Abstract
A detailed functional characterization of reconstituted sarcoplasmic reticulum (SR) vesicles with similar lipid content as normal SR was obtained by studies of ATPase activity and calcium transport in transient state, steady state, and equilibrium conditions. For this purpose, enzyme phosphorylation with ATP, hydrolytic activity, calcium transport, phosphorylation with Pi, and ATP synthesis by reversal of the pump were measured, and utilized to demonstrate function and orientation of catalytic sites. The preparations used in these studies displayed the highest activity reported for reconstituted sarcoplasmic reticulum systems. The rates of phosphoenzyme formation from ATP and hydrolysis as well as steady state levels matched the values obtained with normal SR vesicles. Calcium transport and repeated cycles of ATP synthesis by reversal of the pump were also obtained. However, the efficiency of transport and ATP synthesis from a Ca2+ gradient was approximately three times lower than in native vesicles. This deficiency could not be attributed to passive calcium leak from the reconstituted vesicles but, in part, can be explained by the bidirectional alignment of the calcium pump in reconstituted SR. It is suggested that vectorial transport requires a more complex level of protein structure than that for sustaining simple ATPase activity. Time resolution of the phosphorylation reaction by rapid quench methods can be used to estimate the orientation of the calcium pump in the membrane. Such studies indicate that the calcium pump protein is largely bidirectionally oriented in reconstituted SR vesicles.
Highlights
Reversal of the pump were obtained. ,the efficiency of transport and ATP synthesis froamCa2+ gradient was approximately three times lower than native vesicles
Sarcoplasmic reticulum was isolated as described by Meissner et al (8).The method of reconstitution was essentially that of Meissner iacnonndceFnletriasctihoenr
Time resolutioonf the phosphorylation reaction byrapid quench methodscan be used to estimate the orientatioofnthe calcium pump in the membrane. Such studies indicate that the calcium pump protein is largely bidirectionally oriented in reconstituted SRvesicles
Summary
Reversal of the pump were obtained. ,the efficiency of transport and ATP synthesis froamCa2+ gradient was approximately three times lower than native vesicles. Rate constants for phosphoenzyme decay were determined following quenching of the phosphorylation reaction with ATPand again found to be identical in native and reconstituted vesicles (Fig. 3).
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