Abstract
A two-dimensional polyacrylamide gel electrophoresis system which is suitable for the analysis of protein methylation reactions in cells incubated with l-[ methyl- 3H]methionine is described. The procedure separates proteins under primarily acidic conditions by isoelectric focusing in the first dimension and by sodium dodecyl sulfate electrophoresis at pH 2.4 in the second dimension. The low pH is essential for preserving protein [ 3H]methyl esters, but it limits the effective separating range of this system to proteins with isoelectric points between 4 and 8. With this system, we have shown that most, if not all, erythrocyte membrane and cytosolic proteins can act as substoichiometric methyl acceptors for an intracellular S-adenosylmethionine-dependent carboxyl methyltransferase and that protein carboxyl methylation reactions may be the major methyl transfer reaction in erythrocytes. These results are most consistent with the generation of protein substrate sites for the carboxyl methyltransferase by spontaneous deamidation and racemization reactions.
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