Protein phosphorylation and dephosphorylation in liver plasma membrane. Effect of inorganic phosphate.
When a plasma membrane preparation isolated from rat liver was incubated with [gamma-32P]ATP and Mg2+, protein-bound 32P increased rapidly, followed by a gradual decrease. The time course suggested the existence of membrane-bound kinase(s) and phosphatase(s) phosphorylating and dephosphorylating endogenous proteins. The extent of phosphorylation was not affected by inclusion of cyclic AMP in the reaction mixture. The extent of the maximum phosphorylation was dependent on membrane concentration, owing to rapid hydrolysis of ATP by the membrane-bound ATPase activity. Thus, phosphorylation proceeded further on repeated addition of ATP. Both phosphorylation and dephosphorylation were stimulated by Mg2+, an effective rate of phosphorylation being obtained at 15 mM. Pi up to 20 mM stimulated phosphorylation with little effect on the rate of dephosphorylation. At higher phosphate concentrations, the maximum 32P-incorporation decreased again, and at 100 mM, dephosphorylation was prevented significantly. Autoradiography after polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate and urea revealed six main phosphorylated bands, two of which (Band 3 and 5) were partly extractable with 1 M NaCl. In the presence of 100 mM Pi, very strong phosphorylation of Band 5 (about 23,000 daltons) was noted, and a new strongly labeled band (Band P, about 20,000 daltons) was observed. It was concluded that the phosphoproteins in the membrane may be turned over at different rates and high concentrations of Pi may affect the turnover rate of some phosphoproteins, probably through interference with the phosphatase.
D000255:Adenosine Triphosphate / Q000378:metabolism; D000818:Animals; D002462:Cell Membrane / Q000201:enzymology; D004591:Electrophoresis, Polyacrylamide Gel; D066298:In Vitro Techniques; D008099:Liver / Q000201:enzymology*; D008297:Male; D008565:Membrane Proteins / Q000378:metabolism*; D010710:Phosphates / Q000494:pharmacology*; D010749:Phosphoprotein Phosphatases / Q000378:metabolism; D010750:Phosphoproteins / Q000378:metabolism*; D010766:Phosphorylation; D011494:Protein Kinases / Q000378:metabolism; D051381:Rats; D000254:Sodium-Potassium-Exchanging ATPase / Q000378:metabolism
D008565:Membrane Proteins; D010710:Phosphates; D010750:Phosphoproteins; D000255:Adenosine Triphosphate; D011494:Protein Kinases; D010749:Phosphoprotein Phosphatases; D000254:Sodium-Potassium-Exchanging ATPase