Solubilization and characterization of phosphoprotein phosphatase(s) from bovine corpus-luteum plasma membranes.
Plasma membrane fractions I and II isolated from bovine corpus luteum contain phosphoprotein phosphatases. Enzyme activities associated with both membrane fractions showed pH optima in the neutral range and were most active with phosphoprotamine as the exogenous substrate. The enzyme activity was partially inhibited by Co2+, Zn2+ and Fe2+. Dithioerythritol, glutathione (reduced) and 2-mercaptoethanol stimulated the enzyme activity, whereas N-ethylmaleimide and N-phenylmaleimide were inhibitory. Similarly, various cyclic nucleotides and nuclsoside triphosphates also inhibited phosphoprotein phosphatase activities. The phosphatase activity was also observed with endogenous phosphorylated membrane proteins as substrate. The endogenous phosphorylation of membranes was rapid and attained a maximal level after 15--20 min of incubation. Initially endogenous dephosphorylation was also very rapid, but did not reach completion. In addition to phosphoprotein phosphatase, membrane preparations also possessed very active cyclic-AMP-dependent protein kinase activity. Phosphoprotein phosphatase activity from plasma membranes was solubilized by ionic and nonionic detergents. Optimal solubilization was achieved with 0.1% sodium deoxycholate. Sucrose density gradient centrifugation of deoxycholate-solubilized fraction I and fraction II membranes resolved phosphoprotein phosphatase activity into two species with apparent sedimentation coefficients of 6.7 S (Mr 130000) and 4.8 S (Mr 90000). Cyclic-AMPstimulated protein kinase activity sedimented as a broad peak with a sedimentation coefficient of 5.5 S (Mr 110000).
D000818:Animals; D002417:Cattle; D002462:Cell Membrane / Q000201:enzymology; D003338:Corpus Luteum / Q000201:enzymology*; D003902:Detergents; D005260:Female; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D010750:Phosphoproteins; D010744:Phosphoric Monoester Hydrolases / Q000302:isolation & purification / Q000378:metabolism*; D011494:Protein Kinases / Q000378:metabolism; D012995:Solubility
D003902:Detergents; D010750:Phosphoproteins; D011494:Protein Kinases; D010744:Phosphoric Monoester Hydrolases