Characteristics of Protein Carboxyl Methylation in the Rat Hypothalamus

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Abstract
The formation of methyl‐labeled S‐adenosylmethionine (AdoMet) and methyl esters of endogenous methyl‐acceptor proteins (MAPs) was studied in a synaptosomal preparation from the rat hypothalamus labeled with L‐[methyl‐3H]methionine. Incubation of synaptosomes with l‐[methyl‐3H]methi‐onine resulted in a rapid labeling of the AdoMet pool and a less rapid formation of 3H‐methyl‐MAPs. Accumulation of 3H‐methyl‐MAPs was linear over a 30‐min period. The effects of various inhibitors of AdoMet‐dependent trans‐methylation reactions on the formation of carboxylmethylated MAPs 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 μm), 3H‐methyl‐MAP formation was inhibited by approximately 70%. 100 μm‐l‐homocysteine thiolactone (HTL) as well as 100 μm‐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 μm‐adenosine or 10 μm‐HTL each produced an approximately 40% inhibition of 3H‐methyl‐MAP formation: the inhibitory effect of the two 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. However, these compounds were potent inhibitors of 3H‐methyl‐MAP formation in lysed synaptosomes incubated with [3H‐methyl]AdoMet. These results demonstrate that hypothalamic synaptosomes are capable of methio‐nine activation and protein carboxyl methylation.