Glutathione biosynthesis in Escherichia coli K 12 properties of the enzymes and regulation

Biochimica et Biophysica Acta, 399 (1975) 1--9 © Elsevier Scientific Publishing Company, Amsterdam -- Printed in The Netherlands

BBA 27689

GLUTATHIONE BIOSYNTHESIS IN E S C H E R I C H I A COLI K 12 PROPERTIES OF THE ENZYMES AND REGULATION

P. APONTOWEIL and W. BERENDS

Biochemical and Biophysical Laboratory, Delft University of Technology, Julianalaan 67, Delft (The Netherlands) (Received February 21st, 1975)

Summary The synthesis of glutathione in Escherichia coli K 12 was studied in crude, cell-free extracts. The pH optima and the apparent Km values for the substrates have been determined for both synthesizing enzymes, 7-glutamylcysteine synthetase and glutathione synthetase. 7-Glutamylcysteine synthetase was found to be approximately twice as active as glutathione synthetase. In a growing culture, the cellular level of GSH showed a considerable increase up to 6.6 pmol per ml cell pellet in the stationary growth phase. GSSG was not detectable. The levels of the enzymes remained constant, indicating that glutathione biosynthesis depends at least in the beginning on the availability of the component amino acids. The pathway is controlled by feedback inhibition and not by repression. Introduction

The tripeptide glutathione (GSH) is known to be present in all kinds of cells [1,2] and generally represents the major component of the low-molecular-weight thiol fraction in the cell. In Escherichia coli, 25% of the sulfur is found in glutathione [3]. Relatively high concentrations (up to 10 mM) are found in yeast, liver and eye lens [4,5,6]. The main biological function of glutathione, which could justify its wide distribution, may be the protection of SH groups of proteins. It is difficult to demonstrate whether such protection would be necessary in the intact cell. The congenital disturbance in GSH biosynthesis in erythrocytes of patients with mild hemolytic anemia provides the only indication so far that the protective function of intraceUular GSH is significant [7,8]. Studies of GSH levels and the regulation of glutathione synthesis are of biological interest, but in order to reveal what the intracellular occurrence of GSH means for a cell under different

conditions, such studies should probably be coupled with a genetic study on glutathione synthesis. We u n d e r t o o k both lines of investigation in E. coli K 12. It is known that the biosynthesis of glutathione from its c o m p o n e n t amino acids is accomplished in two steps, each catalyzed by an individual enzyme and requiring ATP. First, a peptide bond is formed between glutamic acid and cysteine b y 7-glutamylcysteine synthetase (EC 6.3.2.2). Then, the second peptide bond is formed between the product 7-glutamylcysteine and glycine by glutathione synthetase (EC 6.3.2.3}. glutamic acid + cysteine + ATP ~ 7-glutamylcysteine • ADP + Pi

(1)

7-glutamylcysteine + glycine + ATP -~ glutathione + ADP + Pi

(2)

The overall synthesis of glutathione in extracts of E. coli was studied by Samuels [9]. The present study provides some information a b o u t the separate activities of the t w o synthetases and their regulation. Materials and Mbthods

The E. coli K 12 strain AB 1157 (thr, leu, proA, his, argB, thi, strA) was used. The genetic symbols are those used by Taylor [10]. Media The minimal medium used was that described by Vogel and Bonnet [ 11 ], supplemented with 0.2% glucose. The necessary growth factors were added in the following concentrations (pg/ml): threonine 25, leucine 50, proline 25, arginine 50, histidine 10 and thiamine 1. Minimal medium without any supplements was used for washing procedures and for diluting cell suspensions. For incorporation of 3 s S into bacterial cells, minimal medium was used with MgC12 instead of MgSO4 and supplemented with 118 mg/1 Na23 s SO4 (spec. act. 6 Ci/mol). Prepara tion o f ex tracts Cultures grown aerobically at 37°C were harvested in the early stationary phase of growth (unless otherwise explicitly stated) by centrifuging. Growth was followed turbidimetrically in a Klett-Summerson colorimeter. The cells w e r e washed once with cold minimal medium. Suspensions of 1 g wet weight cells in 5 ml minimal medium were treated with an MSE 100 Watt Ultrasonic Disintegrator for 5 min at 0 ° C. The sonicated cell suspensions were centrifuged to give cell-free extracts. The supernatant fractions were used in enzyme assays immediately or chilled and stored at - 2 0 ° C. The protein concentration was determined by the method of Lowry et al. [12] with bovine serum albumin as standard. Assays o f 7-glutamylcysteine synthetase The formation of 7-glutamylcysteine on incubation at 37°C for 15 min was measured. The incubation mixture (4 ml, final pH 8.5) had the following composition: 20 pmol ATP, 40 pmol phosphoenolpyruvate, 0.032 mg pyruvate

kinase, 40 #mol MgSO4, 400 pmol KC1, 60 pmol cysteine, 60 pmol glutamic acid, 0.4 ml 1 M diethanolamine/HC1 buffer pH 9.15, 4.0 mg bovine serum albumin and 0.8 ml cell-free extract. After incubation at 37°C, 6.6 ml of ice-cold 3.2% sulphosalicylic acid was added. The suspension was allowed to stand at 0°C for 20 min and then centrifuged at 3000 × g for 10 min. The supernatant was electrolytically reduced by the method of Dohan and Woodward [13] and assayed for ~-glutamylcysteine colorimetrically with the thiol reagent 5,5'-dithiobis-(2-nitrobenzoic acid), after removal of the cysteine by reaction with glyoxylic acid as described by Jackson [14]. Absorbances were read in a Zeiss PMQ II spectrophotometer. The assay described above was less suitable for studying the inhibitory effects of GSH and GSSG on enzyme activity. In these experiments, the incubation mixture (final volume 1 ml) contained [U -~ 4C] glutamic acid (spec. act. 120 pCi/mmol) and the enzyme reaction was terminated by the addition of 0.1 ml 1 M trichloroacetic acid. Denatured protein was removed by centrifugation. To determine the ~-glutamylcysteine formed, 20-/~1 portions of the supernatant were subjected to electrophoresis on Whatman 3MM paper strips for 16 h at 13 V/cm and 4°C in a buffer consisting of acetic acid/pyridine/water ( 7 0 : 1 5 : 2000), pH 3.9. Under these circumstances glutamic acid moved 7--11 cm in the direction of the positive electrode. ~-Glutamylcysteine and its oxidized form diglutamylcystine were found 20--26 cm from the origin in the same direction. The paperstrips were dried, cut into 1-cm sections and counted in a toluenebased scintillation fluid in a Nuclear-Chicago liquid scintillation counter.

Assay of glutathione synthetase The assay conditions were based on the method of Mooz and Meister [15] for crude extracts, with some modifications. The rate of formation of labeled GSH from "/-glutamylcysteine and [U-14 C] glycine was determined. ~/-glutamylcysteine was prepared by the enzymic method of Strumeyer and Block [16]. The reaction mixture (0.5 ml, final pH 8.5) contained: 2.5 pmol ATP, 5 pmol phosphoenolpyruvate, 0.004 mg pyruvate kinase, 2.5 pmol 3,-glutamylcysteine, 7.5 # m o l [U-14C]glycine (spec. act. 60 pCi/mmol), 5 pmol MgSO4, 50 #mol KCI, 0.05 ml 1 M diethanolamine/HC1 buffer pH 9.05, 0.5 mg bovine serum albumin and 0.1 ml cell-free extract. After incubation at 37°C for 30 min, the reaction was stopped by the addition of 0.05 ml 1 M trichloroacetic acid and the mixture then centrifuged. The supernatant was analysed by paper electrophoresis as described above. Electrophoresis was carried o u t at 20°C for 120 min. GSH moved 2--3 cm towards the anode and glycine moved 0.5--1.5 cm in the opposite direction. If complete reaction mixtures were employed, the plot of activity versus enzyme concentration was linear.

Determination of glutathione level Relative amounts of GSH were estimated b y measuring the radioactivity of the trichloroacetic acid-soluble fraction of 3 s S,labeled cells. 80--160 mg wet weight of 3 s S-labeled cells was suspended in 4 ml 5% trichloroacetic acid. The suspension was allowed to stand at 4°C for 30 min and then centrifuged. The sediment was dried overnight at l l 0 ° C . Trichloroacetic acid was removed by shaking the supernatant with ether. Aliquots (25 pl) of the aqueous phase were

pipetted on to Whatman No. 1 paper discs, dried and counted as described above. The GSH concentration was measured enzymically by the method of Klotzsch and Bergmeyer [17]. Cell pellets, sedimented at 8000 × g in 20 min, were suspended in minimal medium and sonicated as described above. Approx. 5% of the GSH was converted to GSSG by the perchloric acid. The total of GSH and GSSG found by this method was used in the calculation of the GSH concentration. The method of Srivastava and Beutler [18] was used to estimate the " t r u e " GSSG concentration.

Chemicals GSH and L-amino acids were obtained from Merck; GSSG, ATP, phosphoenolpyruvate, pyruvate kinase (200 U/mg), NADPH, glyoxalase 1 and glutathione reductase from Boehringer; Na23SSO4, L-[U-14C]glutamic acid and [ U- 14 C] glycine from The Radiochemical Centre, Amersham; carboxypeptidase A from Worthington Biochemical Corporation; methylglyoxal from the Sigma Chemical Co.; 5,5'-dithiobis-(2-nitrobenzoic acid) from K and K Laboratories; thiamine hydrochloride from BDH Chemicals. Results

pH optima The effect of the pH on the activity of 7-glutamylcysteine synthetase and glutathione synthetase is illustrated in Fig. 1. Since the incubation mixtures contained weakly acid compounds, useful buffer action was obtained only in the upper pH range of the buffer systems. The pH/activity profiles of both enzymes were broad. Both enzymes showed maximum activity at pH 8.5.

Stability Several successive freezings and thawings of the cell-free extract had no effect on the activities of the enzymes. No reduction in the activity of 7-gluta-

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mylcysteine synthetase was observed after storage a t - - 2 0 ° C for t w o months, while glutathione synthetase lost 20% of its activity under these conditions. Fig. 2 shows the progress curves of the enzyme-catalysed reactions. The reaction catalysed by glutathione synthetase was reasonably linear with time up to a b o u t 30 min. The rate of synthesis of ~/-glutamylcysteine was less stable at early times. It can be seen further that under the incubation conditions employed, the initial velocity of 7-glutamylcysteine synthesis is approx, two times higher than that of the reaction catalysed by glutathione synthetase. Substrate saturation kinetics

Figs 3 and 4 show the substrate saturation curves of the enzymes. The

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curves were o f the usual hyperbolic form, except for 7-glutamylcysteine. Concentrations o f 7-glutamylcysteine higher than 2 mM had an inhibitory effect on glutathione synthetase, probably due to the oxidized form diglutamylcystine, which is present in sufficient quantity at that concentration. Lineweaver-Burk plots are given as insets in the figures. The Km values calculated from the intercepts on the abscissa for glutamic acid, cysteine, 7glutamylcysteine and glycine were 1.1 " 10 -3, 8.0 " 10 -4, 6.3 • 10 -4 and 4.0 " 10 -4 M respectively.

GSH and enzyme levels Glutathione levels and the activities of the two synthesizing enzymes were determined at different phases of growth. The results shown in Fig. 5 demonstrate a marked increase in the glutathione c o n t e n t during the log phase and up to the beginning of the stationary phase. The enzyme levels, however, remained constant during the whole growth cycle. In this experiment the radioactivity of the trichloroacetic acid-soluble fraction of washed 3 s S-labeled cells was taken as the glutathione level. It has been shown by Roberts et al. [3] with E. coil B that in this fraction at least 95% of the 3 s S is in glutathione. Our chromatographic analysis of the trichloroacetic acid-soluble fraction of strain AB 1157 is in good agreement with this finding. It is known that in the cells a small percentage of glutathione may exist in the oxidized form. We measured the concentrations of GSH and GSSG enzymically in cell pellets. The amounts of GSH found in mid-log-phase cells and stationary-phase cells were 1.15 mg and 2.23 mg per ml cell pellet respectively, which correspond to 3.5 mM and 6.6 mM. GSSG was undetectable in mid-log-phase cells and stationary-phase cells. Repression o f enzyme synthesis The effect of GSH and GSSG in the growth medium on the levels of both enzymes was examined.

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Fig. 5. G l u t a t h i o n e c o n t e n t a n d activities of (~ ~) 7 - g l u t a m y l c y s t e i n e s y n t h e t a s e a n d (o o) g l u t a t h i o n e s y n t h e t a s e as a f u n c t i o n o f g r o w t h phase. G S H c o n t e n t (= s ) is e x p r e s s e d as r a d i o a c t i v i ty o f 25/~1 of t h e t r i c h l o r o a c e t i c acid-soluble f r a c t i o n p e r m g d r y w e i g h t o f cells. T h e e n z y m e activities are e x p r e s s e d as t h e a m o u n t s of 7 - g l u t a m y l c y s t e i n e f o r m e d in 1 5 rain a n d o f G S H f o r m e d in 3 0 rain p e r m g o f p r o t e i n in t h e e x t r a c t s . T h e d i f f e r e n t g r o w t h p h a s e s at w h i c h t h e cells w e r e h a r v e s t e d axe i n d i c a t e d b y t h e d o t t e d line, r e p r e s e n t i n g t h e t u r b i d i m e t r i c a t l y m o n i t o r e d g r o w t h c u r v e .

The maximum deviation in activity from that found in cells grown in unsupplemented minimal medium was 14%. It was concluded from the results presented in Table I that no significant repression of the enzymes occurred in the presence of GSH or GSSG. Especially at the l o w concentrations of GSH (0.13 mM) and GSSG (0.08 mM), stimulation rather than inhibition was observed. It was of interest to k n o w h o w the concentration of GSH or GSSG outside the cells was reflected by the intracellular concentration at the time of harvesting. Measurements of GSH concentrations revealed that the E. coli strain TABLE I E F F E C T O F V A R Y I N G C O N C E N T R A T I O N S O F G S H A N D G S S G IN M I N I M A L G R O W T H M E D I U M ON T H E A C T I V I T I E S O F 9 " - G L U T A M Y L C Y S T E I N E S Y N T H E T A S E A N D G L U T A T H I O N E S Y N T H E T A S E A N D ON T H E C O N C E N T R A T I O N O F G S H I N C E L L P E L L E T S Cells w e r e h a r v e s t e d in t h e e a r l y s t a t i o n a r y p h a s e . Specific a c t i v i t y is g i v e n as ~tmol of p e p t i d e f o r m e d p e r m g p r o t e i n in 15 m i n f o r 7 - g l u t a m y l c y s t e i n e s y n t h e t a s e a n d in 30 m i n f o r g l u t a t h i o n e s y n t h e t a s e . G S H is e x p r e s s e d i n / ~ m o l / m l cell pellet. Addition to minimal growth medium

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Fig. 6. I n h i b i t i o n o f 7 - g l u t a m y l c y s t e i n e s y n t h e t a s e and g l u t a t h i o n e s y n t h e t a s e b y G S H or G S S G . The i n c u b a t i o n m i x t u r e s w e r e s u p p l e m e n t e d w i t h GSH or G S S G to t h e c o n c e n t r a t i o n s i n d i c a t e d o n the abscissa. E n z y m e activities are e x p r e s s e d as p e r c e n t a g e s o f t h e s p e c i f i c a c t i v i t y o b t a i n e d w i t h the c o n t r o l sample.

has the ability to accumulate larger pools of GSH when cultured in the presence of GSH. GSSG remained undetectable in the cells under these growth conditions. It is unlikely that repressed conditions for glutathione synthesis already exist in cells growing in minimal medium. No derepression was observed in mutant strains which were blocked in one of the enzymes [ 1 9 ] .

Inhibition of enzyme activity The activities of 7-glutamylcysteine synthetase and glutathione synthetase were measured in cell-free extracts prepared from early stationary phase cells grown in minimal medium. Enzyme assays were carried out in the absence and in the presence of GSH or GSSG in the concentration range 1 - 10 mM. The results are shown in Fig. 6. It can be seen that 7-glutamylcysteine synthetase was inhibited by GSH (50% at 5.5 mM). Glutathione synthetase was inhibited by GSSG (50% at 1.5 mM) but not by GSH. Discussion The activity of ~/-glutamylcysteine synthetase is roughly twice that of glutathione synthetase, under the incubation conditions employed. This finding agrees with the observation of Minnich et al. [20] and Sass [21] in human erythrocytes. It suggests that the second step in the synthesis of glutathione is rate limiting, but the intermediate ~/-glutamylcysteine has never been found in any type o f cell. The Km values obtained for glutathione synthetase are lower than those for 7-glutamylcysteine synthetase, but these results cannot be adequately discussed without knowledge of other kinetic constants of the enzymes and endogenous pool levels of the three constituent amino acids.

The increase in glutathione content during growth up to the beginning of the stationary phase, without increase in the levels of the synthesizing enzymes, suggests that the availability of the amino acids is an important factor for glutathione synthesis. Moreover, it indicates that there exists a reserve capacity for glutathione synthesis during active growth. This was also demonstrated in erythrocytes by Minnich et al. [ 2 0 ] . GSH was found to inhibit 7-glutamylcysteine synthetase, while GSSG inhibited the activity of glutathione synthetase. It is very likely that the inhibition by GSH is of physiological significance. The concentration of 5.5 mM GSH needed in the incubation mixture to produce 50% inhibition is comparable with the intracellular level measured in stationary-phase cells (6.6 pmol/ml of packed cells). In this connection we may note the obse~ation of Roberts et al. [3] that glutathione synthesis from [ 3 s S] sulphate was greatly reduced when both [ 3 s S] sulphate and nonradioactive glutathione (0.31 mM) were present in the medium. This cannot be due to the repression, as is shown in Table I. The control of the pathway by feedback inhibition and not by repression is consistent with the possible role of glutathione in the protection of cells against toxic agents. It would be important for cells which lose their glutathione under the conditions of a chemical challenge, to have the ability to replenish GSH immediately. Acknowledgements The authors thank Dr J.W.M. Noordermeer and Ir W.R. van Dijk for their collaboration in this investigation and Mr B.W. Groen for his skillful technical assistance. References 1 Jo celyn, P.C. (1959) S y m p o s i u m o n G l u t a t h i o n e (Crook, E.M., ed.), Vol. 16, p. 43, Biochem. Soc. Symp. Cambridge, U.K. 2 Knox, W.E. (1960) in The E n z y m e s ( B o y e r , P.D., Lardy, H. and Myrb//ck, K., eds), 2nd edn, Vol. 2, p. 253, A c a d e m i c Press, N e w Y o r k 3 Roberts, R.B., Abelson, P.H., Cowie, D.B., Bolton, E.T. and Britten, R.J. (1955) Studies o f Biosynthesis in E s c h e r i c h i a c o l i Carnegie Inst. Wash. Publ. 607, p. 318, W a s h i n g t o n 4 Schroeder, E.F. and W o o d w a r d , G.E. (1939) J. Biol. Chem. 129, 283 5 Bhattacharya, S.K., Robson, J.S. and Stewart, C.P. (1956) Blochem. J. 62, 12 6 Harding, J.J. (1970) Biochem. J. 117, 957 7 Boivin, P. and Galand, C. (1965) Nouv. Rev. Ft. H~matoL 5, 707 8 Prins, H.K., Oort, M., Loos, J.A., Z(ircher0 C and Beckers, T. (1966) Blood 2 7 , 1 4 5 9 Samuels, P.J. (1953) Blochem. J. 55, 441 10 Taylor, A.L. (1970) BacterioL Rev. 34, 155 11 Vogel, H.J. a n d B o n n e t , D.M. (1965) J. Biol. Chem. 218, 97 12 Lowry, O.H., Rosebrough, N.J., Farr, A.L. and Randall, R.J. (1951) J. Biol. Chem. 193, 265 13 Dohan, J.S. and W o o d w a r d , G.E. (1939) J. Biol. Chem. 129, 393 14 Jackson, R.C. (1969) Biochem. J. 111, 309 15 Mooz, B.D. and Meister, A. (1967) Biochemistry 6, 1722 16 Strumeyer, D. and Block, IC (1962) Bloehem. Prep. 9, 52 17 Klotzsch, H. and Bergmeyer, H.U. (1962) in M e t h o d e n der E n z y m a t i s c h e n A n a l y s e ( B e r g m e y e r , H.U.. ed.), p. 363, Veriag Chemie, W e i n h e i m 18 Srivastava, S.K. and Beutler, E. (1968) Anal. B l o c h e m . 25, 70 19 Apontoweil, P. and Berends, W. (1975) Blochim. B l o p h y s . A c t a 399, 10---22 20 Minnich, V.0 Smith, M.B., Brauner, M.J. and Majerus, P.W. (1971) J. Clin. Invest. 50, 507 21 Sass, M.D. (1968) Clin. Chim. Aeta 22, 207