Z. Succiny[ - N- cystearnine
]Z, D i s u l f i d e or d[mer
Fig. 5. P o s t u l a t e d m e c h a n i s m o f p H - d e p e n d e n t r e a r r a n g e m e n t o f t h e t h i o e s t e r o f s~ceinic a c i d a n d cyste-
a m i n e . T h e t h i o e s t e r o f succinic acid a n d c y s t e a m i n e (I) u n d e r g o e s a n i n t e r n a l n u c l e o p h f l i c a t t a c k b y t h e
t e r m i n a l a m i n o g r o u p at p H 5 . 0 - - 1 0 , r e s u l t i n g in f o r m a t i o n of N - s u c c i n y l - c y s t e a m i n e (H). This t h i o l is
f u r t h e r o x i d i z e d 4o t h e s t a b l e disulfide d i m e r ( I [ D .

stance underwent rearrangement to the more stable N-succinyl-cysteamine,

which, since it has no free amino group, was n o t retained on Dowex 50 at pH
3.9. The material which was eluted by alkali and exhibited acid-base properties
of 8-aminolevulinic acid was the disulfide form of N-succinylamine o f cyste-
amine (Fig. 5).
It would be expected that succinyl-CoA, enzymatically formed in liver
homogenates from added succinate, would undergo the same pathway of cleav-
age and rearrangement as chemically synthesized. When succmyl-CoA was
genrated from added 14C-labelled succinate in the presence of a relatively large
a m o u n t of liver homogenate, high levels of endogenous substrates present in
the liver homogenate contributed to the formation of stable end products con-
taining 14C, which according to Ebert's m e t h o d [11] also exhibited acid-base
properties of 8-aminolevulinic acid. These products formed only traces of pyr-
role (see Table I), and their formation was independent of added glycine; there-
fore the presence of significant amounts of 8-aminolevulinic acid in this system
was readily ruled out. The identity of the products formed from succinate and
endogenous substrates was n e t further clarified, except their non-identity with
8-aminolevulinic acid was indicated by results shown in Table I. Briggs et aL
[16], employing the technique of Ebert et al. [11] reported the appearance
of significant (40%) quantities of a product formed from succinate which was
eluted simultaneously with 8-aminolevulinic acid. It was stated t h a t this
5-aminolevulinic acid-like product was formed only by heart or adrenal homo-
genates and required Mg2+ [15]. According to our results liver homogenates can
also catalyze the formation of an 5-aminolevulinic acid-like product from suc-
cinate (Table I), which from its acid-base properties alone can be mistaken for
5-aminolevulinic acid, but yields only 12% pyrrole. It is apparent from the
results reported in this paper t h a t at least two types of product can be obtained
 425

which resemble 5-aminolevulinic acid by the Ebert technique [11] from suc-
cinyl-CoA or succinate, respectively.

Acknowledgments

This work was supported by U.S.P.H.S. grants ES-01343, HL-06285 and

GM-20552. Ernest Kun is the recipient of the Research Career Award of the
United States Public Health Service. Takeyoshi Minaga is a Research Fellow of
the Bay Area Heart Association.

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