Abstract
The synaptosomal plasma membrane Ca2+-ATPase (PMCA) purified from pig brain was reconstituted with liposomes prepared by reverse phase evaporation at a lipid to protein ratio of 150/1 (w/w). ATP-dependent Ca2+ uptake and H+ ejection by the reconstituted proteoliposomes were demonstrated by following light absorption and fluorescence changes undergone by arsenazo III and 8-hydroxy-1,3, 6-pyrene trisulfonate, respectively. Ca2+ uptake was increased up to 2-3-fold by the H+ ionophore carbonyl cyanide p-trifluoromethoxyphenylhydrazone, consistent with relief of an inhibitory transmembrane pH gradient (i.e. lumenal alkalinization) generated by H+ countertransport. The stoichiometric ratio of Ca2+/H+ countertransport was 1.0/0.6, and the ATP/Ca2+ coupling stoichiometry was 1/1 at 25 degrees C. The electrogenic character of the Ca2+/H+ countertransport was demonstrated by measuring light absorption changes undergone by oxonol VI. It was shown that a 20 mV steady state potential (positive on the lumenal side) was formed as a consequence of net charge transfer associated with the 1/1 Ca2+/H+ countertransport. Calmodulin stimulated ATPase activity, Ca2+ uptake, and H+ ejection, demonstrating that these parameters are linked by the same mechanism of PMCA regulation.
Highlights
We purified the plasma membrane Ca2؉-ATPase (PMCA) from pig synaptosomes by calmodulin column chromatography [5, 6] and obtained proteoliposomal reconstitution by the method originally developed by Rigaud and coworkers (12, 14 –18)
The pig brain PMCA was purified from solubilized synaptic vesicles by calmodulin affinity chromatography
We found that the purified enzyme had an ATPase activity of 1.10 Ϯ 0.10 IU in the presence of pure Egg phosphatidylcholine (EPC), as compared with 0.063 Ϯ 0.011 IU in the solubilized synaptosomal vesicles
Summary
We purified the PMCA from pig synaptosomes by calmodulin column chromatography [5, 6] and obtained proteoliposomal reconstitution by the method originally developed by Rigaud and coworkers (12, 14 –18). The main advantages of this procedure were: (i) optimal protein incorporation and orientation,. (ii) very low permeability of the proteoliposomal membrane to electrolytes, and (iii) large intravesicular volume which delays back inhibition by high concentrations of lumenal Ca2ϩ [8]. We studied the functional properties of the reconstituted synaptosomal PMCA, measuring ATP-dependent Ca2ϩ-uptake activity, Hϩ ejection, and formations of transmembrane electrical potential
Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have