Protein Phosphorylation and Dephosphorylation in Liver Plasma Membrane. Effect of Inorganic Phosphate

Abstract

When a plasma membrane preparation isolated from rat liver was incubated with [γ- ³² P]ATP and Mg ²⁺ , protein-bound ³² P 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 Mg ²⁺ , an effective rate of phosphorylation being obtained at 15 mM. P ₁ up to 20 mM stimulated phosphorylation with little effect on the rate of dephosphorylation. At higher phosphate concentrations, the maximum ³² P-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 P ₁ , 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 P ₁ may affect the turnover rate of some phosphoproteins, probably through interference with the phosphatase.