THE MECHANISM OF INHIBITION OF THE CA2-ATPASE OF SKELETAL-MUSCLE SARCOPLASMIC-RETICULUM BY THE CROSS-LINKER O-PHTHALALDEHYDE()

Labelling the Ca2+-ATPase of skeletal-muscle sarcoplasmic reticulum wi th o-phthalaldehyde (OPA) results in loss of ATPase activity at a 1:1 molar ratio of label to ATPase. The affinity of the ATPase for Ca2+ is unaffected, as is the E1/E2 equilibrium constant. The rate of dissoci ation of Ca2+ from the Ca2+-bound ATPase is also unaffected and Mg2+ i ncreases the rate of dissociation, as for the unlabelled ATPase. Effec ts of Mg2+ on the fluorescence intensity of the ATPase labelled with 4 -(bromomethyl)-6,7-dimethoxycoumarin are also unaffected by labelling with OPA, consistent with the fluorescence change reporting on Mg2+ bi nding at the gating site on the ATPase. The affinity of the ATPase for ATP is reduced by labelling, as is the rate of phosphorylation. The r ate of phosphorylation is independent of the concentration of ATP abov e 25 mu M ATP, so that the slow step is the first-order rate constant for phosphorylation by bound ATP. The rate of the back reaction betwee n phosphorylated ATPase and ADP is little affected, suggesting that th e slow step in phosphorylation could be the slow conformation step bef ore phosphoryl transfer. The rate of dephosphorylation of the phosphor ylated ATPase is also decreased, suggesting that a similar conformatio n change could be involved in the dephosphorylation step. The rate of the Ca2+ transport step appears to be unaffected by labelling. The net result of these changes is that the labelled ATPase is present predom inantly in a Ca2+-free, phosphorylated form at steady state in the pre sence of ATP.