Characteristics of Protein Carboxyl Methylation in the Rat Hypothalamus

Abstract

The formation of methyl-labeled S-adenosylmethionine (AdoMet) and methyl esters of endogenous methyl-acceptor proteins (MAP) were studied in a synaptosomal preparation from the rat hypothalamus labeled with L-[methyl-3H]methionine. Incubation of synaptosomes with L-[methyl-3H]methionine resulted in a rapid labeling of the AdoMet pool and a less rapid formation of 3H-methyl-MAP. Accumulation of 3H-methyl-MAP was linear over a 30-min period. The effects of various inhibitors of AdoMet-dependent transmethylation reactions on the formation of carboxylmethylated MAP were examined. When hypothalamic synaptosomes were preincubated with L-[methyl-3H]methionine and subsequently incubated for 30 min in the presence of S-adenosyl-L-homocysteine (AdoHcy, 100 .mu.M), 3H-methyl-MAP formation was inhibited by approximately 70%. 100 .mu.M-L-homocysteine thiolactone (HTL) and 100 .mu.M-3-deazaadenosine (c3Ado) also caused a 60-70% inhibition of 3H-methyl-MAP formation; the combination of both c3Ado and HTL produced a slightly but not significantly greater inhibition than either agent alone. 10 .mu.M-adenosine or 10 .mu.M-HTL each produced an approximately 40% inhibition of 3H-methyl-MAP formation: the inhibitory effect of the 2 agents in combination was additive. Sinefungin and A9145C, potent inhibitors of bovine adrenomedullary protein carboxyl methylase, had no effect on 3H-methyl-MAP formation in hypothalamic synaptosomes at concentrations up to 1 mM. These compounds were potent inhibitors of 3H-methyl-MAP formation in lysed synaptosomes incubated with [3H-methyl]AdoMet. Hypothalamic synaptosomes are capable of methionine activation and protein carboxyl methylation.