Endogenous phosphorylation and dephosphorylation of rat liver plasma membrane proteins, suggesting a 18 kDa phosphoprotein as a potential substrate for alkaline phosphatase.
Purified rat liver plasma membranes were incubated for 0-60 min with [gamma-32P]ATP and analysis of 32P-labeled proteins by means of sodium dodecyl sulfate-polyacrylamide gel electrophoresis and autoradiography revealed the presence of two shifted kinetic phenomena. The use of 1-(5-isoquinolinylsulfonyl)-2-methylpiperazine (H7), a potent inhibitor of protein kinases, allowed the identification of one as the endogenous protein phosphorylation. The other was shown to be the labeling of two phospho-intermediate forms of alkaline phosphatase (orthophosphoric monoester phosphohydrolase (alkaline optimum, EC 3.1.3.1.], which have apparent molecular masses of 151 and 135 kDa. Bromolevamisole, a potent inhibitor of the enzyme, stabilized these phospho-intermediates, and consequent on this inhibition the labelling of a 18 kDa phosphoprotein was augmented. So, when alkaline phosphatase was studied in its native plasma membrane environment, a specificity of this enzyme over the endogenous phosphoproteins was established.
Sarrouilhe
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D
Groupe de Recherche et d'Etude d'Analogues de Médicaments, Faculté de Médecine et de Pharmacie, Poitiers, France.
Lalégerie
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P
Baudry
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M
D019307:1-(5-Isoquinolinesulfonyl)-2-Methylpiperazine; D000469:Alkaline Phosphatase / Q000037:antagonists & inhibitors / Q000378:metabolism*; D000818:Animals; D001345:Autoradiography; D002462:Cell Membrane / Q000201:enzymology; D004591:Electrophoresis, Polyacrylamide Gel; D007546:Isoquinolines / Q000494:pharmacology; D007978:Levamisole / Q000494:pharmacology; D008099:Liver / Q000166:cytology / Q000201:enzymology / Q000378:metabolism*; D008297:Male; D008565:Membrane Proteins / Q000378:metabolism*; D010750:Phosphoproteins / Q000378:metabolism*; D010766:Phosphorylation; D010879:Piperazines / Q000494:pharmacology; D051381:Rats; D011919:Rats, Inbred Strains; D013379:Substrate Specificity
D007546:Isoquinolines; D008565:Membrane Proteins; D010750:Phosphoproteins; D010879:Piperazines; D007978:Levamisole; D019307:1-(5-Isoquinolinesulfonyl)-2-Methylpiperazine; D000469:Alkaline Phosphatase