Characterization of sarcoplasmic reticulum calcium(2+) ATPase by membrane reconstitution with preformed liposomes

Abstract

The Ca{dollar}\sp{lcub}2+{rcub}{dollar} transport ATPase of sarcoplasmic reticulum (SR) is an enzyme that catalyzes the transport of cytoplasmic Ca{dollar}\sp{lcub}2+{rcub}{dollar} into the SR lumen upon ATP hydrolysis. The most commonly used experimental system is the SR vesicles prepared from the muscle homogenate. The native SR vesicles have high protein content and electrolyte leakage. These properties have made it difficult to study some aspects of the enzyme mechanism, namely counter-ion transport, electrogenicity and the coupling ratio of Ca{dollar}\sp{lcub}2+{rcub}{dollar}/ATP. The SR Ca{dollar}\sp{lcub}2+{rcub}{dollar} ATPase was reconstituted in unilamellar liposomes prepared by reverse phase evaporation. The size of the resulting proteoliposomes was similar to that of native SR vesicles, but with much lower protein/lipid ratio. The proteoliposomes sustained ATP dependent Ca{dollar}\sp{lcub}2+{rcub}{dollar} uptake to an asymptotic level of 2-3 {dollar}\mu{dollar}mole/mg protein without Ca{dollar}\sp{lcub}2+{rcub}{dollar} precipitating agents. The low permeability of the proteoliposomal membrane permitted direct demonstration of Ca{dollar}\sp{lcub}2+{rcub}{dollar}/H{dollar}\sp+{dollar} countertransport and electrogenicity by parallel measurements in the same experimental system. Countertransport of 1 H{dollar}\sp+{dollar} per 1 Ca{dollar}\sp{lcub}2+{rcub}{dollar} was demonstrated. Consistent with the countertransport stoichiometry, net positive charge displacement produced by Ca{dollar}\sp{lcub}2+{rcub}{dollar} transport was observed. The steady-state electrical potential observed under optimal conditions was approximately 50 mV. The estimated charge transfer associated with Ca{dollar}\sp{lcub}2+{rcub}{dollar} and H{dollar}\sp+{dollar} countertransport were well accounted for by the initial rate and steady-state values of membrane potentials. The coupling ratio of Ca{dollar}\sp{lcub}2+{rcub}{dollar}/ATP was determined by parallel measurements of both Ca{dollar}\sp{lcub}2+{rcub}{dollar} uptake and the ATP hydrolysis. Under optimal condition a 2:1 ratio was observed at early stage of filling of proteoliposomes. However, when maximal loading was achieved, the significant rate of ATP hydrolysis still remained with negligible Ca{dollar}\sp{lcub}2+{rcub}{dollar} uptake. The effects of different anions on Ca{dollar}\sp{lcub}2+{rcub}{dollar} ATPase functions as well as membrane permeability were studied using the reconstitution system. The pH dependence of Ca{dollar}\sp{lcub}2+{rcub}{dollar}/H{dollar}\sp+{dollar} countertransport indicates the direct exchange of H{dollar}\sp+{dollar} for Ca{dollar}\sp{lcub}2+{rcub}{dollar} on ATPase acidic residues which are involved in cation translocation. The observation of futile cycle of ATP hydrolysis shows that as the concentration of lumenal Ca{dollar}\sp{lcub}2+{rcub}{dollar} rises above its dissociation constant, the phosphoenzyme still undergoes significant rates of hydrolytic cleavage even though retaining bound Ca{dollar}\sp{lcub}2+{rcub}{dollar}.