Methionine metabolism in BHK cells: The regulation of methionine adenosyltransferase

Abstract

The regulation of methionine adenosyltransferase (MAT), an enzyme involved in SAM biosynthesis was studied in baby hamster kidney cells. A 4-fold increase of the specific activity of MAT gradually occurred when cells were transferred to a medium containing low amounts of methionine. In a methionine rich medium the MAT specific activity of previously induced cells slowly decreased (half inactivation time: 30 hours). Actinomycin D and cycloheximide blocked the induction of the enzyme. The analysis of the mechanism of repression of MAT biosynthesis by methionine strongly suggested that a posttranscriptional process of regulation was involved. The induction of MAT was not affected by the growth phase of cells.No evidence of a direct correlation between the intracellular amounts of aminoacylated tRNAmet and MAT repression was shown. A clone showing a reduced MTS activity was isolated by a (3H-methyl) methionine suicide technique. The regulation of MAT remained unaffected in this clone. Cycloleucine, an inhibitor of in vivo SAM biosynthesis, induced the biosynthesis of MAT in a methionine rich medium. These results suggest that MAT is regulated by SAM, or a derivative of SAM, in hamster cells.A Cycloleucine resistant clone was isolated. This clone showed increased intracellular SAM pool and MAT activity. No evidence for a structural modification of the enzyme was shown. A regulatory mutation might be involved in this clone.Studies on regulatory aspects of methionine biosynthesis and SAM biosynthesis in baby hamster kidney cells have shown that exogenous methionine exerts a potent repression on the synthesis of methionine adenosyltransferase (EC 2.5.1.6), an enzyme involved in the conversion of methionine to SAM (Caboche, '76). The aim of this work was to further characterize the regulation mechanism of this enzyme, as it plays a key role in the control of intracellular SAM levels and of methylations.The action of various inhibitors on the induction of MAT was first studied. As the growth phase of cells might affect the inducibility of MAT (Martin and Tomkins, '70) we further tested the induction of MAT in synchronized cells.Besides methionine, the two activated forms of this amino acid; i.e., methionyl-tRNA and SAM were postulated as likely candidates for the effector of the regulation. Using specific inhibitors of tRNA activation and SAM biosynthesis we partially tested the role of these metabolites in the regulation of MAT. We also selected for mutant clones affected in MTS and MAT activities. Results presented here show that in baby hamster kidney cells, SAM or one of its derivatives is involved in the regulation of MAT. MATERIALS AND METHODS ChemicalsL (Carboxyl '*C) methionine (49 mCi/ mmol); L (methyl 14C) methionine (50 mCi/ mmol) and L (methyl 3H) methionine (5 Ci/ mmol) were obtained from C.E.A. L-methio-

Relative effects of S-adenosylmethionine depletion on nucleic acid methylation and polyamine biosynthesis

“…That this increase in enzyme activity is related to AdoMet depletion is strongly suggested by the finding that exogenous AdoMet partially prevents the effect in L-cisAMB-treated cells (Table 1). These findings relate directly to previous studies by Caboche (1977) and Jacobsen et al (1980), which indicate that AdoMet synthetase activity increases severalfold when methionine in media is restricted. Further, Jacobsen et al (1980) suggest that the increase is related to a post-transcriptional derepression of enzyme synthesis, which is mediated by methionine or one of its metabolites.…”

“…It is also possible that, because L-cisAMB is a reversible inhibitor of AdoMet synthetase, it might also stabilize L-cisAMB treatment (4 h) AdoMet (nmol/107 cells) the enzyme against degradation and thereby extend its half-life in a manner similar to the effect of methylglyoxal bis(guanylhydrazone) on AdoMet decarboxylase (Fillingame & Morris, 1973;Pegg et al, 1973). However, because AdoMet synthetase appears to have a relatively long half-life [-30 h;Caboche (1977)], the contribution of this phenomenon would probably be minimal over a 24 h period. The increased effectiveness of L-cisAMB in depleting AdoMet pools under conditions of decreased methionine in media (Fig.…”

Cycloleucine blocks 5'-terminal and internal methylations of avian sarcoma virus genome RNA

“…Cycloleucine acts by reducing the intracellular concentration of SAM (Caboche, 1977), whereas STH probably acts as an inhibitor in the enzymatic transfer of methyl groups from SAM to the RNA.…”

Methylthioadenosine toxicity and metabolism to methionine in mammalian cells

“…However, we found the same enzyme activity in Raji and CCL39 cells cultured in 100 /LM-Met (5.2 + 0.5 and 5.5 + 0.5 nmol/h per mg of protein respectively), in accordance with the results of Oden et al (1983), who found no difference in methionine adenosyltransferase activity between Met-dependent and -independent cells. Previous studies by Caboche (1977) have shown that exogenous Met exerts a potent repression on the synthesis of methionine adenosyltransferase, presumably via an increase in the concentration of AdoMet itself. To characterize such a repression with Met and with its precursor, MeSAdo, we compared methionine adenosyltransferase activity in CCL39 cells cultured for 20 h in media with various (Met + MeSAdo) concentrations.…”

“…MeSAdo phosphorylase activity was measured as previously described (Christa et al, 1983), and methionine adenosyltransferase activity was assayed by the method of Caboche (1977), modified as follows: the incubation mixture contained, in a total volume of 0.2 ml, 0.15 M-Pipes (dipotassium salt), pH 7.2, 0.067 M-MgCl2, 0.010 M-ATP (dipotassium salt) and 67 /M (0.2 gCi) [carboxy-'4C]Met. After 15, 30 or 45 min incubation at 37°C, 0.05 ml samples were transferred into 0.025 ml of 0.3 M-HC104.…”