417

Biochimica et Biophysica Acta, 538 (1978) 417—425

O Elsevier/North-Holland Biomedical Press

BBA 28414

ENZYMATIC DEGRADATION OF SUCCINYL-COENZYME A BY RAT

LIVER HOMOGENATES

TAKEYOSHI MINAGA, MANOHAR L. SHARMA, ERNEST KUN * and WALTER N.

PIPER *
Department of Pharmacology and the Cardiovascular Research Institute, University of
California, San Francisco, Calif. 94143 (U.S.A.)
(Received June 13th, 1977)

Summary

When a dilute suspension of the mitochondrial fraction of rat liver

homogenates was incubated with chemically synthesized succinyl-CoA, a
product was rapidly formed which was retained at pH 3.9 on Dowex 50 (H+).
Although its acid-base properties were indistinguishable from those of Ö-
aminolevulinic acid, the product did not form a pyrrole with acetylacetone,
nor was its enzymatic formation dependent on added glycine. The enzyme
which cleaved succinyl-CoA to the Ö-aminolevulinic acid-like product was
inhibited by phenylmethyl sulfonylfluoride. The first substance formed by
the peptidase was the unstable thioester of succinic acid and cysteamine
which underwent rearrangement to the more stable N-succinyl cysteamine
above pH 4.0.
It is apparent that the assay of ö-aminolevulinic acid synthetase (EC 2.3.1.37) by
the ion-exchange method 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 out for less than 25 min.

Introduction

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

acid from succinyl-CoA and glycine is an important rate-limiting step in
heme biosynthesis [1]. Subcellular localization of the enzyme ö-
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.

418
Abbreviations: DTNB, 5,5'-dithiobis(2-nitrobenzoic acid); HEPES, 4-(2-hydroxyethyl)-l-
piperazineethanesulfonic acid.
ceptibility to induction by a large variety of drugs 4—8], analyses of •j
•aminolevalinic acid synthetase content of tissues provide a sensitive test
for induced derepression of chromatin template activity for RNA
synthesis, a problem which led us to study assay procedures for
&aminolevalinic acid syn thetase in some detail, The colorimetric method for
assay of Ö-aminolevulinic acid synthetase is based on the condensation of
Naminolevulinic acid with ace tylacetone to form a pyrrole, which is then
detected colorimetrically by its subsequent reaction with Ehrlich's reagent
[9,101, This method is relatively insensitive, and the radiochemical assay
developed by Ebert et aln appeared to offer significant advantages. The
procedure of Ebert et alg con* sists of radiochemical detection of ö-
aminolevulinic acid, which is selectively adsorbed on Dowex 50(H*) ion-
exchange resin at pH and subsequently eluted by a strong base. Unreacted
succinyl-CoA and glycine should not he bound to the resin under these
conditions; thus the method promises a simple procedure for separating
substrates from the product, Other laboratories have reported results with
Ebert's method for the assay of 5-aminolevulinic acid synthetase [12—16] under
conditions where succinyl-CoA was generated enzymatically from its precursors,
although it was stated [11] that chemically synthesized succinyl-CoA was also
suitable as a directly added substrate for the assay of ö-aminolevulinic acid
synthetase in crude tissue preparationso Since it is possible that drugs can
indirectly influence apparent ö-aminolevulinic acid synthetase activity of
tissue preparations by primarily acting cn the succinylCoA-generating system
[17], we decided to evaluate the use of chemically syn w thesized 14C-labelled
succinyl-CoA. It was observed that a substance which was not ö-aminolevulinic
acid but behaved exactly like ö-aminolevulinic acid on Dowex was rapidly
formed from succinyl-CoA by liver homogenates cr isolated mitochondria. The
present report deals with the identification of this product of succinyl-CoA
degradation,

Materials and Methods

Labelled anhydride (9.3 Ci/mol) and 434 0-labelled ö-

aminolevulinic acid (25.4 Ci/mol) were obtained from New England Nuclear,
Boston, Mass. (19 Ci/mol) was purchased from ICN, Irvine}
Calif., and COA, glycine, pyridoxal 5'-phosphate, ß-mercaptoethanol, EDTA,
phenylmethyl sulfonylfluoride and 5,5P -dithiobis(2-nitrobenzoic acid) (DTNB)
from Sigma, St. Louis) Moe The resin AG 50W-X8 (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 body 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 HEPES and 2 mM Tris HO,
pH 72 [18] or in 10 mlV1 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 [18,191. Protein was assayed either
by a biuret method [20] or by the method of Lowry et
al. [21] G
 419
14 14
Synthesis of [ C]succinyl-CoÄ, [ 0] Succinic anhydride (3e1 mg)
and COA (31 mg) were dissolved in 3 ml of cold water, and the pH
adjusted to 7.3 with
5% NaHC03 (aqueous) as described by 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 HCI and the product was
immediately lyophilized. The product was then dissolved in 28 ml of cold
water and the pH adjusted to 1.0 with HCI. The solution of [1,4- 14
C]succinyl-CoA (28 umol;
1.44 • 10 7 cpm/gmol) was divided into 120-gl aliquots, and stored frozen (—
150 0) until used.
Conversions of AG 50W-X8 f-I+ to the Na+ or NT4 form. The resin
(about 300 g) was washed three times in succession with 1 1 aliquots of 1 M
HOI, sufficient distilled water to remove Cl-, 1 1 of 1 M NaOH (or NH40H)
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 of the ö-aminolevulinic acid-like enzymatic product.
Enzymatic formation of products from 14 C-labelled succinyl-CoA (0.1
Ci/100 M) was determined in a reaction mixture (100 WI) which contained
liver homogenate (200 protein), 100 mM Tris HCI, pH 7.4, 100 pyridoxal 5'-
phosphate, and 10 mM EDTA, in the presence and absence of 100 mM
glycine. Incubations were performed at 300 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 1
M sodium (or ammonium) acetate buffer of pH 3.9. Products were separated
by ion-exchange chromatography as follows. Small columns (1 X 2 cm) were
filled with resin (Na+ or form adjusted to pH 3.9). After deproteinization
the supernatant (at pH 3.9) was d.irectly 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 NHfi form)
the columns were finally washed with 2 ml of 0.01 M HCI or acetic acid,
respectively. Thereafter, final elution was carried out with 3 ml 1 M NaOH
(or NH40H) 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 I of
toluene/Triton X-100 (2 : 1, v/v).
Pyrrole formation. The pH of the samples was adjusted to 4.6 with 1 M
acetate buffer. Acetylacetone (200 gl) was added to the samples in glass-
stoppered tubes, which were immersed into a boiling water bath for 20 min.
After cooling, the pyrrole was extracted with ethyl acetate that had been
420

saturated with water. The radioactivity of the ethyl acetate extract was
determined.
Determination of -SH content. The disulfide form of the product after
lyophylization was treated with ß-mercaptoethanol (1 ml aqueous solution of
the disulfide +20 mercaptoethanol) for 6 h at 25 0 C. The sample was then
lyophylized and the dry material redissolved in 1 ml 5 mM Tris • HCI (pH
72). The colorimetric assay for -SH was carried out with DTNB by the
method of Deakin et al. [23].
Results and Discussion
When dilute liver homogenates were incubated with succinyl-CoA, a
product was rapidly formed, which according to the method cf Ebert et
behaved exactly like ö-aminolevulinic acid on a Dowex 50
column, The reaction was linear with time only up to 5—6 min (Fig.
1), but 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 min. Addition
of glycine failed to alter product formation, A direct propor
tionality between initial (3 min) rates and protein concentration was
obtained (Fig. 2)-
The time course of the reaction was similar when the activities of
total homogenates and lysosomem and microsome-free [18,191
mitochondria twere compared. Product formation was not altered by the
addition of glycineo There was no detectable enzymatic activity in
the particle-free cytoplasmic fraction (supernatant after
centrifugation at 100 000 X g for 1 h). When the activi+y of total
homogenates and lysosome-free mitochondria were compared on a mg
protein basis, it was found that they were virtually identical.
*T'hese seemingly paradoxical results suggest that a significant
portion of enzymatic activity is probably lysosomal, because these
particles were absent in the pyeparation of purified mitochondria
while total liver homogenates contain disrupted lyso„ somes. The
precise subcellular 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 breakdown of succinyl-CoA to a product which by the
method of Ebert et al. was indistinguishable from ö-
aminolevulinic acid.
Since the presence of glycine did not influence product formation
from succinyl-CoA in both homogenates and mitochondria, it seemed
unlikely that the substance retained on the column at pH 3.9 was
Naminolevulinic acid. The
 421

36
Time of incubation( minutes)
Fig. 1. Apparent rates of product formation from succinyl-CoA as a function of time of incubation with
liver homogenate. Liver homogenate (200 ug protein) was incubated in the presence of 100 mM Tris HCI, pH
7.4, 100 PM pyridoxal phosphate, 10 mM EDTA and 100 PM succiny2-CoA (051 with 100 mM glycine
cr without glycine (0- - - - - -o). The product was isolated and determined as described in Materials
and Methods,

IOO 4
00
pg protein
Fig. 2. Linear relationship between rates of product formation and concentration of liver homogenate.
Varying amounts of liver homogenate were incubated in the presence of 100 mM Tris • HOI, pH 7.4,
100
1.1M pyridoxal phosphate, 10 mM EDTA, 100 mM glycine and 100 'JM [ 14 C] succinyl-CoA (0.1
WCi) for
0

3 min at 300. The product was isolated and determined as described in Materials and Methods.

estimated endogenous glycine content of the amount of liver homogenates

used was too low to be contributory as the second substrate of ö-
aminolevulinic acid synthetase because the Km for glycine is about 10 mM
[24]. When initial velocities were extrapolated to hourly rates, the estimated
apparent ö-aminolevulinic acid synthetase activity of liver homogenates with
succinyl-CoA as substrate 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 Ö-aminolevulinic
acid synthetase. The product formed from succinyl-CoA could not be
extracted into ethyl acetate after treatment with acetylacetone (Table I),
422

providing further evidence that it was not ö-aminolevulinic acid.

Furthermore, injection of rats with the well-known

TABLE 1
PYRROLE FORMATION FROM ö-AMINOLEVULINIC ACID AND ITS ABSENCE IN THE ENZYMATIC
PRODUCTS OF SUCCINYL-CoA OR SUCCINATE
250 VM (1 uCi) [ 14 C]succinate was incubated in 2 ml volumes with 10 mg (protein liver homogenate,
50 rnM Tris HCI buffer (pH 7.3), 100 pyridoxal phosphate, 10 rnM EDTA and 20 mM MgC12 (cf. ref.
13) in the absence and presence of 100 mM glycine. After incubation for 5 min (at 30 0 C) the reaction
was terminated by 1 ml 15% trichloroacetic acid and chromatographic procedures on Dowex 50 were
performed as described in Materials and Methods. The final eluates with NH40H from the columns
were lyophilized and adjusted to pH 4.6 with 1 M sodium acetate buffer. Acetylacetone (200 PI) was
added and the mixture immersed in a boiling water bath for 20 min. After cooling, the ö-aminolevulinic
acid-pyrrole when present was extracted from the aqueous phase into ethyl acetate. Authentic ö-amino[
14
]levulinic acid was present in controls. Each value represents the average of three experiments.
Solvent phase 8 -Amino [ 14 levulinic 14
C-labelled product from:
acid
[14 C] Succinyl-CoA [ 14 C]
Succinate •
With 100 rnM Without With 100 mM Without
glycine glycine glycine glycine

Ethyl acetate 93% 13% 8% 12%

A queous 87% 92% 88% 96%
inducer of B-aminolevulinic acid synthetase, allylisopropylacetamide, had
augmenting effect on the measured reaction rates, All these results strongay
indicated that products other than ö-aminolevulinic acid weze assayed by the
technique of Ebert et alo [11],
It seemed possible that a peptidase present in liver homogenates could
hydrolyze succinyl-CoAB A peptidase (or amidase) would be expected to act on
succinyl-CoA at two possible sites, either at the cysteamine„pantothenyl or
the ß-alanyl site. The hydrolytic products formed from peptidase activity at
either site are thioesters containing terminal amino groups. Thus, their ionic
properties would be similar to 5-aminolevulinic acido
The mechanism of the enzymatic formation of the product of C 4 C]succmyiCOA
was partly elucidated by the powerful inhibitory effect of phenylmethyl
sulfonylfluoride, as shown in Figa 3, About 98% inhibition was obtained Again,
even in the presence of this inhibitor, added glycine did not alter the
reaction rate- However, phenylmethyl sulfonylfluoride markedly depressed the
total degradation of succinyl-CoA, indicating that the inhibitor-sensitive e
nzy e 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 thicester of succinic
acid and cysteamine and the thioester of alanylcysteamine and
succinic acid was carried out as follows. The alkaline elution
product of the Dowex column obtained from reaction mixtures using
succinyl-CoA as substrate was hydrolyzed in 6 M HOI at 120 0 0 for 24
h. Amino acid analyses of the hydrolyzate revealed no ß-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 m ester of succinic acid and cysteamine.
However, the Naminolevulinic acid-like product eluted with 1 M NaOH
 423
(or NH40H) could not be readsorbed on the cation-exchange resin at pH
309, but was retained on an anion (Dowex 1 Cl-) exchange resin- These
aci&base properties of the pmoduct suggested that the thioester of
succinic acid and cysteamine might have undergone a pH-depen

{00

0-5 2-0
mg protein
Fig. 3. Inhibition of producz forrnation from succinyl-CoA by phenyl-methyl sulfonylfluoride. Vaxying
amounts of liver homogenate were incubated for 3 rnin in the presence 100 mM Q ris • HCIE pH 7 100 VM
pyridoxal phosphate, 10 mM EDTA and 100 PM [ 14 C]succiny1-CoA (0.1 uCi) with 100 mM glycihe (0—0)
without glycine (o- - - The same experiments were pea*formed in the presence of phenylmethyl
sulfonylfluoride (10 ¯4 M) with 100 mM glycine or without glycine
 42 3

dent rearrangement to a product which had lost its NH; group and could not
be retained on Dowex 50.
Experimental demonstration of the pH-dependent rearrangement is shown
in Fig. 4. Aliquots of the acidic eluate of the product 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 Dowex 50 column.
The percent retention on the column was followed by 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 by Dowex 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 product which was eluted with 2 M HCI
(see legend of Fig. 4) contained no thiol groups. When the alkaline eluate
was treated with ß-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-

IOO

6 7 8 9 10

pH
Fig. 4. pH-depende%t binding of the enzymatically formed product of succinyl-CoA on Dowex 50.
Incubation of [ 14 C] succinyl-CoA for 10 min at 3000 was carried out as described in Materials and
Methods. The reaction (5 ml) containing liver homogenate (10 mg protein), 100 mM Tris • HOI, pH
7.4, 100 gM pyridoxal phosphate, 10 mM EDTA and 100 WM [ 14 C] succinyl-CoA (5 gCi) was
terrninated by 1 ml 30% trichloroacetic acid. After separation of precipitated proteins by
centrifugation, the pH of the supernatant was adjusted to 3.9 and applied to a Dowex 50 resin (NHa
form, 1 X 20 cm, equilibrated to pH 3.9). The column was washed with 50 ml of 0.1 M ammonium
acetate buffer (pH 3.9), 50 ml of methanol/O.1 M ammonium acetate (2 : 1, v/v, pH 3.9), 25 ml of 0.01
M ammonium acetate and then eluted with 50 ml 2 M HOI. Portions (1 ml each) of the 2 M HCI eluate
were adjusted to different pH values with NH4 OH, ranging between pH 1 and 10. These samples were
maintained at 250 C for 10 h to insure equilibration. The pH of all samples was then readjusted to pH
3.9 and they were reapplied to small Dowex columns (1 X 2 cm) equilibrated to pH 3.9. The amounts
of radioactive materiel retained on the columns were determined by elution with 1 M NH4 OH and by
radiochemical analyses of eluates.
4 24

o c
c
CH2
CH2

SH

CR2—CH2 CH2

1
L. Succinyl-cysteamine
thioester Succinyl — N-
cysteamine
Disulfide or dimer
Fig. 5. Postulated mechanism of pH-dependent rearrangement of the thioester of succinic acid and
cysteamine. The thioester of succinic acid and cysteamine (I) undergoes an internal nucleophilic attack
by the terminal amino group at pH 5.0—10, resulting in formation of N-succinyl•cysteamine (Il). This
thiol is further oxidized to the stable disulfide dimer (Ill).

stance underwent rearrangement to the more stable N-

succinyl-cysteamine, which, since it has no free amino
group, was not retained on Dowex 50 at pH 3.9. The
material which was eluted by alkali and exhibited acid-
base properties of ö-aminolevulinic acid was the
disulfide form of N-succinylamine of cysbeamine (Fig-
5),
It would be expected that succinyl-CoA, enzymatically formed in
liver homogenates from added succinate, would undergo the same
pathway of cleavage and rearrangement as chemically synthesized.
When succinyl-CoA was genrated from added 14C-labelled succinate in
the presence of a relatively large amount of liver homogenate, high
levels of endogenous substrates present in the liver homogenate
contributed to the formation of stable end products containing which
according to Ebert's method [11] also exhibited acid-base properties
of ö-aminolevulinic acid. These products formed only traces of
pyrrole (see Table I), and their formation was independent of added
glycine; therefore the presence of significant amounts of ö-
aminolevulinic acid in this system was readily ruled out, The
identity of the products formed from succinate and endogenous
substrates was not further clarified, except their non-identity with
ö-aminolevulinic acid was indicated by results shown in Table I.
Briggs et ala [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 ö-
aminolevulinic acid. It was stated that this ö-aminolevulinic acid-
like product was formed only by heart or adrenal hom.o genates and
required Mg2 + [15]. According to our results liver homogenates can
also catalyze the formation of an ö-aminolevulinic acid-like product
from succinate (Table I), which from its acid-base properties alone
can be mistaken for ö-aminolevulinic acid, but yields only 12%
pyrrole. It is apparent from the results reported in this paper that
at least two types of product can be obtained
425

which resemble ö-aminolevulinic acid by the Ebert technique [11] from

succinyl-CoA or succinate, respectively.

Acknowledgnents
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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