Purification and characterization of a membrane-bound protein carboxyl methyltransferase from rat kidney cortex.
Class II protein carboxyl methyltransferases (EC 2.1.1.77) are known to exist predominantly in a soluble form in all cells studied so far. These enzymes have been purified to homogeneity from the cytosols of many mammalian tissues but not from membranes. We describe here the purification to apparent homogeneity of a membrane-associated protein carboxyl methyltransferase from the brush border membrane of rat kidney. The enzyme was purified by fast protein liquid chromatography on Superdex 75 and Mono-Q and by preparative sodium dodecyl sulfate-polyacrylamide gel electrophoresis and consists of a single 27,300 polypeptide. The purified enzyme recognizes exogenous substrate proteins such as ovalbumin and gamma-globulins as well as synthetic peptides containing a L-isoaspartyl residue but not a synthetic peptide containing a farnesylated C-terminal cysteine (S-farnesyl-LARYKC). The Km for S-adenosyl-L-methionine with ovalbumin as the substrate is 1.5 microM and the purified enzyme is sensitive to inhibition by S-adenosyl-L-homocysteine (Ki = 0.3 microM). Peptide map obtained after Staphylococcus aureus V8 protease digestion of brush border membrane protein carboxyl methyltransferase showed a fragmentation pattern that was identical to that obtained for a soluble protein carboxyl methyltransferase purified according to the same procedure, indicating a high degree of homology. These results support the notion that class II protein carboxyl methyltransferases are not restricted to a cytosolic localization and show that the membrane-bound form of this enzyme shares many characteristics with known cytosolic protein carboxyl methyltransferases.
Boivin
D
D
Laboratoire de Membranologie, Université du Québec à Montréal, Canada.
Gingras
D
D
Béliveau
R
R
D000595:Amino Acid Sequence; D000818:Animals; D002462:Cell Membrane / Q000201:enzymology; D006863:Hydrogen-Ion Concentration; D007672:Kidney Cortex / Q000201:enzymology*; D008297:Male; D008745:Methylation; D008969:Molecular Sequence Data; D010449:Peptide Mapping; D010455:Peptides / Q000737:chemistry / Q000378:metabolism; D011497:Protein O-Methyltransferase / Q000737:chemistry / Q000302:isolation & purification* / Q000378:metabolism; D051381:Rats; D017207:Rats, Sprague-Dawley; D012995:Solubility; D013379:Substrate Specificity; D013696:Temperature
D010455:Peptides; D011497:Protein O-Methyltransferase