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Biochimica et Biophysica Acta, 418 (1976) 195--203 © Elsevier Scientific Publishing Company, Amsterdam -- Printed in The Netherlands

BBA 98489

REGULATION OF TERNARY [Met-tRNAf • GTP • EUKARYOTIC INITIATION FACTOR 2] PROTEIN SYNTHESIS INITIATION COMPLEX FORMATION BY THE ADENYLATE ENERGY CHARGE

GORDON M. WALTON and GORDON N. GILL

Department of Medicine, Division of Endocrinology, University of California, San Diego, School of Medicine, La Jolla, Calif. 92093 (U.S.A.) (Received July 21st, 1975)

Summary Formation of the ternary [Met-tRNAf • GTP • eukaryotic initiation factor 2] protein synthesis initiation complex in rabbit reticulocyte ribosomal eluates is dependent on the GTP: GDP ratio and on the adenylate energy charge. The elements controlling ternary initiation complex formation have been studied in a reconstituted system containing eukaryotic initiation factor 2 and nucleoside diphosphate kinase purified from the ribosomal eluate. The concentration of GTP required for half maximal formation of the ternary initiation complex is 2.5 • 10 -6 M; GDP is a p o t e n t competitive inhibitor with Ki = 3.4 • 10 -7 M. Sensitive control of ternary initiation complex formation by the adenylate energy charge occurs through nucleoside diphosphate kinase regulation of the GTP : GDP ratio. Over a wide range of GTP : GDP ratios, 50% of maximal ternary initiation complex formation is observed at an adenylate energy charge of 0.85--0.90 resembling that seen in the unfractionated system. Small changes in adenylate energy charge near this value result in significant changes in the extent of ternary initiation complex formation. Since GTP is continually hydrolyzed to GDP during protein synthesis and since GDP is a competitive inhibitor of GTP binding to several of the protein factors necessary for mRNA translation, the synthetic process provides sensitive control by product inhibition. Ribosome-associated nucleoside diphosphate kinase control of GTP regeneration in response to the adenylate energy charge provides one mechanism for linking protein synthesis to the nutrient state and energy charge of the cell.

Introduction Guanosine nucleotides are involved in m a n y protein biosynthetic reactions. GTP is the primary source of energy for the synthesis of polypeptides

196 with hydrolysis of GTP to GDP + Pi OCCUlTing during peptide bond formation [1,2]. Several factors involved in eukaryotic protein synthesis including eukaryotic initiation factor 2*, and eukaryotic elongation factors 1 and 2 have guanosine nucleotide binding sites [3--19]. The first step in the eukaryotic protein synthesis initiation sequence is formation of a ternary complex containing Met~tRNA~, GTP, and eukaryotic initiation factor 2 [20--22]. GTP remains intact through formation of the [Met-tRNAf • 40S • m R N A ] complex b u t is hydrolyzed upon addition of the 60 S subunit [21]. The concentration of GTP required for half maximal formation of the ternary initiation complex is 2.5 • 10 .6 M; GDP is a potent competitive inhibitor of ternary initiation complex formation with K i = 3.4 • 10 -7 M [7]. Since eukaryotic initiation factor 2 has a higher affinity for GDP than for GTP, the ratio of the two nucleotides is an important factor in controlling initiation of protein synthesis. Studies from the laboratory of Atkinson have indicated that the energy charge of the adenylate pool is an important metabolic regulatory parameter [23--28]. The adenylate energy charge is defined as half the average number of anhydride-bound phosphate groups per adenine moiety and in terms of concentrations equals ([ATP] + 1 / 2 [ A D P ] / [ A T P ] + [ADP] + [AMP] ). The adenylate energy charge may regulate the concentration of all the nucleoside triphosphates through the action of nucleoside diphosphate kinase (EC 2.7.4.6) which catalyzes the general reaction NITP + N2DP ~ N1DP + N2TP [29--33]. We have recently observed an ATP-dependent stimulation of ternary initiation complex formation using partially purified initiation factor preparations [7]. The ATP-dependent stimulation is mediated by nucleoside diphosphate kinase which serves to remove inhibitory levels of GDP by phosphorylation with ATP. The present studies indicate that nucleoside diphosphate kinase present in the 0.5 M KC1 ribosomal extract provides an efficient system for regeneration of GTP and removal of inhibitory levels of GDP. This reaction responds to the adenylate energy charge. Since the formation of the ternary [Met-tRNAf • GTP • eukaryotic initiation factor 2] initiation complex is dependent on the GTP : GDP ratio, the available energy in the adenylate pool through nucleoside diphosphate kinase may indirectly regulate the rate of initiation of protein synthesis. Elongation factors may respond to the GTP : GDP ratio in a similar manner providing for increased protein synthesis when the energy charge of the cell is high and decreased protein synthesis when the energy charge falls. Materials and Methods

[3H]Met-tRNA~ (1540 cpm/pmol) was synthesized under conditions where only the t R N A M et species is acylated [34,35]. [~,_32p] GTP was synthesized and purified free of [ 3 2 p ] o r t h o p h o s p h a t e as previously described [7].

* I n the p r e s e n t r e p o r t the n o m e n c l a t u r e for initiation f a c t o r s c o r r e s p o n d s t o that u s e d b y Schreier and S t a e h e l i n [ 6 ] .

197 Unlabeled GTP was purified free of the GDP by the procedure of Moffatt [36]. Nucleotide purity was determined b y thin-layer chromatography on polyethyleneimine-cellulose with 4.0 M sodium formate buffer, pH 3.4 [37]. [ 3HI GTP purified b y this procedure was contaminated with 0.03% GDP. Protein purification. Eukaryotic initiation factor 2 was purified from rabbit reticulocyte ribosomal eluates through the phosphocellulose chromatography step yielding preparations that b o u n d 650--700 pmol of Met-tRNAf per mg of protein [7]. Nucleoside diphosphate kinase was prepared from the reticulocyte ribosomal eluate by ammonium sulfate fractionation, DEAE-cellulose and Sephadex G-150 chromatography [7]. This preparation containing 21 • 10 -2 units per mg protein was stored in small aliquots at --70°C for up to 12 months without apparent loss of activity. In some experiments, beef liver nucleoside diphosphate kinase was substituted for the rabbit reticulocyte ribosome derived enzyme with equivalent results. Assays. GTP-dependent [3H]Met~tRNAf binding to eukaryotic initiation factor 2 was determined by retention of the complex on 24 mm cellulose-ester filters, 0.45 p m (Millipore Corporation) as previously described [4--7,20]. Ternary complex formation is complete after 10 min at 30°C and is a linear function of eukaryotic initiation factor 2 protein concentration. Nucleoside diphosphate kinase activity was determined by the method of Chiga et al. [31] and Mourad and Parks [32] as previously described [7]. A unit of activity is one pmol of dGTP produced per min per mg of protein. Pyruvate kinase was assayed according to the method of Bucher and Pfleiderer [38]. A unit of activity is one pmol of ATP produced per min per mg of protein. Adenylate kinase activity was determined from ADP production from AMP and ATP utilizing the coupled assay system of Bucher and Pfleiderer [38]. [7 -32P] GTP hydrolysis was determined with modification of the method of Hershey et al. [7,39]. Results

Optimal ternary [Met-tRNAf • GTP. eukaryotic initiation factor 2] initiation complex formation The binding of Met-tRNAf by eukaryotic initiation factor 2 is completely dependent on GTP; GDP is a p o t e n t competitive inhibitor of Met-tRNAf binding. An ATP-dependent stimulation of ternary initiation complex formation observed in partially purified initiation factor preparations is due to nucleoside diphosphate kinase which catalyzes phosphorylation of GDP from ATP [7]. When ternary complex formation was examined in crude initiation factor preparations as a function of the adenylate energy charge, a steep response indicative of sensitive control was obtained above a charge of 0.8 (Fig. 1). The curve which is upwardly concave and steepest in the region of high energy charge resembles those found for several enzyme reactions involved in energy consuming biosynthetic processes which utilize ATP [24]. Though purified GTP was utilized in the experiment shown in Fig. 1, GTPase activity was sufficient to generate inhibitory levels of GDP. Direct assay of GTP hydrolysis in the initiation factor fraction indicated a rate of 53 pmol of GTP hydrolyzed/min/mg of protein. In the assays described, such

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Fig. 1. T e r n a r y c o m p l e x f o r m a t i o n in u n f r a c t i o n a t e d r i b o s o m a l e x t r a c t s as a f u n c t i o n o f a d e n y l a t e e n e r g y charge. M e t - t R N A f b i n d i n g w a s d e t e r m i n e d as d e s c r i b e d u n d e r M e t h o d s using 0.1 m M GTP purified b y D E A E - S e p h a d e x c h r o m a t o g r a p h y , and 18 p g o f t h e 0 - - 7 0 % ( N H 4 ) 2 S O 4 f r a c t i o n o f t h e 0 . 5 M KC1 r i b o s o m a l e x t r a c t . T h e a d e n y i a t e e n e r g y charge w a s e s t a b l i s h e d b y u t i l i z i n g A M P a n d / o r A T P t o give a t o t a l c o n c e n t r a t i o n o f 2 m M . A d e n y l a t e k i n a s e (2 # g ) w a s a d d e d and e q u i l i b r i u m o f t h e a d e n y l a t e p o o l e s t a b l i s h e d w i t h a 1 0 m i n i n c u b a t i o n at 3 0 ° C prior to t h e a d d i t i o n o f [ 3 H ] M e t - t R N A f , GTP and t h e i n i t i a t i o n f a c t o r f r a c t i o n . Similar results w e r e o b t a i n e d w i t h o u t a d d i t i o n o f e x o g e n o u s a d e n y l a t e kinase. Fig. 2. O p t i m a l t e r n a r y c o m p l e x f o r m a t i o n as a f u n c t i o n o f GTP c o n c e n t r a t i o n . M e t - t R N A f b i n d i n g w a s d e t e r m i n e d as d e s c r i b e d u n d e r M e t h o d s using 4 ~tg o f e u k a r y o t i c initiation f a c t o r 2 purified t h r o u g h t h e p h o s p h o c e l l u l o s e c h r o m a t o g r a p h y step, GTP at 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 , and w h e n a d d e d , 1 m M A T P and 4.2 • 10 -4 units o f n u c l e o s i d e d i p h o s p h a t e k i n a s e purified f r o m t h e r e t i c u l o c y t e r i b o s o m a l e x t r a c t t h r o u g h t h e S e p h a d e x G - 1 5 0 c h r o m a t o g r a p h y step d e s c r i b e d u n d e r M e t h o d s . (X X), GTP purified b y D E A E - S e p h a d e x c h r o m a t o g r ~ ' p h y free o f the d e t e c t a b l e GDP; (© o), u n p u r i f i e d GTP c o n t a m i n a t e d w i t h a p p r o x . 3% GDP; (v, o), u n p u r i f i e d G T P , p l u s A T P and n u c l e o s i d e d i p h o s p h a t e kinase.

activity produces GDP concentrations of 5 • 10 -7 M which are inhibitory to ternary complex formation. Nucleoside diphosphate kinase is present in the initiation factor preparation at levels (2.7 • 10 -2 units/mg protein) sufficient to catalyze GTP regeneration at a rate controlled by the adenylate energy charge [ 7 ] . Adenylate kinase which is required to catalyze equilibrium of the adenylate pool was also present at an activity sufficient to produce 0.32 gmol of ADP from AMP and ATP per min per mg of protein. To quantitate the regulatory elements involved in optimal ternary complex formation in ribosomal extracts, the 0--70% ammonium sulfate fraction of the 0.5 M KC1 extract o f ribosomes was fractionated as described under Methods. Using eukaryotic initiation factor 2 purified by phosphocellulose chromatography free of Mg2+-dependent GTPase activity, the concentration of GTP required for half-maximal formation of the ternary complex was determined as 2.5 • 10 -6 M (Fig. 2). When GTP obtained from commercial sources is used, the extent of complex formation is reduced even when a large excess of GTP is present (Fig. 2). Since commercial GTP contains approx. 3% GDP, a standard assay concentration of 10 -4 M GTP represents a GDP concentration which is strongly inhibitory to ternary complex formation. The addition of nucleoside diphosphate kinase purified from the reticulocyte ribosomal extract plus ATP completely overcomes the inhibitory effect of contaminating GDP and provides

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GDP ( MxlO 4} Fig. 3. T e r n a r y c o m p l e x f o r m a t i o n as a f u n c t i o n o f G D P c o n c e n t r a t i o n . M e t - t R N A f b i n d i n g w a s d e t e r m i n e d as d e s c r i b e d u n d e r M e t h o d s u s i n g 2 ~tg o f e u k a r y o t i c i n i t i a t i o n f a c t o r 2 p u r i f i e d t h r o u g h p h o s p h o c e l l u l o s e c h r o m a t o g r a p h y , G D P at 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 , a n d , w h e n a d d e d , 1 m M A T P a n d 1.6 • 1 0 -3 units o f b e e f liver n u c l e o s i d e d i p h o s p h a t e ( N D P ) k i n a s e (o o) o r 3 m M p h o s p h o e n o l p y r u v a t e (PEP) a n d 0 . 1 2 u n i t s of r a b b i t m u s c l e p y r u v a t e kinase (~ A). T h e c o n t r o l r e p r e s e n t s a n a s s a y i n c u b a t e d w i t h o u t the a d d i t i o n o f n u c l e o s i d e d i p h o s p h a t e k i n a s e or p y r n v a t e k i n a s e (X X). E q u i v a lent r e s u l t s w e r e o b t a i n e d using n u c l e o s i d e d i p h o s p h a t e k i n a s e p u r i f i e d f r o m t h e r e t i c u l o c y t e r i b o s o m a l e x t r a c t as d e s c r i b e d u n d e r M e t h o d s .

a system equivalent to that obtained with purified GTP and eukaryotic initiation factor 2. The efficiency of the GTP regeneration system is evident from the observation that, in the presence of ATP and nucleoside diphosphate kinase, GDP can be utilized for ternary complex formation (Fig. 3). As shown from the lower curve, GDP alone does not support ternary complex formation. The optimal concentration of GDP in the regeneration system is approx. 10 -s M, approximating that observed for GTP. Below 10 -s M limiting concentrations of GTP are provided, while above 10 -4 M some GDP remains and inhibition is observed. Optimal activity achieved with ATP and nucleoside diphosphate kinase exceeds that obtained with a phosphoenolpyruvate- and pyruvate kinase-regenerating system. Optimal ternary complex formation was observed with 4 • 10 -s units of nucleoside diphosphate kinase activity, whereas pyruvate kinase in units 100-fold greater than nucleoside diphosphate kinase was unable to overcome inhibition of eukaryotic initiation factor 2 activity. As indicated previously, pyruvate kinase activity was not detectable in ribosomal eluates [7l. Ternary initiation complex formation as a function o f the adenylate energy charge

Ternary initiation complex formation in response to the adenylate energy charge was examined at various ratios of GDP and GTP using purified reaction components (Fig. 4). With GTP alone (Fig. 4A, broken line), eukaryotic initiation factor 2 activity is proportional to the adenylate energy charge. This response approaches linearity over the entire adenylate energy charge range and provides little sensitivity to variations which may occur under physiological conditions. Increasing the GDP : GTP ratio increases inhibition of ternary

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ENERGY CHARGE Fig. 4. T e r n a r y c o m p l e x f o r m a t i o n in r e s p o n s e to t h e a d e n y l a t e e n e r g y c h a r g e in a r e c o n s t i t u t e d s y s t e m . M e t - t R N A f b i n d i n g w a s p e r f o r m e d as d e s c r i b e d u n d e r M e t h o d s u s i n g 2 p g o f e u k a r y o t i c i n i t i a t i o n f a c t o r 2 p u r i f i e d b y p h o s p h o c e l l u l o s e c h r o m a t o g r a p h y , 1 . 6 • 1 0 - 3 u n i t s o f b e e f liver n u c l e o s i d e d i p h o s p h a t e k i n a s e , a n d G D P a n d p u r i f i e d G T P at t h e ratios i n d i c a t e d to a t o t a l g u a n o s i n e n u c l e o t i d e c o n c e n t r a t i o n o f 0 . 1 r a M . T h e a d e n y l a t e e n e r g y c h a r g e in t h e p r e s e n c e o f a d e n y l a t e k i n a s e w a s e s t a b l i s h e d at 2 m M as d e s c r i b e d in Fig. 1; t h e a d e n y l a t e e n e r g y c h a r g e in t h e a b s e n c e o f a d e n y l a t e k i n a s e w a s e s t a b l i s h e d b y a d d i n g A M P , A D P , a n d A T P in t h e a p p r o p r i a t e r a t i o s to a final a d e n y l a t e c o n c e n t r a t i o n o f 2 r a M . A. T e r n a r y c o m p l e x f o r m a t i o n in r e s p o n s e t o t h e a d e n y l a t e e n e r g y c h a r g e at various r a t i o s o f G D P a n d G T P in t h e a b s e n c e o f n u c l e o s i d e d i p h o s p h a t e k i n a s e . B. T e r n a r y c o m p l e x f o r m a t i o n in t h e p r e s e n c e o f n u c l e o s i d e d i p h o s p h a t e k i n a s e . S y m b o l s c o r r e s p o n d to t h e G D P : G T P ratios i n d i c a t e d in p a n e l A ; t h e b r o k e n l i n e c o r r e s p o n d s to t h e e x p e r i m e n t o f p a n e l A p e r f o r m e d in t h e a b s e n c e o f n u c l e o s i d e d i p h o s p h a t c k i n a s e a n d G D P . C. E f f e c t o f a d e n y l a t c k i n a s e o n t h e r e s p o n s e o f t e r n a r y c o m p l e x f o r m a t i o n t o energy charge. (3 ...... ,q), w i t h o u t a d e n y l a t c k i n a s e , a n d w i t h o u t n u c l c o s i d e d i p h o s p h a t e k i n a s e ; (l m), w i t h a d e n y l a t e k i n a s e ; ( o - o), with adenylate kinase and with nucleoside diphosphate kinase.

complex formation. At a ratio of 1 : 1, eukaryotic initiation factor 2 activity is strongly inhibited and relatively unresponsive to energy charge. When nucleoside diphosphate kinase is added to the system, a steep upward concave response curve is observed (Fig. 4B). At higher adenylate energy charge {0.8--1.0) ternary initiation complex formation becomes responsive to small changes in the adenylate pool over a wide range of GDP : GTP ratios. Approx. 50% of maximal ternary initiation complex formation observable occurs at an adenylate energy charge of 0.85, resembling that seen in the unfractionated system (Fig. 1). Small changes in adenylate energy charge near this value result in significant changes in the extent of ternary initiation complex formation. However, complete reversal of GDP inhibition was not achieved below a charge of 1.0, reflecting the strong control exercised by the adenylate pool on nucleoside diphosphate kinase activity. The apparent linear response of ternary initiation complex formation to adenylate energy charge in the absence of GDP and nucleoside diphosphate kinase is due to adenylate kinase and not to competitive inhibition by adenosine nucleotides. In the absence of adenylate kinase, ternary initiation complex formation is unresponsive to adenylate energy charge (Fig. 4C, upper curve). In the presence of adenylate kinase, changes in ternary complex formation are proportional to changes in adenylate energy charge {Fig. 4C, middle curve). This response is the probable result of adenylate kinase-catalyzed production of GDP from GTP and AMP. At lower charges GTP can effectively compete with ATP for adenylate kinase so that at zero charge almost complete conversion of

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GTP to GDP occurs. Since the adenylate pool (2 mM) exceeds the guanylate pool (0.1 mM) by 20-fold, less than 5% of the former is involved. The resulting GDP : GTP ratio is reflected in the amount of ternary initiation complex formed. When the non-hydrolyzable analog of GTP, ~, ~/-methylene-guanosine triphosphate, is used, adenylate kinase-dependent inhibition does not occur, indicating that GTP hydrolysis is required in order to observe inhibition. The addition of nucleoside diphosphate kinase provides a more sensitive response to energy control (Fig. 4C, lower curve). Though pure GTP was used, further GTP hydrolysis occurred yielding higher GDP : GTP ratios without substantive changes in the total adenylate pool. At a charge of 0.9, 50% inhibition of ternary complex formation is observed. Thus, as shown in Fig. 4B, the response of ternary complex formation to the adenylate energy charge is the same regardless of the initial GDP : GTP ratio. The flow of phosphate between the adenylate and guanylate pools and the formation of the ternary initiation complex is thus under sensitive control of the adenylate energy charge. Discussion

Formation of the ternary [Met-tRNA~ • GTP • eukaryotic initiation factor 2] initiation complex, the first step in the sequential assembly of the eukaryotic protein synthesis initiation complex, is subject to sensitive regulation by the adenylate energy charge. By using an in vitro system reconstituted from components of rabbit reticulocyte ribosomal extract, this regulation was shown to depend on adenylate kinase and on nucleoside diphosphate kinase. The reported ATP and nucleoside diphosphate kinase stimulation of [Met-tRNA~ • 40 S] complex formation indicates that subsequent initiation steps also reflect the response of ternary initiation complex formation to adenylate energy charge [7]. Sensitive control of ternary complex formation by the adenylate energy charge is mediated through regulation of the GTP : GDP ratio. Nucleoside diphosphate kinase catalyzes GTP regeneration from GDP providing GTP essential for sustained polypeptide synthesis and removing inhibitory levels of GDP produced during repetitive initiation and elongation cycles. It has been demonstrated that the nucleoside diphosphate kinase catalyzed rephosphorylation of GDP from ATP is sensitively responsive to variations in adenylate energy charge [28]. Nucleoside diphosphate kinase which has been characterized as utilizing two substrate reaction kinetics is uniquely capable of phosphorylating low concentrations of GDP in response to higher concentrations of ATP [32,33]. Since cellular concentrations of ATP exceed those of other nucleoside triphosphates, changes in the adenylate energy charge may regulate the regeneration of other nucleoside triphosphates and, therefore, control reactions in which GTP, CTP, and UTP participate. Transphosphorylation of anhydride phosphate between phosphate donor and phosphate acceptor within the adenylate pool or within the guanylate pool is non-productive and without effect on nucleotide concentration. Phosphate transfer between pools catalyzed by nucleoside diphosphate kinase and adenylate kinase occurs in a manner which may effectively conserve energy. When the adenylate charge is low, the GTP : GDP ratio is lowered and use of GTP in polypeptide synthesis is decreased; when the adenylate charge is high the GTP : GDP is raised and GTP

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utilization in polypeptide synthesis is increased. It has been demonstrated that nucleoside diphosphate kinase is present in rabbit reticulocyte ribosomal eluates; a GTP-GDP transphosphorylase activity which is also responsive to ATP has been observed in eukaryotic elongation factor 2 preparations and a role in GTP regeneration suggested [18]. As shown in Fig. 4B, small changes in the adenylate energy charge in the range demonstrated to occur in vivo [40,41] sensitively regulate ternary initiation complex formation over a wide range of GTP : GDP ratios. The GTP : GDP ratio may similarly regulate the rate of elongation. GDP is a competitive inhibitor of GTP binding to a number of the protein factors involved in translation of m R N A into protein. While the binding of GTP is required for activity of the protein factors during the translation sequence, GDP binding results in an inactive protein configuration. This pattern of response has been observed with prokaryotic initiation factor 2 [42,43] and elongation factor Tu [2,44], and with eukaryotic initiation factor 2 [4,7] and eukaryotic elongation factors 1 [45] and 2 [17,18]. Where studied, the affinity of the guanosine nucleotide binding sites for GDP equals or exceeds, by 1 to 2 orders of magnitude, that for GTP. Since GTP is continually hydrolyzed during initiation and elongation steps, the protein synthetic process provides adequate GDP for control by p r o d u c t inhibition. Several of the translation factors including prokaryotic initiation factor 2 [42] and factor G [2], eukaryotic initiation factor 2 [46] and eukaryotic elongation factor 2 [13,47] are in fact ribosome-dependent GTPases. GTP regeneration and removal of GDP are thus required for continued protein synthesis. We suggest that regulation of GTP regeneration by the adenylate pool provides one mechanism for linking protein synthesis to energy charge so that increased synthesis of proteins may occur under high nutrient conditions and decreased synthesis of proteins may occur under poor nutrient conditions. Acknowledgements This investigation was supported in part by NIH research grant number AM13149 from the National Institute of Arthritis, Metabolism and Digestive Diseases. G.N.G. is an NIH Research Career Development Awardee from the National Institute of Arthritis, Metabolism and Digestive Diseases. References 1 S k o u l t c h i , A., O n o , Y., W a t e r s o n , J. a n d L e n g y e l , P. ( 1 9 6 9 ) C o l d S p r i n g H a r b o r S y m p . Q u a n t . Biol. 34, 437--454 2 L u c a s - L e n ~ r d , J . a n d L i p m a n n , F. ( 1 9 7 1 ) A n n u . Rev. B i o c h e m . 4 0 , 4 0 9 - - 4 4 8 3 C h e n , Y.C., W o o d l e y , C . L . , Bose, K . K . and Gupta, N . K . ( 1 9 7 2 ) B i o c h e m . B i o p h y s . Res. C o m m u n . 4 8 , 1--9 4 D e t t m a n , G . L . a n d S t a n l e y , J r , W.M. ( 1 9 7 2 ) B i o c h i m . B i o p h y s . A c t a 2 8 7 , 1 2 4 - - 1 3 3 5 L e v i n , D . H . , K y n e r , D. a n d Acs, G. ( 1 9 7 3 ) P r o c . N a t l . A c a d . Sci. U.S. 7 0 , 4 1 - - 4 5 6 S c h r e i e r , M.H. a n d S t a e h e l i n , T. ( 1 9 7 3 ) N a t . N e w Biol. 2 4 2 , 3 5 - - 3 8 7 W a l t o n , G . M . a n d Gill, G . N . ( 1 9 7 5 ) B i o c h i m . B i o p h y s . A c t a 3 9 0 , 2 3 1 - - 2 4 5 8 R i c h t e r , O. ( 1 9 7 0 ) B i o c h e m . B i o p h y s . Res. C o m m u n . 3 8 , 8 6 4 - - 8 7 0 9 Collins, J . F . , M o o n , H.M. a n d M a x w e l l , E.S. ( 1 9 7 2 ) B i o c h e m i s t r y 1 1 , 4 1 8 7 - - 4 1 9 6

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