Cellular localization of α-ketoglutarate: Glyoxylate carboligase in rat tissues

321

Biochimica et Biophysica Acta, 499 (1977) 321--328

© Elsevier/North-Holland Biomedical Press

BBA 28316 C E L L U L A R LOCALIZATION OF a - K E T O G L U T A R A T E : G L Y O X Y L A T E CARBOLIGASE IN R A T TISSUES JAMES V. O'FALLON and RONALD W. BROSEMER Program in Biochemistry and Biophysics and Department of Chemistry, Washington State University, Pullman, Wash. 99164 (U.S.A.)

(Received February 28th, 1977) Summary ~-Ketoglutarate : glyoxylate carboligase activity has been reported by other laboratories to be present in mitochondria and in the cytosol of mammalian tissues; the mitochondrial activity is associated with the ~-ketoglutarate decarboxylase moiety of the ~-ketoglutarate dehydrogenase complex. The cellular distribution of the carboligase has been r e e x a m i n e d here using marker enzymes of known localization in order to m o n i t o r the composition of subcellular fractions prepared by differential centrifugation. Carboligase activity paralleled the activity of the mitochondrial matrix enzyme citrate synthase in subcellular fractions prepared from rat liver, heart and brain as well as from rabbit liver. Whole rat liver mitochondria upon lysis released both carboligase and citrate synthase. The activity patterns of several other extramitochondrial marker enzymes differed significantly from that of carboligase in rat liver. In addition, the distribution pattern of carboligase was similar to that of ~-ketoglutarate decarboxylase and of ~-ketoglutarate dehydrogenase complex. The data indicate that ~-ketoglutarate : glyoxylate carboligase activity is located exclusively within the mitochondria of the rat and rabbit tissues investigated. There is no evidence for a cytosolic form of the enzyme. Thus the report from another laboratory that the molecular etiology of the human genetic disorder hyperoxaluria type I is a deficiency of cytosolic carboligase must be questioned.

Introduction ~-Ketoglutarate : glyoxylate carboligase is a thiamine pyrophosphate-dependent enzyme that catalyzes the reaction: H

H

-OOC-CH2CH2 C-*COO- + C-COO- + H ÷ -~ -OOC-CH2CH2 C-C-COO- + *CO2 il il I[I O O OOH ~-ketoglutarate glyoxylate ~-hydroxy-~-keto-adipate A b b r e v i a t i o n : TPP, t h i a m i n e p y r o p h o s p h a t e .

322 The carboligase has been found in microorganisms, plants and animals [1,2,3]. Carboligase activity has been reported to occur in both the mitochondrial and the cytosolic fractions of mammalian cells. Scblossberg et al. [1] and Saito et al. [4] have convincingly demonstrated that the mitochondrial carboligase activity is associated with the a-ketoglutarate dehydrogenase complex (EC 1.2.4.2) of the citric acid cycle. Saito et al. also showed that the carboligase activity is catalyzed by the first of the three enzymes in the dehydrogenase complex, a-ketoglutarate decarboxylase. Koch et al. [5] reported that about one-half of the carboligase activity in human liver, spleen and kidney is found in the soluble fraction and that the genetic disorder hyperoxaluria type I is associated with a deficiency of the soluble, but not the mitochondrial, activity. Bourke et al. [6] also reported both a particulate and soluble carboligase activity in human striated muscle, although the activity in neither fraction was depressed in mucles of patients with hyperoxaluria. The studies on the subcellular distribution of carboligase, however, have not been conclusive. Schlossberg et al. [1] reported that almost all the activity in rat heart, kidney, liver and brain is particulate. In addition the ratio of carboligase to a-ketoglutarate decarboxylase activities remains essentially constant during purification from a beef heart homogenate. This would not be expected if a portion of the carboligase were cytosolic, since the first fractionation step is the isolation of mitochondria. The methods used by Koch et al. [5] and Bourke et al. [6] to fractionate tissues could have readily led to disruption of mitochondria with concomitant release of matrix enzymes into the supernatant fraction. The one rat tissue reported by Schlossberg et al. [1] to contain less than 50% of the carboligase activity in the particulate fraction is skeletal muscle; however, the difficulty of disrupting muscle tissue by standard homogenization techniques usually results in disruption of a large portion of the mitochondria. The lack of assay of marker enzymes in order to monitor the release of mitochondrial proteins into the soluble fraction during homogenization results in inconclusive interpretation of the previous studies on the existence of a soluble form of the carboligase. The present study was undertaken in order to determine whether there is a cytosolic carboligase activity in mammalian liver and other tissues. This is of special interest because of the reported deficiency of such an activity associated with hyperoxaluria. In addition, thiamine deficiency results in greatly elevated blood and tissue glyoxylate levels [7]; a-ketoglutarate : glyoxylate carboligase is the only known thiamine-pyrophosphate-dependent enzyme directly involved in glyoxylate metabolism. Materials and Methods Chemicals. Glyoxylic acid monohydrate, cacodylic acid, and triethanolamine were products of J.T. Baker Chemical Co. 5,5'-Dithiobis(2-nitrobenzoic acid} was acquired from Aldrich Chemical Co. [U-14C]Glyoxylic acid, sodium salt, 5.8 Ci/mol, was obtained from Amersham/Searle Corp. and a-[14C1]ketoglutarate, 57 Ci/mol from New England Nuclear. All other biochemicals were purchased from Sigma Chemical Company.

323 E n z y m e assays. The following enzymes were assayed with previously reported procedures: citrate synthase, EC 4.1.3.7 [8]; glucosephosphate isomerase, EC 5.3.1.9 [9]; glucose 6-phosphatase, EC 3.1.3.9 [10]; catalase, EC 1.11.1.6 [11]. ~-Galactosidase (EC 3.2.1.23) was assayed by the method of Sellinger e t a ] . [12], except that the assay buffer was 0.1 M acetate (pH 5.0), 0.1% (v/v) Triton X-100 and 5 mM p-nitrophenyl-fl-D-galactoside. a-Ketoglutarate : glyoxylate carboligase was assayed by a modification of the procedure of Koch et al. [5]. The incubation mixture (2.0 ml) containing 55 mM K H 2 P O J 5 5 mM triethanolamine (pH 6.5), 2.5 mM MgC12, 0.1 mM TPP and enzyme was placed in a 25 ml flask which was capped with a serum stopper. After preincubation for 5 min at 37°C, the reaction was started by injection of 0.5 ml of 5 mM a-ketoglutarate and 10 mM [U-14C]glyoxylate ( 1 0 0 0 0 0 cpm). After one hour the reaction was terminated by injection of a mixture containing 0.4 ml 0.2 M 4-amino-antipyrine and 0.2 ml 13% (w/v) trichloroacetic acid; the evolved 14CO~ was absorbed in 0.2 ml ethanolamine in a minivial. The ethanolamine/carbonate was transferred to 0.7% (w/v) 2,5-diphenyloxazole (PPO) in 30% (v/v) absolute ethanol/70% toluene. The radioactivity was counted in a Beckman LS-230 liquid scintillation system. The assay values were corrected for p r o d u c t formation in the absence of either enzyme or a-ketoglutarate; the blank reaction was the same in both cases. The amount of product formed was directly proportional to incubation time up to at least 60 min and to a m o u n t of added extract up to an enzyme activity of 2 pmol/h. Since preliminary experiments indicated that carboligase activity was slightly more stable in the presence of 0.2 mM TPP, this level of TPP was added to solutions used in the homogenization and fractionation of tissues. a-Ketoglutarate dehydrogenase complex [13] and a-ketoglutarate decarboxylase [14] were assayed by methods used for the analogous pyruvate enzymes, except that a-[14C~]ketoglutarate replaced pyruvate as substrate. In addition, the release of ~4CO~ as described above was used to monitor both activities and fluoride was n o t included in the assay mixtures. Cell fractionation. Unless otherwise noted, the fractionation of rat or rabbit liver was as follows. The liver from Sprague-Dawley male rats (340--390 g) or from adult rabbits were homogenized in 5 volumes of 0.25 M sucrose, 0.2 mM TPP, 5 mM NaH2PO4 (pH 7.0) with five passes of a Potter-Elvehjem homogenizer. The homogenate was strained through four layers of cheesecloth and fractionated by the m e t h o d of Morr~ [15] into crude nuclear, mitochondrial, lysosomal, microsomal and cytosolic fractions. All pellets were resuspended in 5 volumes of the 0.25 M sucrose buffer and all fractions were frozen and stored at --80 ° C. The fractionation of rat brain and heart was similar to the method described above, except that the centrifugal forces and times were those described in the Results section. Protein. The protein content of each fraction was determined by the method of L o w r y et al. [16] with bovine serum albumin as standard. Results

Carboligase in three rat tissues Initial experiments were designed to characterize the "soluble" form of

324

~-ketoglutarate : glyoxylate carboligase in various rat tissues. Heart, brain and liver tissues were homogenized and centrifuged at 18 000 × g for 60 min to sediment whole cells, debris, nuclei, mitochondria, lysosomes, and other cellular components of comparable size. The supernatant was then centrifuged at 1 6 0 0 0 0 × g for 60 min to sediment microsomes. As shown in Table I, essentially all of the carboligase activity was present in the low-speed sediment from brain and liver. Citrate synthase activity, which is located in the mitochondrial matrix [17], was also found mainly in the low-speed sediment in these two tissues. In heart tissue, however, about 60% of the carboligase activity was present in the high-speed sediment plus high-speed supernatant. This would suggest a soluble and possibly microsomal form of carboligase in rat heart, except that about 60% of the citrate synthase activity was also released into the low-speed supernatant. Thus the carboligase in the low-speed supernatant may have arisen from mitochondrial lysis during tissue homogenization. The method used to prepare heart mitochondria was the standard method developed for liver tissue. Since heart tissue is much tougher than liver, harsher treatment with the PotterElvehjem homogenizer is required in order to disrupt adequately the heart cells; this leads to increased mitochondrial disruption and release of mitochondrial enzymes. Special techniques have been developed for preparing heart mitochondria [ 18], but these were not utilized in these experiments. The sedimentation of about 35% of the heart carboligase in the microsomal

TABLE I CARBOLIGASE DIFFERENTIAL

AND CITRATE SYNTHASE CENTRIFUGATION

DISTRIBUTION

IN RAT TISSUE FRACTIONS

AFTER

H e a r t , brain and liver (I) w e r e h o m o g e n i z e d in five v o l u m e s o f 0 . 2 5 M s u c r o s e , 0 . 2 m M T P P , 1 0 m M p o t a s s i u m p h o s p h a t e ( p H 6 . 7 ) w i t h f o u r c o m p l e t e passes o f the P o t t e r - E l v e h j e m h o m o g e n i z e r . The h o m o g e n a t e was c e n t r i f u g e d at 1 8 0 0 0 × g f o r 6 0 r a i n ( 1 . 1 • 1 0 6 ~ - m i n ) . L i v e r ( I I ) w a s h o m o g e n i z e d m o r e v i g o r o u s l y than a b o v e ; the h o m o g e n a t e w a s c e n t r i f u g e d at 4 0 0 0 0 X g f o r 6 0 m i n ( 2 . 4 • 1 0 6 g - r a i n ) . All p e l l e t s w e r e r e s u s p e n d e d in 5 v o l u m e s o f h o m o g e n i z a t i o n b u f f e r and s t o r e d at - - 8 0 ° C . I n e a c h case, carboligase and citrate s y n t h a s e w e r e a s s a y e d in the s e d i m e n t ( s ) and in the final s u p e r n a t a n t . R a t Tissue

Carboligase (%)

Citrate S y n t h a s e (%)

Heart 1.1 • 1 0 6 g*min s e d i m e n t 9 . 6 - 1 0 6 g-rain s e d i m e n t 9.6 • 106 g-min supernatant

38 35 27

42 3.1 55

99 1.3 0.1

86 1.4 13

97 0.8 2.3

90 0.7 9.0

53 47

58 42

Brain 1.1 - 1 0 6 g-rain s e d i m e n t 9.6 - 106 g-min s e d i m e n t 9 . 6 - 1 0 6 g-rain s u p e r n a t a n t

Liver (I) 1.1 • 1 0 6 g-rain s e d i m e n t 9 . 6 • 1 0 6 e-rain s e d i m e n t 9.6 • 106 g-min supernatant

Liver ( I I ) 2 . 4 • 1 0 5 g-rain s e d i m e n t 2 . 4 - 1 0 6 g-rain s u p e r n a t a n t

325 fraction is not inconsistent with a mitochondrial origin for the enzyme. Schlossberg et al. [1] and Saito et al. [4] have demonstrated that the mitochondrial activity is associated with the a-ketoglutarate dehydrogenase complex of molecular weight approx. 2.7 X 106 [19]. The solublized dehydrogenase complex, and thus the associated carboligase activity, partially sediment under the centrifugal forces used to prepare the microsomal fraction in this experiment, while the smaller citrate synthase enzyme does not sediment. As also shown in Table I, a large portion of both carboligase and citrate synthase can be solubilized from rat liver tissues if harsher homogenization conditions are used in order to lyse more mitochondria. Again, however, the carboligase activity release paralleled that of the marker matrix enzyme. Subcellular location in rat liver Rat liver homogenates were separated into subcellular fractions and the activities of several enzymes measured in each fraction (Fig. 1). The marker enzymes used to monitor the composition of the fractions were the following: citrate synthase, mitochondrial matrix [17]; glucose 6-phosphatase, microsomes [20];/~-galactosidase, lysosomes [12]; catalase, peroxisomes and soluble fraction [21]; glucosephosphate isomerase, cytosol. The activity of carboligase paralleled that of citrate synthase when either two passes or fifteen passes of the Potter-Elvehjem homogenizer were used to disrupt the tissue. The subcellular distribution of the o t h e r marker enzymes differed significantly from that of the carboligase activity. The activities of both the a-ketoglutarate dehydrogenase complex and the a-ketoglutarate decarboxylase moiety of the complex were measured. Assays of the total complex were complicated by instability of the activity in extracts [22]. In addition, the variability of the decarboxylase assays was greater than that for other enzymes tested, probably because of the non-physiological nature of the assay mixture. Although the results of these two assays are probably a semi-quantitative reflection of actual enzyme levels, the data in Fig. 1 suggest that the subcellular distribution of carboligase does parallel that of the dehydrogenase complex and of the decarboxylase. Since a large fraction of both the carboligase and citrate synthase activities (Fig. 1) were found in the low-speed "nuclear" fraction containing whole cells, cell debris, nuclei and other components, rat liver nuclei were further purified via centrifugation in 2 M sucrose. Carboligase, citrate synthase, a-ketoglutarate decarboxylase and dehydrogenase complex activities in the purified nuclei and in mitochondria are shown in Table II. The purified nuclear fraction was still contaminated with mitochondria, as indicated by the citrate synthase, decarboxylase and dehydrogenase activities. However, the level of all four enzymes was several-fold lower in the purified nuclear fraction than in the mitochondria. Release of carboligase from lysed mitochondria To define further the mitochondrial location of the carboligase, whole mitochondria were prepared and then lysed under conditions where about one-third of the citrate synthese was released (0.25 M sucrose, 5 mM phosphate) and where about 80% of the citrate lyase was released (50 mM phosphate). As

326 Carboligase

Citrate Synthase

a Ketoglutarate Decarboxylase

d2

a Ketoglutarate

Dehydrogenase Complex I

/~ Galactosidase V~5

Glucose - 6- Pose

Glucosephosphale Isomerase

4

3 2

,J

I

0

Catalase

4 3 0

N

M L Mc

C

~-2'o .'o ~o 8'0 ,~o P E R C E N T A G E OF T O T A L

N

o

MLMc

20 40 60

C

80 ,oo

PROTEIN

Fig. 1. S u b c e l l u l a r d i s t r i b u t i o n p a t t e r n of e n z y m e s in r a t liver, as o b t a i n e d b y t h e d i f f e r e n t i a l c e n t r i f u g a t i o n d e s c r i b e d in Materials a n d M e t h o d s . O r d i n a t e s : relative specific a c t i v i t y of f r a c t i o n s ( p e r c e n t a g e t o t a l r e c o v e r e d a c t i v i t y / p e r c e n t a g e t o t a l r e c o v e r e d p r o t e i n ) . Abscissae: relative p r o t e i n c o n t e n t of s u b c e l l u l a r f r a c t i o n . N, n u c l e a r f r a c t i o n ; M, m i t o c h o n d r i a ; L , l y s o s o m e s ; Mc, m i c r o s o m e s ; C, c y t o s o l . L e f t c o l u m n : Tissue g e n t l y h o m o g e n i z e d w i t h 2 passes of the P o t t e r - E l v e h j e m h o m o g e n i z e r . Pellet f r a c t i o n s s u s p e n d e d in 0 . 2 5 M s u c r o s e , 0.2 m M TPP, 5 m M s o d i u m p h o s p h a t e (pH 7.0). R i g h t c o l u m n : Tissue h o m o g e n i z e d with 1 5 Passes of t h e P o t t e r - E l v e h j e m h o m o g e n i z e r . Pellet f r a c t i o n s s u s p e n d e d in 0 . 2 5 M sucrose, 0 . 2 m M TPP, 50 m M s o d i u m p h o s p h a t e ( p H 7 . 0 ) . G l u c o s e 6 - p h o s p h a t a s e c o u l d n o t be a s s a y e d in these f r a c t i o n s d u e to i n t e r f e r e n c e f r o m t h e high p h o s p h a t e c o n c e n t r a t i o n .

shown in Table III, release of mitochondrial carboligase activity via lysis approximately paralleled that of the matrix enzyme citrate synthase and of a-ketoglutarate decarboxylase. Carboligase in rabbit liver Subcellular fractions from rabbit liver were prepared and assayed for carboligase, citrate synthase, decarboxylase and dehydrogenase complex activities; the activity ratios for the four enzymes were relatively constant in each fraction

327 T A B L E II CARBOLIGASE, CITRATE SYNTHASE, ~-KETOGLUTARATE DECARBOXYLASE AND ~-KETOG L U T A R A T E D E H Y D R O G E N A S E C O M P L E X A C T I V I T I E S IN P U R I F I E D N U C L E A R A N D M I T O CHONDRIAL FRACTIONS T h e m i t o c h o n d r i a l a n d c r u d e n u c l e a r f r a c t i o n s w e r e p r e p a r 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 n u c l e i w e r e f u r t h e r p u r i f i e d b y s u s p e n d i n g t h e c r u d e n u c l e a r f r a c t i o n in 2.0 M s u c r o s e a n d c e n t r i f u g i n g a t 1 0 0 0 0 0 X g f o r 6 0 rain [ 2 3 ] Values are in n m o l / m i n p e r g fresh w t . Fraction

Carboligase

Citrate synthase

~-Ketoglutarate decarboxylase

~-Ketoglutarate dehydrogenase complex

Purified nuclei Mitochondria

1.1 12.2

177 841

0.07 0.52

1.3 12.6

TABLE III RELEASE OF CARBOLIGASE, ~-KETOGLUTARATE DECARBOXYLASE THASE FROM MITOCHONDRIA UPON FREEZING AND THAWING

AND CITRATE

SYN-

T h e m i t o c h o n d r i a l f r a c t i o n was p r e p a r e d as d e s c r i b e d in Materials a n d M e t h o d s w i t h t h e final s u s p e n s i o n e i t h e r in (I) 0 . 2 5 M s u c r o s e , 0 , 2 m M TPP, 5 m M p o t a s s i u m p h o s p h a t e ( p H 7.0) or in ( I I ) 0 . 2 rnM T P P , 50 m M K - p h o s p h a t e ( p H 7,0). A f t e r f r e e z i n g a n d t h a w i n g o n c e , t h e s u s p e n s i o n was c e n t r i f u g e d at 1 5 0 0 0 X g for 1 0 rain a n d t h e t h r e e e n z y m e s a s s a y e d in t h e p e l l e t a n d s u p e r n a t a n t . Fraction

Carboligase (%)

~-Ketoglutarate d e c a x b o x y l a s e (%)

Citrate s y n t h a s e (%)

I

85 15 33 67

94 6 33 67

67 33 20 80

Pellet Supernatant II Pellet Supernatant

Carboligase

5

Citrate Synthase

4

3 >P" 2 :> P- I

¢.) u..

0

0

==6 co w >

J w

5

a Ketoglutarate Decarboxylase

aKetoglutarate Dehydrocjenase Complex

3

2

N L 0

ML Mc i i 25 50

C

N

5

= I00

PERCENTAGE

0

OF TOTAL

ML ~5

Mc

C 5j 0

7J5

j I00

PROTEIN

Fig. 2. S u b c e U u l a r d i s t r i b u t i o n p a t t e r n of e n z y m e s in r a b b i t liver. T h e e x p e r i m e n t a l design is t h e s a m e as f o r the left c o l u m n in Fig. 1.

328

{Fig. 2). Thus, as was shown for the rat enzyme, rabbit liver carboligase is located mainly or exclusively in mitochondria. Discussion The experimental data reported here demonstrate that there is no significant activity of a-ketoglutarate : glyoxylate carboligase detectable in cytosolic fractions of rat liver, brain and heart or of rabbit liver. Human tissues were not used in the present studies, but it seems unlikely that there would be cytosolic activity present. Thus the report that the molecular etiology of hyperoxaluria type I is a deficiency of a soluble carboligase [5] must be questioned. On the basis of our findings and those of Schlossberg et al. [1] and Saito et al. [4], it appears that ~-ketoglutarate : glyoxylate carboligase activity in mammalian cells is associated mainly or exclusively with the decarbox,vlase moiety of the mitochondrial a-ketoglutarate dehydrogenase complex. References 1 Schlossberg, M.A., Bloom, R.J., Richert, D.A. and Westerfeld, W.W. (1970) Biochemistry 9, 1148-1153 2 Hixabayashi, T. and Haxada, T. (1972) Agr. Biol. Chem. 36, 1249--1251 3 Prather, C.W. and Sisler, E.C. (1972) P h y t o c h e m i s t r y , 11, 1637--1647 4 Salto, T., Tuboi, S., Nishimttra, Y, and Kikuchi, G. (1971) J. Biochem. 6 9 , 2 6 5 - - 2 7 3 5 Koch, J., S t o l ~ t a d , E.L.R., Williams, H.E. and Smith, Jr., L.H. (1967) Proc. Natl. Aead. Sci. U.S. 57, 1123--1129 6 Bourke, E., Frindt, G., Flynn, P. and Schreiner, G.E. (1972) Ann. Int. Med. 7 6 , 2 7 9 - - 2 8 4 7 Liang, C. (1962) Bioehem. J. 8 2 , 4 2 9 - - 4 3 4 S Srere, P.A, (1969) Methods Enzymol. 13, 3--11 9 No ltman n, E.A. (1964) J. Biol. Chem. 239, 1 5 4 5 - - 1 5 5 0 10 Baginski, E.S., Fo~i, P.P. and Zak, B. (1974) in Methods of Enz yma t i c Analysis (Bergmeyer, H.E., ed.) Vol. 2, pp. 876--880, Academic Press, New Y o r k 11 Aebi, H. (1974) in Methods of Enzymatic Analysis (Bergmeyer, H.U., ed.), Vol. 2, pp. 673--684, Academic Press, New Y o r k 12 Sellinger, O.Z., Beaufay, H., Jacques, P., Doyen, A. and de Duve, C. (1960) Bioehem. J. 74, 450--456 13 Linn, T.C., PeUey, J.W., Pettit, F.H., Hucho, F., Randall, D.D. and Reed, L.J. (19"/2) Arch. Bioehem. Biophys. 148, 327--342 14 Roche, T.E. and Reed, L J . (1972) Biochem. Biophys. Res. C o m m u n . 48, 840--846 15 Morrd, D.J. (1973) in Molecular Techniques and Approaches in Developmental Biology (ChrisPeels, M.J., ed.), pp. 1--27, J o h n Wiley and Sons, New York 16 Lowry, O.H., Rosenbrough, N.J., Farr, A.L. and Randall, R.J. (1951) J. Biol. Chem. 193, 265--275 17 Garland, P.B. (1968) in Biochemical Society S y m p o s i u m No. 27, The Metabolic Roles of Citrate (Goodwin, T.W., ed.), pp. 41--60, Academic Press, New York 1S Tyler, D.D. and Gonze, J. (1967) Methods E n z y m o l . 10, 75--77 19 Tanaka, N., Koike, K., Hamada, M., Otsuka, K., Suematsu, T. and Koike, M. (1972) J. Biol. Chem. 247, 4 0 4 3 - - 4 0 4 9 20 De Duve, C., Pressman, B.C., Gianetto, R., Wattiaux, R. and Appelmans, F. (1955) Biochem. J. 60, 604---617 21 Scott, P.J., Visentin, L.P. and Allen, J.M. (1969) Ann. N.Y. Acad. Sci. 168, 244--264 22 Linn, T.C. (1974) Arch. Bioehem. Biophys. 1 6 1 , 5 0 5 - - 5 1 4 23 Chauveau, J., Moul4, Y. and Rouiller, C. (1956) Expt. Cell Res. 11, 317--321