Isolation of a plasma-membrane fraction from gastric smooth muscle. Comparison of the calcium uptake with that in endoplasmic reticulum

Abstract

A plasma membrane fraction was isolated from the smooth muscle of the pig stomach by using differential and sucrose-density-gradient centrifugations. When the centrifugation was carried out after preloading the crude microsomal fraction with Ca2+ in the presence of oxalate, the contamination of the plasma membrane fraction by endoplasmic reticulum was decreased and a fraction enriched in endoplasmic reticulum vesicles filled with calcium oxalate crystals was obtained. The plasmalemmal and endoplasmic reticulum membranes could be distinguished by differences in the activity of marker enzymes and in the cholesterol content and by their different permeability to oxalate and phosphate. Oxalate and phosphate stimulated the Ca2+ uptake in the endoplasmic reticulum much more than in the plasmalemmal vesicles. In the plasma membrane vesicles 40 mM-phosphate was more effective for stimulating the Ca2+ uptake than was 5 mM oxalate, but there reverse was seen in the endoplasmic reticulum. The high cholesterol/phospholipid ratio of the crude microsomal fraction suggests that the majority of the vesicles present in the crude microsomal fraction are of plasmalemmal origin. The Ca2+ pump of the plasmalemmal and endoplasmic reticulum vesicles could be differentiated by their different sensitivities to calmodulin. However, the 2 Ca2+-transport ATPases did not differ by their sensitivity to vanadate nor by the energization of the Ca2+ transport by different nucleoside triphosphates.