Tubulin and high molecular weight microtubule-associated proteins as endogenous substrates for protein carboxymethyltransferase in brain

Abstract

The endogenous substrate for protein carboxymethyltransferase in brain was examined. Several polypeptides were methylated when brain slices were incubated with L-methionine or when subcellular fractions of brain, such as the cytosolic fraction, were incubated with S-adenosyl L-methionine. Two methyl-accepting proteins in the cytoplasm were identified as tubulin and high molecular weight microtubule-associated proteins (300 kDa), which are components of microtubules. Tubulin behaved as a 43 kDa protein in acidic polyacrylamide gel electrophoresis, but as a 55 kDa protein in SDS-polyacrylamide gel electrophoresis. the methyl moiety transferred to these proteins from L-methionine was labile at alkaline pH. The high molecular weight microtubule-associated proteins showed higher methyl-accepting activity than tubulin or ovalbumin, which was used as a standard substrate: about 20 mmol of high molecular weight microtubule-associated proteins, 2 mmol of tubulin and 10 mmol of ovalbumin were methylated per mol of each protein in 30 min under the experimental conditions used.