A study of the phosphorylated intermediate of sarcoplasmic reticulum atpase

Incorporation of ATP terminal phosphate into sarcoplasmic reticulum (SR) occurs very rapidly on ice. Ca²⁺ is a requirement and half-maximal activation is obtained in the presence of 1 × 10^-7, m Ca²⁺. The activating Ca²⁺ concentrations are identical for phosphate incorporation and ATPase, while excess Ca²⁺ only inhibits ATPase. The rate of enzymatic degradation of the ³²P-membrane complex, in the presence of Mg²⁺, is comparable to that of ATP hydrolysis. Mg⁺⁺ accelerates the P- membrane complex turnover. The pH stability of the phosphorylated species is different in membranes denatured at acid pH (trichloroacetic acid), as opposed to membranes denatured at neutral pH (Salyrgan, acetone) and then exposed to various pH values. ATPase activity and steady-state levels of ³²P-membrane complex are reduced when SR is incubated in the presence of hydroxylamine. However, SR incubated with hydroxylamine in optimal conditions for formation of P-membrane complex, regains full activity after washings. This lack of permanent inactivation suggests that hydroxylamine does not form a stable hydroxamate at the active site. Degradation of trichloroacetic acid-treated ³²P-membrane complex is accelerated by hydroxylamine. On the other hand, 0-(¹⁴C) methylhydroxylamine is bound by trichloroacetic acid-treated SR, independent of its previous incubation in the presence or in the absence of ATP. This indicates that the hydroxylamine effect may not be related to a specific reaction with an acylphosphate. Solubilization of ³²P-membrane complex with Triton X 100 at acid pH, and gel chromatography, yield phosphorylated particles with 60-80% of the original specific activity. On the other hand, particles solubilized with deoxycholate at neutral pH retain only traces of ³²P. A mechanism is proposed for SR ATPase, in which Ca²⁺ binding to the membrane (a) occurs as a consequence of site modification induced by ATP binding, and (b) activates phosphoryl transfer and formation of a phosphorylated intermediate.