. The recombinant MAT II ␣ 2 and r␤ subunit associated spontaneously either in cell-free system or in COS-1 cells coexpressing both subunits. Analysis of nickel-agarosepurified His-tagged r␣ 2 subunit from COS-1 cell extracts showed that the ␤ subunit co-purified with the ␣ 2 subunit. Furthermore, the ␣ 2 and ␤ subunits co-migrated in native polyacrylamide gels. Together, the data provide evidence for ␣ 2 and ␤ MAT subunit association. In addition, the ␤ subunit regulated MAT II activity by reducing its K m for L-Met and by rendering the enzyme more susceptible to feedback inhibition by AdoMet. We believe that the previously described differential expression of MAT II ␤ subunit may be an important mechanism by which MAT activity can be modulated to provide different levels of AdoMet that may be required at different stages of cell growth and differentiation.

This paper reports clinical and metabolic studies of two Italian siblings with a novel form of persistent isolated hypermethioninaemia, i.e. abnormally elevated plasma methionine that lasted beyond the first months of life and is not due to cystathionine beta-synthase deficiency, tyrosinaemia I or liver disease. Abnormal elevations of their plasma S-adenosylmethionine (AdoMet) concentrations proved they do not have deficient activity of methionine adenosyltransferase I/III. A variety of studies provided evidence that the elevations of methionine and AdoMet are not caused by defects in the methionine transamination pathway, deficient activity of methionine adenosyltransferase II, a mutation in methylenetetrahydrofolate reductase rendering this activity resistant to inhibition by AdoMet, or deficient activity of guanidinoacetate methyltransferase. Plasma sarcosine (N-methylglycine) is elevated, together with elevated plasma AdoMet in normal subjects following oral methionine loads and in association with increased plasma levels of both methionine and AdoMet in cystathionine beta-synthase-deficient individuals. However, plasma sarcosine is not elevated in these siblings. The latter result provides evidence they are deficient in activity of glycine N-methyltransferase (GNMT). The only clinical abnormalities in these siblings are mild hepatomegaly and chronic elevation of serum transaminases not attributable to conventional causes of liver disease. A possible causative connection between GNMT deficiency and these hepatitis-like manifestations is discussed. Further studies are required to evaluate whether dietary methionine restriction will be useful in this situation.

Methionine adenosyltransferase (MAT) catalyzes the biosynthesis of S-adenosylmethionine (AdoMet), a key molecule in transmethylation reactions and polyamine biosynthesis. The MAT II isozyme consists of a catalytic ␣2 and a regulatory ␤ subunit. Down-regulation of the MAT II ␤ subunit expression causes a 6 -10-fold increase in intracellular AdoMet levels. To understand the mechanism by which the ␤ subunit expression is regulated, we cloned the MAT2B gene, determined its organization, characterized its 5-flanking sequences, and elucidated the in vitro and in vivo regulation of its promoter. Transcription of the MAT2B gene initiates at position ؊203 relative to the translation start site. Promoter deletion analysis defined a minimal promoter between positions ؉52 and ؉93 base pairs, a GC-rich region. Inclusion of the sequences between ؊4 and ؉52 enhanced promoter activity; this was primarily because of an Sp1 recognition site at ؉9/؉15. The inclusion of sequences up to position ؊115 provided full activity; this was attributed to a TATA at ؊32. The Sp1 site at position ؉9 was key for the formation of protein⅐DNA complexes. Mutation of both the Sp1 site at ؉9 and the TATA at ؊32 reduced promoter activity to its minimal level. Supershift assays showed no effect of the anti-Sp1 antibody on complex formation, whereas the anti-Sp3 antibody had a strong effect on protein⅐DNA complex formation, suggesting that Sp3 is one of the main factors binding to this Sp1 site. Chromatin immunoprecipitation assays supported the involvement of both Sp1 and Sp3 in complexes formed on the MAT2B promoter. The data show that the 5-untranslated sequences play an important role in regulating the MAT2B gene and identifies the Sp1 site at ؉9 as a potential target for modulating MAT2B expression, a process that can have a major effect on intracellular AdoMet levels. Methionine adenosyltransferase (MAT)1 (ATP⅐L-methionine S-adenosyltransferase) (EC 2.5.1.6) catalyzes the biosynthesis of S-adenosylmethionine (AdoMet) (1, 2). AdoMet is the major methyl donor in transmethylation reactions and the propylamine donor in the biosynthesis of polyamines (3-5). Furthermore, AdoMet participates as a co-factor in key metabolic pathways (3-5). Most species studied to date have more than one MAT isozyme (6). In mammals, the two major MAT isozymes are designated MAT I/III and MAT II (7-10). The MAT I/III isozymes are, respectively, a tetramer and dimer of a catalytic ␣1 subunit that is expressed only in liver (7, 11-13). The MAT II isozyme is expressed in all tissues including the liver (9, 14 -21). Previous studies from our group (15, 22) have determined that MAT II from human leukemic T and B cells, as well from activated human lymphocytes, is a hetero-oligomer that consists of ␣2 (53 kDa), ␣2Ј (51 kDa), and ␤ (38 kDa) subunits. The ␣2/␤ hetero-oligomeric composition of MAT II was also determined in bovine brain, Ehrlich ascites tumor, and calf thymus (15,19). The ␣2 subunit is responsible for the enzyme catalytic activity and is post-translationally modifie...

Here we show that in extrahepatic methionine adenosyltransferase replacement of a single amino acid (glycine 120) by cysteine is sufficient to create a functional nitric oxide binding site without affecting the kinetic properties of the enzyme. When wild-type and mutant methionine adenosyltransferase were incubated with S-nitrosoglutathione the activity of the wild-type remained unchanged whereas the activity of the mutant enzyme decreased markedly. The mutant enzyme was found to be S-nitrosylated upon incubation with the nitric oxide donor. Treatment of the S-nitrosylated mutant enzyme with glutathione removed most of the S-nitrosothiol groups and restored the activity to control values. In conclusion, our results suggest that functional S-nitrosylation sites can develop from existing structures without drastic or large-scale amino acid replacements.z 1999 Federation of European Biochemical Societies.

We exploited the fact that leukemic cells utilize significantly higher levels of S-adenosylmethionine (SAMe) than normal lymphocytes and developed tools that selectively diminished their survival under physiologic conditions. Using RNA interference gene silencing technology, we modulated the kinetics of