Activation and binding volumes of the sarcoplasmic reticulum transport enzyme activated by calcium or strontium

Abstract

The effect of pressure on the hydrolysis of dinitrophenyl phosphate (DnpP) and p-nitrophenyl phosphate (NpP) by the sarcoplasmatic reticulum transport enzyme in permeabilized and native closed vesicles activated by calcium or strontium, respectively, in aqueous and Me2SO-containing media has been studied. At atmospheric pressure, the enzyme in permeabilized vesicles, saturated with respect to substrates and activating ions, hydrolyzes DnpP ten times faster than NpP; for both substrates, calcium activation exceeds that by strontium only a little (20%). In aqueous media the enzyme displays, under all activating conditions, an almost identical curvilinear relationship between the logarithm of enzyme activity and pressure. The data were analysed on the basis of a simplified reaction scheme, in which two unidirectionally proceeding substrate-driven pressure-dependent reactions (k2, k4) cyclically transfer high-affinity into low-affinity binding sites which are assumed to be in equilibrium with either calcium or strontium. The fitting procedure yielded two sets of positive activation volumes delta V2* = 90-110 ml/mol and delta V4* = 15-25 ml/mol. Substrate specificity, as well as the effect of temperature, are exclusively localized in the pressure-independent rate constants k'2 and k'4. Considerable different pressure/activity relations characterized by a single activation volume of 20 ml/mol were obtained for the strongly suppressed substrate hydrolysis of native closed vesicles. At atmospheric pressure DnpP hydrolysis of open vesicles is inhibited by Me2SO, while NpP hydrolysis is considerably activated, irrespective of its activation by calcium or strontium. In the presence of 22.5% Me2SO, the activation volumes are reduced by 50-70 ml/mol. The rate constants of DnpP and NpP hydrolysis are either augmented or reduced by rising Me2SO concentrations, depending on the corresponding supporting substrate. Me2SO has only a slight effect on the pressure dependence of substrate hydrolysis by native vesicles. The small activation volume observed for the activity of native vesicles could be assigned on account of the simplified reaction scheme of the slow reaction step k4, by which the enzyme is transferred from its low-affinity into its high-affinity binding state. Volume changes connected with the binding of calcium or strontium to the luminal binding site of the enzyme were deduced from the observed activation volume and the computed volume change of the slow reaction step (delta V4*).