Stoichiometric methylation of porcine adrenocorticotropin by protein carboxyl methyltransferase requires deamidation of asparagine 25. Evidence for methylation at the alpha-carboxyl group of atypical L-isoaspartyl residues.

Protein carboxyl methyltransferase facilitates conversion of atypical L-isoaspartyl peptides to normal L-aspartyl peptides.

Purification and molecular identification of two protein methylases I from calf brain. Myelin basic protein- and histone-specific enzyme.

Protein carboxymethylase (EC 2.1.1.24) from cytosol of bovine brain was found to exist as two apparent isozymes that could be separated by chromatography on DEAE-cellulose at pH 8.0. Rechromatography of the two forms, designated PCM I and PCM II, indicated that they are not interconvertible. Both enzymes have a molecular weight of 24,300 by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, PCM I consists mainly of one isoelectric form, pI 6.5, whereas PCM II resolves into two forms of pI 5.6 and 5.7. The relative amounts of PCM I and PCM II show a marked tissue dependence. Brain has approximately twice as much PCM I as II, whereas liver contains only the type II enzyme. The two enzymes were found to have similar substrate specificities when tested with five different methyl-accepting proteins. Synapsin I, a basic protein associated with synaptic vesicles, was found to be an excellent methyl-accepting protein with regard to its Km (1.2 microM), but it exhibited a low stoichiometry of methyl incorporation.

Purification and characterization of two distinct isozymes of protein carboxymethylase from bovine brain.

We studied the regulation of 1-aminocyclopropane-1-carboxylate (ACC) synthase activity in tomato ( Lycopersicon esculentum Mill.) fruit tissue and attempted the purification of this enzyme. The increase of ACC synthase activity in wounded tomato pericarp was inhibited by cordycepin and cycloheximide. Density labeling studies showed a 0.75% increase in the buoyant density of ACC synthase isolated from tomato pericarp tissue that had been incubated on 2 H 2 O as compared to ACC synthase from H 2 O-treated tissue. These data are consistent with the hypothesis that ACC synthase is synthesized de novo following wounding of tomato pericarp tissue. SDS-gel electrophoresis and fluorography showed that the pattern of incorporation of l-[ 35 S]methionine into protein changed with time after wounding of the tissue. Radioactive protein bands that were not detected 1 hour after wounding, became apparent 2 to 3 hours after wounding. Gel filtration on Sephadex G-100 gave a molecular weight estimate for ACC synthase of 57,000 ± 1,500 daltons. Hydrophobic interaction chromatography on phenyl-Sepharose yielded a 60- to 70-fold purification of the enzyme. SDS-gel electrophoresis of this preparation indicated the presence of one intense band at 57,000 daltons and several less intense bands. Affinity chromatography was of limited usefulness in the purification of ACC synthase since the enzyme could not be eluted specifically from any of the affinity gels tried. Purification methods that involved pH changes led to a rapid loss of ACC synthase activity. ACC synthase was estimated to comprise less than 1% of the total protein in tomato pericarp tissue.

Walter Chin

Papers

A rapid method for the purification of S-adenosylmethionine: Protein-carboxyl O-methyltransferase by affinity chromatography