DIRECT DEMONSTRATION OF AN ACID-LABILE PHOSPHOENZYME IN THE CYCLE OF THE SARCOPLASMIC-RETICULUM CA-2+-DEPENDENT ADENOSINE-TRIPHOSPHATASE

The Ca+-dependent adenosinetriphosphatase (Ca2+-ATPase) from the sarcoplasmic reticulum (SR) of rat skeletal muscles is phosphorylated by inorganic phosphate (Pi) in the absence of Ca2+. The reaction can be described by the following simplified scheme: {A figure is presented} where E-P is a covalent, acid-stable and ADP-insensitive phosphoenzyme, and E · Pi is a noncovalent and acid-labile complex. The reaction is Mg2+-dependent. Membrane fragments deposited on Millipore filters were successively perfused with two solutions, at constant flow. The effluent samples were analyzed. The perfused solutions were Ca2+ free and always contained 40% dimethylsulfoxide (DMSO), plus other reactants. Following the successive perfusion of solutions without and with [32P]Pi, 32P binding is only detected in the presence of Mg2+, indicating the formation of the phosphoenzymes (E · Pi and E-P). Following perfusions of the phosphoenzymes with 5% trichloroacetic acid, 32P release indicates the amount of the acid-labile moiety (E · Pi). After phosphorylations, the filters were washed with acid and unlabeled Pi, and the remaining radioactivity was measured to evaluate the acid-stable phosphoenzyme (E-P). The acid-labile and acid-stable phosphoenzymes amounted, respectively, 0.72 ± 0.12, and 1.48 ± 0.10 nmol of Pi/mg of protein (±S.E., n = 5), after phosphorylations with 20 μM Pi. The results indicate: (1) The method allowed the evaluation of the acid-labile intermediate of the SR Ca2+-ATPase cycle. Keq = k2 k-2 in the above scheme, approaches 2.0. (2) The substrate of the phosphorylation reaction, in the presence of DMSO, is likely to be the Mg · Pi complex, since Mg2+ is necessary for step 1 in the above scheme. © 1990.