Phosphorylation from Inorganic Phosphate and ATP Synthesis of Sarcoplasmic Membranes

Abstract

The incorporation of Pi in the fragmented rabbit skeletal muscle sarcoplasmic membranes induced by the removal of Ca2+ bound to high affinity binding sites at the cytoplasmic surface of the membranes gives rise to the formation of 2 species of phosphoenzyme. The properties of the phosphorproteins formed depend on the absence or the presence of a gradient of Ca2+ across the membranes. The phosphoenzymes differ by the affinity of the protein for phosphate, the enthalpy of formation, the kinetics of phosphate incorporation and by the sensitivity to ionophores and ADP. In the absence of a Ca gradient less than 0.5 nmol phosphoenzyme/mg protein are formed in media containing < 5 mM phosphate at pH 7 and 10.degree. C. Under the same conditions .apprxeq. 2 nmol of phosphoenzyme/mg protein are formed with an initial rate of 0.5 nmol mg-1 .cntdot. s-1 when a Ca gradient exists. When the gradient is abolished by the addition of the ionophore X537A [lasalocid], the level of phosphoprotein drops to the same value as observed in the absence of a gradient. On addition of ADP at concentrations increasing from 0.3 to 10 .mu.M continuous ATP formation is activated to its maximum rate, and simultaneously, the level of phosphoprotein declines. These concentrations of ADP scarcely affect phosphorprotein formed in the absence of a gradient, the phosphoryl residue of which is displaced when the concentration of ADP exceeds 10 .mu.M without the formation of an equivalent amount of ATP. Minimum mechanisms for the formation of gradient-independent and gradient-dependent phosphoprotein are discussed.