Purification and Characterization of Two Distinct Isozymes of Protein Carboxymethylase from Bovine Brain

Abstract

Protein carboxymethylase (EC 2.1.1.24) from cytosol of bovine brain existed as 2 apparent isozymes that could be separated by chromatography on DEAE-cellulose at pH 8.0. Rechromatography of the 2 forms, designated PCM I and PCM II, indicated that they are not interconvertible. Both enzymes have a MW of 24,300 by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. PCM I consists mainly of 1 isoelectric form, pI 6.5, whereas PCM II resolves into 2 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, liver contains only the type II enzyme. The 2 enzymes were found to have similar substrate specificities when tested with 5 different methyl-accepting proteins. Synapsin I, a basic protein associated with synaptic vesicles, was an excellent methyl-accepting protein with regard to its Km (1.2 .mu.M), but it exhibited a low stoichiometry of methyl incorporation.