Protein carboxyl methylation in kidney brush-border membranes.
Protein carboxyl methylation activity was detected in the cytosol and in purified brush-border membranes (BBM) from the kidney cortex. The protein carboxyl methyltransferase (PCMT) activity associated with the BBM was specific for endogenous membrane-bound protein substrates, while the cytosolic PCMT methylated exogenous substrates (ovalbumin and gelatin) as well as endogenous proteins. The apparent Km for S-adenosyl-L-methionine with endogenous proteins as substrates were 30 microM and 4 microM for the cytosolic and BBM enzymes, respectively. These activities were sensitive to S-adenosyl-L-homocysteine, a well known competitor of methyltransferase-catalyzed reactions, but were not affected by the presence of chymostatin and E-64, two protein methylesterase inhibitors. The activity of both cytosolic and BBM PCMT was maximal at pH 7.5, while BBM-phospholipid methylation was predominant at pH 10.0. Separation of the = methylated proteins by acidic gel electrophoresis in the presence of the cationic detergent benzyldimethyl-n-hexadecylammonium chloride revealed distinct methyl accepting proteins in the cytosol (14, 17, 21, 27, 31, 48, 61 and 168 kDa) and in the BBM (14, 60, 66, 82, and 105 kDa). Most of the labelling was lost following electrophoresis under moderately alkaline conditions, except for a 21 kDa protein in the cytosol and a 23 kDa protein in the BBM fraction. These results suggest the existence of two distinct PCMT in the kidney cortex: a cytosolic enzyme with low selectivity and affinity, methylating endogenous and exogenous protein substrates, and a high-affinity BBM-associated methylating activity.
Gingras
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Département de chimie-biochimie, Université du Québec à Montŕeal, Canada.
Ménard
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Béliveau
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D000818:Animals; D002462:Cell Membrane / Q000378:metabolism; D003600:Cytosol / Q000378:metabolism; D004591:Electrophoresis, Polyacrylamide Gel; D005780:Gelatin / Q000378:metabolism; D006863:Hydrogen-Ion Concentration; D007672:Kidney Cortex / Q000201:enzymology / Q000378:metabolism*; D007700:Kinetics; D007930:Leucine / Q000031:analogs & derivatives / Q000494:pharmacology; D008297:Male; D008745:Methylation; D008871:Microvilli / Q000201:enzymology / Q000378:metabolism*; D009842:Oligopeptides / Q000494:pharmacology; D010047:Ovalbumin / Q000378:metabolism; D011497:Protein O-Methyltransferase / Q000037:antagonists & inhibitors / Q000378:metabolism*; D051381:Rats; D011919:Rats, Inbred Strains; D012436:S-Adenosylmethionine / Q000378:metabolism
D009842:Oligopeptides; D012436:S-Adenosylmethionine; D005780:Gelatin; D010047:Ovalbumin; C002101:chymostatin; D011497:Protein O-Methyltransferase; D007930:Leucine; C024974:E 64