Characterization of endogenous protein phosphorylation in isolated rat liver lysosomes.
Membranes prepared from highly purified rat liver lysosomes contain endogenous protein-phosphorylation activities. The transfer of phosphate to membrane fractions from [gamma-32P]ATP was analyzed by gel electrophoresis under acidic denaturing conditions. Two phosphopeptides were detected, with molecular weights of 3,000 and 14,000. Phosphorylation of these proteins was unaffected by the addition of cAMP, cGMP, or the heat-stable inhibitor of cAMP-dependent protein kinase. No additional phosphorylation was observed when cAMP-dependent protein kinase was included in the reaction or when exogenous protein kinase substrates were added. The 14,000-dalton 32P-labeled product was formed rapidly in the presence of low concentrations (250 microM) of either Ca2+ or Mg2+. This product was labile under both acidic and alkaline conditions, suggesting that this protein contains an acyl phosphate, present presumably as a catalytic intermediate in a phosphotransferase reaction. The lower molecular weight species required a high concentration (5 mM) of Mg2+ for phosphorylation, and micromolar concentrations of Ca2+ stimulated the Mg2+-dependent activity. The addition of Ca2+ and calmodulin stimulated the phosphorylation reaction to a greater extent than with Ca2+ alone. This activity was strongly inhibited by 0.2 mM LaCl3 and to a lesser extent by 50 microM chlorpromazine or trifluoperazine. These results suggest that the 3000-dalton peptide may be phosphorylated by a Ca2+, calmodulin-dependent kinase associated with the lysosomal membrane.
D000818:Animals; D000242:Cyclic AMP / Q000494:pharmacology; D006152:Cyclic GMP / Q000494:pharmacology; D007425:Intracellular Membranes / Q000378:metabolism; D007700:Kinetics; D008099:Liver / Q000201:enzymology*; D008247:Lysosomes / Q000201:enzymology*; D008565:Membrane Proteins / Q000378:metabolism; D008862:Microsomes, Liver / Q000201:enzymology; D010766:Phosphorylation; D011494:Protein Kinases / Q000378:metabolism*; D051381:Rats
D008565:Membrane Proteins; D000242:Cyclic AMP; D011494:Protein Kinases; D006152:Cyclic GMP