Purification and properties of protein methylase II from wheat germ.
Protein methylase II (S-adenosylmethionine: protein-carboxyl O-methyltransferase, EC 2.1.1.24) was purified from wheat germ approximately 1200-fold with a yield of 4.6% by employing gel filtration. The enzyme, using S-adenosyl-L-methionine, methyl-esterifies the free carboxyl group of protein. Since the enzymatic product was unstable and hydrolyzed non-enzymatically to yield methanol, methanol was identified from the hydrolysate by the formation of the methyl ester of 3.5-dinitrobenzoate. The pH optimum for the wheat germ enzyme was 7.0 in contrast to the 6.0 for the methylase II of calf brain. The wheat germ enzyme had a molecular weight of 41 000 compared to 25 000 for the corresponding rat erythrocyte enzyme [S. Kim (1975) Arch. Biochem. Biophys. 161, 652-657]. The enzyme also differed with the mammalian enzyme in protein substrate preference: the mammalian enzyme showed equal preference to histone and immunoglobulin G while the wheat germ enzyme transferred a methyl group 4.5 times more to histone than to immunoglobulin G. The Km values for histone and S-adenosyl-L-methionine were 0.2 mM and 5 microM. S-Adenosyl-L-homocysteine and its analogues, sinefungin and A9145C, were competitive inhibitors with Ki values 1.5 microM, 0.4 microM and 0.1 microM, respectively. To investigate the identity of endogenous substrate(s) for protein methylase II, the crude wheat germ extract was incubated with S-adenosyl-L-[methyl-3H]methionine and the methylated proteins were analyzed by acid/urea and sodium dodecyl sulfate/polyacrylamide gel electrophoresis. Among other methyl acceptors in the wheat germ, histone polypeptides were the major endogenous substrates.
D007700:Kinetics; D008970:Molecular Weight; D010944:Plants / Q000201:enzymology*; D011496:Protein Methyltransferases / Q000302:isolation & purification*; D011497:Protein O-Methyltransferase / Q000302:isolation & purification* / Q000378:metabolism; D013379:Substrate Specificity; D014908:Triticum / Q000201:enzymology
D011496:Protein Methyltransferases; D011497:Protein O-Methyltransferase