Tripolyphosphatase associated with S-adenosylmethionine synthetase isozymes from rat liver

Biochimica et Biophysica Acta 883 (1986) 293-298 Elsevier

293

BBA 22423

Tripolyphosphatase associated with S-adenosylmethionine synthetase isozymes from rat liver Keiko Shimizu, Izumi Maruyama, Shigeyuki Iijima and Kinji Tsukada * Department of Pathological Biochemistrp, Medical Research Institute, Tokyo Medical and Dental University, Kandasurugadai, Chiyoda-ku, Tokyo 101 (Japan) (Received April 10th, 1986)

Key words: S-Adenosylmethionine synthetase isozyme; Methionine adenosyltransferase; Tripolyphosphatase; Integrated enzyme; (Rat liver)

S-Adenosylmethionine (AdoMet) synthetase a and/~ were purified to homogeneity, as judged by SDS-polyacrylamide gel electrophoresis from rat liver. When the purified enzymes were applied onto Sephacryl S-200, each synthetase was eluted together with a tripolyphosphatase. The activities of these isozymes in synthesizing AdoMet and in hydrolyzing tripolyphosphate decreased in parallel with increasing amounts of rabbit anti-(/3-form) IgG. The activity of the/I-form isozyme was markedly stimulated by the addition of tripolyphosphate, whereas that of the a-form isozyme was inhibited. The tripolyphosphatase activity of both the a- and the ~-form was markedly stimulated by the addition of AdoMet. The tripolyphosphatases of each isozyme showed some other similar properties. Introduction Enzymatic synthesis of S-adenosylmethionine (AdoMet) is catalyzed by AdoMet synthetase (ATP: L-methionine S-adenosyltransferase, EC 2.5.1.6): Methionine + ATP ~ AdoMet + PP~ + Pi As reported by Cantoni and Durell [1,2], the three phosphates of ATP are cleaved. The 5'-deoxyadenosyl group of ATP is transferred to one free electron pair from the sulfur atom of L-methionine and, at the same time, tripolyphosphate is formed. This then generates pyrophosphate and inorganic phosphate; the former originates from a- and t-phosphate groups, and the latter from the yphosphate of ATP. The enzyme-bound intermediate, tripolyphosphate, is cleaved by a tripolyphosphatase that is associated with purified AdoMet synthetase from yeast [3] and E. coli [4].

* To whom correspondence should be addressed.

The same reaction mechanism is thought to operate in AdoMet synthetase of mammalian tissues [5]. Kinetic studies of the AdoMet synthetase reaction have been reported in detail [3,6-11]. Three isozymes, termed a, t , and y, of AdoMet synthetase have been demonstrated in mammalian tissues [12-17]. At least two isozymes, the a- and t-forms are present in normal liver, and the y-form is found in kidney, fetal liver [14] and some other tissues, except liver [13]. The t-form was purified to homogeneity from rat liver cytosol [17,18], and antiserum against the t-form was shown to crossreact with the a-form and the B-form, but not with the y-form [17]. Thus the a-form was purified to homogeneity using an immunoabsorbent against the t-form [18]. Tripolyphosphatase activity was observed intrinsically associated with these isozymes. Recently, an AdoMet synthetase corresponding to the y-form has been isolated from human lymphocytes [19], and this enzyme also was shown to be associated with a tripolyphosphatase.

0304-4165/86/$03.50 © 1986 Elsevier Science Publishers B.V. (Biomedical Division)

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To understand the regulation mechanism of AdoMet synthetase isozymes in mammalian liver, we investigated the properties of the tripolyphosphatase of each isozyme from rat liver. Materials and Methods

Chemicals. All the reagents were as described in preceding papers [13,17]. All other reagents were of analytical grade. Assay of AdoMet synthetase. The synthetase was assayed (in a total volume of 0.1 ml) in 0.1 M Tris-HCl (pH 9.0), 20 mM MgC12, 0.15 M KC1, 5 mM dithiothreitol, 10 mM ATP and 25 # M L-[methyl-3H]methionine (0.25 #Ci). Assays were initiated by the addition of enzyme, the mixture was incubated for 10 rain at 37°C, and the reaction was terminated by the addition of 20 #1 2 M HC104/5 mM methionine and by chilling the reaction tubes in an ice-water bath. The resulting precipitate was removed by centrifugation at 1000 x g for 5 min, and a 100 #1 aliquot of the supernatant was spread onto a cellulose phosphate paper disc (diameter 2.5 cm). The disc was dropped into a beaker containing 5 mM phosphate buffer (pH 7.0) and was extensively washed with the same buffer. The washed paper was transferred to a counting vial and was immersed in 1 ml 1.5 M NH4OH. The radioactivity was measured in Triton X-100/toluene scintillation fluid. Protein was determined by the method of Lowry et al. [20] with bovine serum albumin as standard. One unit of the enzyme activity was defined as the formation of 1 n m o l / m i n of AdoMet in the incubation mixture. Analytical gel filtration. Gel filtration was performed on a Sephacryl S-200 column (1.9 x 79 cm) with 10 mM Tris-HCl (pH 7.9) containing 0.5 m M dithiothreitol and 0.1 mM EDTA (buffer A) and 0.1 M KC1 as eluting buffer. Assay of tripolyphosphatase. The activity was measured by the production of phosphate from tripolyphosphate. Phosphate was determined by the procedure of Baginski et al. [21] as modified by Lombardini et al. [5]. The reaction mixture (0.1 ml) containing 50 mM Tris-HC1 (pH 7.8), 100 mM KCI, 10 mM MgC12, 2 mM sodium tripolyphosphate, 0.5 mM dithiothreitol and enzyme. After a 10 min incubation at 30°C, to each tube the

following additions were made rapidly: 0.3 ml 10% (w/v) trichloroacetic acid/2% (w/v) ascorbic acid/0.1% EDTA; 0.2 ml water; 0.15 ml 2% (w/v) ammonium molybdate; 0.3 ml 2% (w/v) sodium arsenate/4% ( w / v ) sodium citrate/10% trichloroacetic acid. Each tube was thoroughly mixed on a vortex mixer after addition of each reagent. After 20 min, the absorbance of the solution was determined at 724 nm and concentrations were calculated with a standard curve. Sodium dodecyl sulfate gel electrophoresis. Electrophoresis in the presence of sodium dodecyl sulfate was performed by the procedure of Laemmli [22]. Protein samples were subjected to electrophoresis at 7 mA per tube (0.5 x 8 cm) on a 7.5% polyacrylamide get containing 0.1% sodium dodecyl sulfate. Gels were stained with Coomassie brilliant blue and were destained according to the method of Weber and Osborn [23]. Sucrose gradients. Linear 4.5 ml gradients of 5-20% sucrose were prepared in 20 mM Tris-HC1 (pH 7.5) containing 0.5 mM dithiothreitol and 0.1 mM EDTA. Samples containing the purified enzyme (1.0 unit) in 100 /~1 were layered on to gradients and centrifuged at 40000 rev./min for 15 h at 5°C in a Hitachi SW 50 rotor. Catalase (S20,w = 11.3 S), bovine serum albumin (S20,w = 4.4 S) and cytochrome c (S20,w = 1.7 S) were used as external marker proteins. Lactate dehydrogenase (S20,w = 7.0 S) was used as an external and internal marker protein. Purification of AdoMet synthetase isozymes. The fl-form of AdoMet synthetase was purified to homogeneity from rat liver cytosol [17,18], and the a-form was isolated from rat liver cytosol as a single protein using immunoabsorbent against the fl-form [18]. To purity the a-form the following steps were followed: calcium phosphate gel, ammonium sulfate (50% saturation), phenyl-Sepharose, DEAE-cellulose and immunoaffinity chromatography [18]. Immunochemical procedure. Rabbit antisera were prepared against homogeneous AdoMet synthetase fl, and the immunoglobulin fraction was isolated as described previously [17,24]. Control immunoglobulin fraction was prepared from nonimmunized rabbit.

295 ~4

I

I

I

I

,

I

3

i

I

z

[

+ I LZ,

+ ~0

5

I0

15 20 25 30 35 Fraction number

Fig. 1. Evidence for association of tripolyphosphatase with AdoMet synthetase isozymes from rat liver. A Sephacryl S-200 column (1.9 × 79 cm) was equilibrated with buffer A containing 0.1 M KC1. The purified fl-form (20 units) or a-form (16 units), which was dialyzed against the same buffer, was applied to the above column and the column eluted with the equilibrating buffer at a flow rate of 10 ml/h. The eluate was collected in 1.0 ml fractions. Aliquots of 10 #1 were removed from each fraction and assayed for the synthetase and tripolyphosphatase activities. The arrow shows the molecular weight estimation, where myoglobin (1) (M r 17200), ovalbumin (2) (M r 45000), lactate dehydrogenase (3) (M r 145000) and catalase (4) (M r 244000) were used.

the activity of t r i p o l y p h o s p h a t a s e in each fraction (Fig. 1). T h e m o l e c u l a r masses of the a - a n d fl-forms were e s t i m a t e d as a p p r o x . 200 a n d 100 k D a , respectively. A specific a n t i s e r u m against the fl-form was shown to cross-react with b o t h the fla n d the a - f o r m s [17]. Various a m o u n t s of antib o d y against the fl-form were tested against a fixed a m o u n t of e n z y m e activity of each isozyme. A s shown in Fig. 2, the ability of each isozyme to synthesize A d o M e t decreased in coincidence with the decreased a b i l i t y to h y d r o l y z e t r i p o l y p h o s phate, with increasing a m o u n t s of the a n t i b o d y . However, the a b i l i t y to synthesize A d o M e t a n d to h y d r o l y z e t r i p o l y p h o s p h a t e was n o t affected b y the increased a m o u n t s of c o n t r o l 7 - I g G . These results i n d i c a t e that the s a m e e n z y m e catalyzes b o t h the A d o M e t synthetase a n d the t r i p o l y p h o s p h a t a s e reaction in b o t h isozymes. T h e p u r i t y of the p o o l e d fractions f r o m the a - f o r m a n d the fl-form was e x a m i n e d b y electrophoresis. S D S - p o l y a c r y l a m i d e gel electrophoresis gave one m a j o r p r o t e i n b a n d on staining with C o o m a s s i e b r i l l i a n t blue. T h e b a n d of the a - f o r m

Results and Discussion T h e purified a - a n d fl-forms were a p p l i e d o n t o S e p h a c r y l S-200 e q u i l i b r a t e d with b u f f e r A containing 0.1 M KC1 a n d eluted with the s a m e buffer. T h e activity o f each of the synthetase isozymes eluted as a s h a r p p e a k c o i n c i d i n g with

:---+

.......

; .......

i- . . . . . .

IgG (pg) Fig. 2. Effects of antibody against/be ~ - f o ~ on the activities of AdoMet syntbetase isozymes from rat liver. AdoMet syn-

thetase a or fl (40 milliunits) was incubated with indicated amounts of anti-(fl-form) IgG or control IgG (. . . . . . ). After incubation at 25°C for 2 h, the supernatant was assayed for the activities of AdoMet synthetase of the a-form (C)) and the fl-form (e), and of tripolyphosphatase of the a-form (zx) and the fl-form (-).

Fig. 3. Determination of molecular weight of AdoMet synthetase isozymes from rat liver by sodium dodecyl sulphatepolyacrylamide gel electrophoresis. The purified a-form (3/tg) and fl-form (7 #g) in Fig. 1 were subjected to electrophoresis. 'M' shows the three marker proteins, bovine serum albumin (M r 67000); ovalbumin (M r 43000); lactate dehydrogenase (M r 35000; A subunit), a, AdoMet synthetase a; /3, AdoMet synthetase ft.

296

700 ~600 ~500

\

%

r

\

i

dl

I

I

2

Trlpol~e

3

(mM)

Fig. 4. Effects of tripolyphosphate on the activity of A d o M e t synthetase isozymes from rat liver. Each isozyme assay was performed in the standard reaction mixture containing the indicated concentration of tripolyphosphate. The purified isozyme (each 2 /~g of protein) from rat liver was used. The enzyme activity at 0 M tripolyphosphate was defined as 100%. O, A d o M e t synthetase ct; O, AdoMet synthetase ft.

coincided with the subunit of the fl-form with a molecular mass of 48 kDa (Fig. 3, Ref. 17). These results suggest that the a-form is composed of four subunits of molecular weight 48000, w~le the fl-form is composed of two subunits of the same molecular size. The sedimentation coefficient of the fl- and a-forms was calculated to be 5.5 S and 8.0 S, L

I

I

6t~

respectively, after sucrose gradient centrifugation with protein markers of known molecular weight and S20.w values. Tripolyphosphate is a powerful inhibitor of AdoMet synthetase and is a competitor of ATP [7-9,25]. The effects of increasing concentrations of tripolyphosphate on both isozymes were examined. Low concentrations of tripolyphosphate activated the fl-form strikingly, whereas the a-form was inhibited, as shown in Fig. 4. The activity of the tripolyphosphatase of the aand fl-forms is markedly stimulated by AdoMet (Fig. 5). The K m values for tripolyphosphate in the absence and presence of AdoMet were calculated to be 31/LM and 80/~M for the a-form, and 38/~M and 90/~M for the fl-form, respectively. The tripolyphosphatases of these isozymes absolutely require divalent cations (Table I).When dithiothreitol was omitted from the standard reaction mixture, the addition of 0.5 mM p-chloromercuribenzoate resulted in inhibition of tripolyphosphatase activities (Table I). When 10% dimethyl sulfoxide (v/v), was added to the reaction mixture of AdoMet synthetase, the ability to synthesize AdoMet in the a- and fl-forms was activated by 1.5- and 17-fold, respectively [13]. As in the case of dimethyl sulfoxide, another cryoprotectant, 20% glycerol (v/v), similarly affected the activity of the synthetase isozymes. TABLE I C A T A L Y T I C PROPERTIES O F T R I P O L Y P H O S P H A T A S E O F AdoMet S Y N T H E T A S E ISOZYMES F R O M R A T LIVER The same a m o u n t s of each isozyme as in Fig. 4 were used. Assay conditions

Relative activity of tripolyphosphatase (% of control)

Control systems - MgCI 2

100 2

P

!l~~°o d.i o'.2 da (raM)

Fig. 5. Effects of AdoMet on the activity of tripolyphosphatase of each synthetase isozyme. Each enzyme assay was performed in the standard reaction mixture containing the indicated concentration of AdoMet. The same a m o u n t s of each enzyme in as Fig. 4 were used in this experiment. The enzyme activity at 0 M AdoMet was defined as 100~. O, AdoMet synthetase a; e , A d o M e t synthetase ft.

- KCI

98

Dithiothreitol -- Dithiothreitol + p-chloromercuribenzoate (0.5 mM) + Glycerol (205~, v / v ) + Dimethyl sulfoxide (10%, v / v ) + Cyelolencine (0.1 raM) + Cycloleucine (1.0 raM)

85

100 3 95 80

20 60 92 96 105

15 45 89 95 103

-

297 However, the activities of the tripolyphosphatase of the isozymes were somewhat inhibited by these reagents. An analogue of L-methionine, cycloleucine, was shown to inhibit AdoMet synthetases from yeast and E. coli [26]. AdoMet synthetase "t was susceptible to inhibition by tripolyphosphate and cycloleucine, as was AdoMet synthetase a [13]. Addition of 0.1 m M cycloleucine to the reaction mixture stimulated the fl-form of the synthetase, whereas the a- and "t-forms were inhibited [13]. However, a wide range of concentrations of this compound was without effect on the tripolyphosphatase activity of the isozymes from rat liver (Table I). AdoMet synthetases which have been purified to homogeneity from E. coli [11] and yeast [10] require divalent and monovalent cations for full activity, and also have a tripolyphosphatase activity which is stimulated by AdoMet. The yeast AdoMet synthetase is a protein of molecular weight 110000 composed of two subunits of molecular weight 60000 and 55000 [10]. AdoMet synthetase with a molecular weight of 185 000 has been purified to apparent homogeneity from human lymphocytes [19]. The enzyme has tripolyphosphatase activity, which is stimulated by AdoMet, and when incubated, the expected products AdoMet, PPi and Pi are formed in equimolar amounts [19]. AdoMet synthetase isozymes from mammalian tissues differ in their ability to synthesize AdoMet, in the number of charges per enzyme molecule and in some characteristics. However, they are not different with respect to tripolyphosphatase activity (Table I, Fig. 5). The activity of tripolyphosphatase in all AdoMet synthetase isozymes is stimulated by exogenous AdoMet. Double-reciprocal plots of the activity of synthetase a and ), against the two substrates, L-methionine and ATP, respectively, exhibit negative cooperativity. However, kinetic patterns of synthetase fl showed positive cooperative for each substrate. AdoMet synthetase may, therefore, be regarded as an allosteric enzyme capable of undergoing conformational changes in the presence of various ligands. The activity of the a-form in rat liver is increased following administration of ethionine plus adenine for two consecutive days [15,16], and after feeding a high methionine diet [27]. The

values of S-adenosylethionine and AdoMet in the cells increased almost in parallel with the change in the activity of the a-form [28]. These results suggest that only the a-form is functional in the liver when a rat receives excess exogenous substrates. In the presence of tripolyphosphate, the activity of the a-form was inhibited, whereas that of the fl-form was activated strongly (Fig. 4). AdoMet stimulated the hydrolysis of tripolyphosphate promoted by both the a- and the fl-form. From these phenomena, we deduced that a relatively low concentration of AdoMet probably plays a role in modulating the synthesis of AdoMet itself. Acknowledgements

This investigation was supported in part by a Grant-in-Aid for Cancer Research from the Ministry of Education, Science and Culture, and from the Ministry of Health and Welfare (60-20), Japan. We wish to thank Miss Yukie Suma and Maki Horii for their technical assistance in part of this work. References

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