Abstract
The electrogenicity and some molecular properties of the sarcoplasmic reticulum Ca2+ pump protein were studied by measuring steady-state Ca2+ pump currents. Ca2(+)-ATPase protein was solubilized from rabbit skeletal muscle sarcoplasmic reticulum membrane preparations and purified by liquid chromatography. The purified Ca(+)-ATPase molecules were reconstituted into proteoliposomes and then incorporated by fusion into a planar bilayer lipid membrane. Short circuit currents across the planar membrane were detected when the ATPase molecules were activated by addition of ATP under optimal ionic conditions. Thus, the electrogenicity of the Ca2+ pump molecules was directly demonstrated. The amplitude of the pump current was dependent on the ATP concentration, and the relation was described by a Michaelis-Menten-type equation. The Michaelis constant was calculated to be 0.69 +/- 0.16 mM, which agrees well with the dissociation constant for a low affinity ATP-binding site deduced previously from the kinetics of ATP hydrolysis and from ATP binding.
Highlights
Measurement of Steady-state Ca2+ Pump Current Caused by Purified Ca2+-ATPase of Sarcoplasmic Reticulum Incorporated into a Planar Bilayer Lipid Membrane*
All the findings described above indicate that the membrane current was observed only in the presence of Ca2’-ATPase molecules activated by appropriate concentrations of ATP
It is noteworthy that this is the first measurement of Ca2+ pump activity obtained in a true steady-state, which was not realized in previous experiments using sarcoplasmic reticulum (SR) membrane vesicles or reconstituted vesicles
Summary
Measurement of Steady-state Ca2+ Pump Current Caused by Purified Ca2+-ATPase of Sarcoplasmic Reticulum Incorporated into a Planar Bilayer Lipid Membrane*. The electrogenicity and some molecular properties of the sarcoplasmic reticulum Ca2’ pump protein were studied by measuring steady-state Ca2+ pump currents. Ca’+-ATPase protein was solubilized from rabbit skeletal muscle sarcoplasmic reticulum membrane preparations and purified by liquid chromatography. The purified Ca2+-ATPase molecules were reconstituted into proteoliposomes and incorporated by fusion into a planar bilayer lipid membrane. Short circuit currents across the planar membrane were detected when the ATPase molecules were activated by addition of ATP under optimal ionic conditions. The electrogenicity of the Ca’+ pump molecules was directly demonstrated. The amplitude of the pump current was dependent on the ATP concentration, and the relation was described by a Michaelis-Menten-type equation. 0.16 mM, which agrees well with the dissociation constant for a low affinity ATP-binding site deduced previously from the kinetics of ATP hydrolysis and from
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