Characterization of Ca 2+ uptake in plasma membrane vesicles isolated from guinea pig ileum smooth muscle

Abstract

We have characterized ATP-dependent Ca 2+ transport into highly purified plasma membrane fraction isolated from guinea pig ileum smooth muscle. The membrane fraction contained inside-out sealed vesicles and was enriched 30–40-fold in 5′-nucleotidase and phosphodiesterase I activity as compared to post nuclear supernatant. Plasma membrane vesicles showed high rate (76 nmol/mg/min) and high capacity for ATP dependent Ca 2+ transport which was inhibited by addition of Ca 2+ ionophore A23187. The inhibitors of mitochondrial Ca 2+ transport, i.e., sodium azide, oligomycin and ruthenium red did not inhibit ATP-dependent Ca 2+ uptake into plasma membrane vesicles. The energy dependent Ca 2+ uptake into plasma membranes showed very high specificity for ATP as energy source and other nucleotide triphosphates were ineffective in supporting Ca 2+ transport. Phosphate was significantly better as Ca 2+ trapping anion to potentiate ATP-dependent Ca 2+ uptake into plasma membrane fraction as compared to oxalate. Orthovanadate, an inhibitor of cell membrane (Ca 2+-Mg 2+)-ATPase activity, completely inhibited ATP-dependent Ca 2+ transport and the Ki was approximately 0.6 μM. ATP-dependent Ca 2+ transport and formation of alkali labile phosphorylated intermediate of (Ca 2+-Mg 2+)-ATPase increased with increasing concentrations of free Ca 2+ in the incubation mixture and the Km value for Ca 2+ was approximately 0.6 – 0.7 μM for both the reactions.