Expression and functional interaction of the catalytic and regulatory subunits of human methionine adenosyltransferase in mammalian cells

Methionine adenosyltransferase (MAT) catalyzes the synthesis of S-adenosylm ethionine (AdoMet). The mammalian MAT II isozyme consists of catalytic alph a(2) and regulatory beta subunits, The aim of this study was to investigate the interaction and kinetic behavior of the human MAT II subunit proteins in mammalian cells. COS-1 cells were transiently transfected with pTargeT v ector harboring fall-length cDNA that encodes for the MAT II alpha(2) or be ta subunits. Expression of the His-tagged recombinant alpha(2) (r alpha(2)) subunit in COS-1 cells markedly increased MAT II activity and resulted in a shift in the K-m, for L-methionine (L-Met) from 15 mu M (endogenous MAT I I) to 75 mu M (r alpha(2)), and with the apparent existence of two kinetic forms of MAT in the transfected COS-I cell extracts, By contrast, expressio n of the recombinant beta (r beta) subunit had no effect on the K-m for L-M et of the endogenous MAT II, while it did cause an increase in both the V-m ax and the specific activity of endogenous MAT. Go-expression of both r alp ha(2) and r beta subunits resulted in a significant increase of MAT specifi c activity with the appearance of a single kinetic form of MAT (K-m = 20 mu M). The recombinant MAT II alpha(2) and r beta suibunit associated spontan eously either in cell-free system or in COS-1 cells coexpressing both subun its, Analysis of nickel-agarose-purified His-tagged r alpha(2) subunit from COS-1 cell extracts showed that the beta subunit co-purified with the alph a(2) subunit. Furthermore, the alpha(2) and beta subunits co-migrated in na tive polyacrylamide gels. Together, the data provide evidence for alpha(2) and beta MAT subunit association. In addition, the beta subunit regulated M AT II activity by reducing its K-m for L-Met and by rendering the enzyme mo re susceptible to feedback inhibition by AdoMet. We believe that the previo usly described differential expression of MAT II beta subunit may be an imp ortant mechanism by which MAT activity can be modulated to provide differen t levels of AdoMet that may be required at different stages of cell growth and differentiation.