MULTIPLE
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GLUTAMINE SYNTHETASE
Summaria, L., Hsieh, B., Groskopf, W. R., and Robbins, K . C. (1969), Proc. Soc. Exp. Bio/. Med. 130,737. Taylor, F. B., and Beisswenger, J. G. (1973), J . Biol. Chem. 248,1127.
Weber, K., and Osborn, M. (1969), J . B i d . Chem. 244,4406. Wulf, R . J., and Mertz, E. T. (1969), Can. J. Biochem. 47,927. Zylber, J., Blatt, W. F., and Jensen, H. (1959), Proc. Soc. Expt. Biol. Med. 102,755.
Multiple Forms of Glutamine Synthetase. Hybrid Formation by Association of Adenylylated and Unadenylylated Subunits1 J. E. Ciardi,S F. Cimino,$ and E. R . Stadtman*
ABSTRACT: Fully adenylylated glutamine synthetase (EE) is rapidly inactivated in the presence of 4 M urea and 0.5 mM ADP, whereas unadenylylated enzyme (Eos) is not. The kinetics of urea inactivation of a partially adenylylated enzyme preparation isolated directly from Eschericliia coli extracts are significantly different from that of a n in L;itro mixture of E a and ET3 having the same average state of adenylylation. After 30-min exposure to inactivation conditions all adenylylated subunit activity is lost from mixtures of E= and EE, but significant adenylylated activity remains in partially adenylylated native enzymes. It is concluded that the native enzyme preparations contain hybrid molecular forms composed of both adenylylated and unadenylylated subunits and that heterologous subunit interactions lead to stabilization of adenylylated subunit activity. Exposure of either E 6 or EE to 7 11 urea at 0” leads to complete dissociation of subunits and complete loss of catalytic activity. Tenfold dilution of the dissociated subunit mixture with Tris. HCl buffer containing KCl, Mgsf, Mn?’, and 2-mercaptoethanol (pH 7.5) results in reassociation of the subunits to produce a 55-65
P
revious studies have shown that glutamine synthetase from Escliericlzia coli has a mol wt of 600,000 and is composed of 12 apparently identical subunits arranged in two superimposed hexagonal rings (Woolfolk et a/., 1966; Shapiro and Ginsburg, 1968; Valentine et a[., 1968). The activity is regulated by the covalent attachment of one 5’-adenylyl group to a unique tyrosyl moiety of each subunit (Shapiro et al., 1967; Kingdon et al., 1967). Adenylylation is accompanied by changes in divalent ion specificity, in the p H optimum, and in susceptibility to inhibition by various products of glutamine metabolism and by other effectors (Kingdon et al., 1967; Stadtman et al., 1968). Since each one of the 12 subunits of glutamine synthetase can be adenylylated, the enzyme may exist in multiple molecular forms that differ from each other with respect to the number (0-12) and orientation of adenylylated subunits within single molecules. M. S. Raff ~
~~~~
t From the National Heart and Lung Institute, National Institutes of Health Laboratory of Biochemistry, Bethesda, Maryland 20014. Receiced .Mrrj, 7 , 1973.
1 Recipient
of L‘. S . Public Health Service Postdoctoral Fellowship.
0 Recipient of a U. S . Public Health Service International Postdoctoral Fellowship. On leave of absence from the Institute of Eiological Chemistry-I1 Chair, School of Medicine, University of Naples, Naples, Italy.
yield of catalytically active dodecameric aggregates that are indistinguishable from the original enzyme. The yield of active reconstituted enzyme is increased by the presence of ATP or A D P in the reassociation mixture and is diminished by the presence of other substrates and feedback inhibitors of glutamine synthetase including: glutamine, hydroxylamine, potassium arsenate, tryptophan, glycine, CTP, alanine, and AMP. Hybrid dodecameric aggregates produced by reversible dissociation of mixtures of Efi and Efi are similar to those present in partially adenylylated native enzyme preparations. Eo2 and EE could not be separated from each other by electrophoresis. With succinylation of approximately 36 amino residues per mol wt 600,000, 7 5 z of the catalytic activity is lost but the derivatized enzyme is readily separated from unmodified enzyme by electrophoresis. Hybrids produced by reversible dissociation of a mixture containing equal amounts of E a a n d succinylated E E a r e readily separated from E r g and succinylated E12 by electrophoresis ; these hybrids are a mixture of partially adenylylated molecules with from four to nine adenylylated subunits per dodecameric aggregate.
and W. C. Blackwelder have calculated that 382 molecular forms of the enzyme are possible (personal communication). Other studies support the conclusion that hybrid forms of the enzyme (;.e., enzyme molecules containing both adenylylated and unadenylylated subunits) d o exist and that heterologous interactions between dissimilar subunits affect catalytic parameters (Ginsburg et a/., 1970; Denton and Ginsburg, 1970; Segal and Stadtman, 1972) and stability characteristics of the enzyme (Stadtman et a/., 1970). When divalent cations are removed from glutamine synthetase (by treatment with EDTA), it undergoes transition to a “relaxed” state in which tryptophan, tyrosine, and sulfhydryl groups become exposed (Shapiro and Stadtman, 1967; Shapiro and Ginsburg, 1968). On exposure to 1 M urea or p H 8.0 the “relaxed” enzyme is dissociated into mol wt 50,000 inactive subunits (Woolfolk and Stadtman, 1967b; Shapiro and Ginsburg, 1968). In earlier studies it was shown that upon adding divalent cations and decreasing the p H , these subunits reassociated to unstable aggregate forms that were similar but not identical in structure to the native enzyme (Woolfolk and Stadtman, 1967b; Valentine et al., 1968). Reaggregation was accompanied by only transient restoration of catalytic activity . B I O C H E M I S T R Y , VOL.
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The present study was initially concerned with the development of conditions by which reaggregation of dissociated subunits would yield catalytically active enzyme preparations comparable to the native enzyme. After establishing these conditions. hybrid species of enzyme were prepared by the reversible dissociation of mixtures of fully adenylylated (El?) and unadenylylated (E0.9) enzymes. E\,idence i i presented indicating that heterologous interactions between adenylylated and unadenylylated subunits in hybrid enzyme molecules increase the stability of adenylylated subunits to denaturing conditions. Preliminary accounts ha\ i: been reported (Ciardi et d.. 1 970: Stadtman ct d , ,1970: Ciardi and Cimino. 1971).
Materials and Methods Cheniiculs. Amino acids. nucleotides. succinic anhydride,
tris(hydroxymethy1)aminomethane. and 2,4.6-trinitrobenzenesulfonic acid were obtained from Sigma Chemical Co. and Calbiochem. Urea (Fisher or Merck) was filtered through 0.45-p Millipore filters and/or recrystallized from 95 %ethanol. Imidazole was from Eaatman Organic Chemicals, Inc., and 2-methylimidazole and 2,4-dimethy1imidaz.de were from Gallard Schlesinger Co. ; solutions were decolorized with activated charcoal before use. Ail other chemicals were reagent grade. Inrrrbiirion Mixtures. (a) A S S ~ biixTuw. Y Total y-glutamyltransferase activity was measured as previously described (Kingdon and Stadtman, lY67b) except that the L I S S L I ~mixiurp was modified to contain a pH 7.15 mixed imidazole. HCI buffer (50 m\i each of imidazole. 2.4-dimethylimidazole, and 2-methylimidazole), 20 n i h i glutamine, 20 nihr potassium arsenate, 20 m q hydroxylamine. HCI. 0.5 nni ADP, and 0.4 m \ i Mn’-. To measure the extent of inhibition by various feedback inhibitors the transfer assay was used as previously described (Cimino et d . ,1970). (b) UREAI N A c T i v . \ T r m LIIXTCJRE. Dit‘ierential inactivation of adenylylated subunits was obtained by incubating the enzyme (0.25 m g m l ) at 37” in the i i m i inacticution riiixrirre containing 1S7 m\i mixed irnidazole buffer (pH 7.1). 0.5 mbi ADP, 25 mhi glutamine. 25 nil{ potassium arsenate. and 4 xi urea. After various periods of time, aliquots were removed and assayed for y-glutamyltransferase activity and the state of adenylylation. (c) DISSOCIATION MIXTURE. Complete dissociation of glutamine synthetase subunits was obtained by incubating the enzyme (0.5-1.0 mg;ml) for 1--3hr at 0”.p H 8.7. in a dissocicition riiixfirre containing 50 mhi Tris. HCI. 1 mxi EDTA, 143 m\i 2-mercaptoethanol, and 7 hi urea. Dissociation was followed by loss of enzyme activity, ultracentrifugation. and electron microscopy (d) RE,\SSOCI.ATION h i i x T u R E . Reassociation of subunits to form catalytically active dodecameric aggregates was initiated at 0” by a tenfold dilution with a reussocicrtion tizisture containing 50 mbi Tris.HCI ,lcrted GIutatiiine Sj,nthetcise. SUCcinylation (Klotz, 1967) of [I 4Cjadenylyl-labeled glutamine synthetase (E,.j) was carried out at pH 7.5 in the presence of a 500 molar excess of succinic anhydride. The succinic anhydride was dissolved in chloroform and added to the reaction vessel.
MULTIPLE FORMS OF GLUTAMINE SYNTHETASE
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FIGURE 1: Effect of urea concentration on the transferase activity of glutamine synthetase of different states of adenylylation. Activity was determined at pH 7.15 in the modified transfer assay mixture (see Materials and Methods) containing from 0 to 4.6 M urea. The amount of y-glutamylhydroxamate formed by the enzyme was measured after 15 min at 37“. The enzyme preparations were obtained as described under Materials and Methods.
Just before addition of the enzyme solution, the chloroform was evaporated with a stream of air. The enzyme (1 mgiml) in 10 mM imidazole’HC1 (pH 7.5) a n d 10 mM MgClz was incubated with the succinic anhydride at 4 ” for 75 min. The succinylated enzyme was then dialyzed against 10 mM imidazole.HCl buffer (pH 7.5) containing 10 mM MgCI?. The number of amino groups modified by succinylation was determined by titrating the unmodified amino groups with trinitrobenzenesulfonic acid (Haynes et al., 1967). Glutamine synthetase (0.67 phi) was incubated for 12 hr with trinitrobenzenesulfonic acid (0.565 niM) in 10 mM imidazole. HC1 buffer (pH 7.5) containing 10 mM MgCI?. The number of free amino groups was estimated from the change in absorbancy a t 344 nm. Disc Gel Electrophoresis. Analytical disc gel electrophoresis was carried out with 5 % gels a t p H 7.2 in bis-Tris. HCl buffer according to the method of Rodbard and Chrambach (1971). Duplicate samples containing about 100 pg of protein were applied to parallel gels; one gel was assayed for y-glutamyltransferase activity and the other stained with Amido Schwarz to detect protein. In other experiments, after electrophoresis the gels were sliced into 2-mm segments and radioactivity and the state of adenylylation were determined. Cellulose Acetate Electrophoresis. Cellulose polyacetate strips (Gelman Sephaphore 111) were wetted with 50 mM potassium phosphate, p H 6.0, and 2-6 pg of protein were applied. The same buffer was placed in the electrode compartments and a constant 200 V was applied for 1 hr. The protein was then fixed and stained with Ponceau S and Nigrosin as described by Meighen and Schachman (1970). Isoelectryfocusing. Isoelectrofocusing was carried out o n a 110-ml LKB column, with a 0-5Z sucrose gradient. A p H gradient of 3-10 was established with 1 ampholyte. A I-W current was maintained for 48-96 hr until focusing was complete and 1-2-ml samples were collected. The pH, absorbancy at 280 nm, y-glutamyltransferase activity, and state of adenylylation of each sample were determined, Sedimentation Coeficients. Sedimentation coefficients were measured with a Spinco Model E ultracentrifuge equipped with a n ultraviolet scanning system. The measurements carried out in urea were corrected to the viscosity and density of water at 30” using the equations of Kawahara and Tanford (1966). Light-Scattering Measurenients. Changes in light scattering were measured in the Hitachi (Perkin-Elmer) fluorospectro-
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? IO 20 30 40 50 60 MINUTES FIGURE 2: Differentiation of glutamine synthetase hybrids from mixtures of adenylylated and unadenylylated enzyme preparations. All enzyme preparations (0.2-0.4 mg/ml). as described under Materials and Methods, were incubated at 37’ in the 4 M urea inactivation mixture (Materials and Methods). The enzyme activity and state of adenylylation as measured i n the modified transfer assay mix in the presence and absence of Mg*? (Materials and Methods) were determined on aliquots removed after various periods of time. The numbers in parentheses on the curves refer to the state of adenylylation at the time indicated: (A) mixtures of EG and EE (see Materials and Methods); (B) “natural” enzymes.
photometer at 350 nm. Solutions of 0.5-1.0 mglml of protein were filtered five times through a 0.22-p Millipore filter. Results Differential Inactivation o f Adenylylated and Unadenj~lj~lated Enzyiies by the Presence of’ Urea in the Assay Mixture. Addition of urea (4.0 M) to the standard y-glutamyltransferase assay mixture leads to inhibition of E12 but not of E=. As shown in Figure 1, with El:! there is a nearly linear decline in activity to 15 of the initial value when the concentration of urea added to the assay mixture is increased from 0 to 3 M. Addition of 4-4.5 XI urea causes almost complete inhibition. In contrast, little or n o inhibition of activity occurs even with 4 M urea; in fact stimulation (up to 2 0 7 3 of activity is obtained with 2-3 \I urea. A mixed enzyme, E= mix (see Materials and Methods) was inactivated by increasing urea concentration as would be predicted o n the assumption that only the E12 component is affected. Effect of Urea on the Stability of Hjibrid Enzjsine Forms. To determine if heterologous interactions between adenylylated and unadenylylated subunits in hybrid molerules affect their stability to inactivation by 4 hi urea, the kinetics of inactivation of nzixed enzyme preparations at various states of adenylylation (see Materials and Methods) were compared with those of natural enzyme preparations. As shown in Figure
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4: Effect of urea concentration on the inactivation of glutamine synthetase. ET, (0.5 mg/ml) was incubated at 0" at pH 8.7 in a solution containing 50 mhq Tris.HC1, 1 mbi EDTA. 143 mal 2-mercaptoethanol, and from 1 to 7 M urea. Control was without urea. Aliquots of incubation mixtures were taken at the times shown and assayed for transferase activity (Materials and Methods).
FIGURE
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t-
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FIGURE 3 : Diagramatic representation of polyacrylamide gel separations of enzyme preparations pretreated with 4 h i urea. Enzymes (1.5-2.5 mg/ml) were incubated for 15 rnin at 37' i n the urea inactivation mixture (Materials and Methods). An equal volume of 60% sucrose was added and samples (75-300 pg of protein) put on 5 % gels at pH 7.2 and current applied for 1 hr (Materials and Methods). Gels were stained with Amido Schwarz to detect protein. The arrows mark the position of the tracking dye at the bottom of each gel. The enzyme preparations ure described in Figure 2. Prior to incubation in the urea inactivation mixture, all preparations exhibited electrophoretic patterns identical with that depicted here for the E, preparation.
2A, incubation of various mixed enzyme preparations in the urea inactivation mixture led to a rapid partial inactivation ; the fraction of enzyme activity ultimately lost was directly proportional to the average number of adenylylated subunits initially present in the mixture. In contrast, as shown in Figure 2B, the natural enzyme preparations were inactivated less rapidly and the extent of inactivation after 10 rnin was not directly proportional to the average state of adenylylation. Figure 2B also shows that the stability of unadenylylated , by deadenylylation of E ?: with snake enzyme ( E g ~ s )prepared venom phosphodiesterase, is about the same as that of Erg enzyme isolated directly from E. coli. As is indicated by the numbers o n the curves in Figure 2A, no adenylylated subunit activity remained after 30-min exposure of mixed preparations to 4 M urea; all residual activity was due to unadenylylated enzyme. This is as expected from the stability characteristics of the Eo3 and E% preparations (Figure 1) and shows that under these conditions there is probably no interaction between fully adenylylated and unadenylylated enzymes to form hybrid molecules. In contrast, as shown in Figure 2B, when the natural enzyme preparations were exposed to the urea inactivation mixture, the activity that remained after 30 and 60 rnin was due to both adenylylated and unadenylylated subunits. Thus, the average states of adenylylation of the ET:luied I"Claden~l)l-t,.(with 2s"; residual trenrferare activit)) ~ 3 mixed s uith an q u a l amount of k j". The mixed enLyme5 acre dirroriateu inlo ruhunm snd the suhun~irruhrequentl! rcasrociaied as dcwlbed under Materiali and 2lethods. The rriultant reconstituted mr)me mixture u a s s u h iecicd to elecirophorc\i~ on an acr!lamdr disc gel at pH 7.2 (Materials and Method
