Subcellular fractionation of pig stomach smooth muscle. A study of the distribution of the (Ca2+ + Mg2+)-ATPase activity in plasmalemma and endoplasmic reticulum.
Isolated membrane vesicles from pig stomach smooth muscle (antral part) were subfractionated by a density gradient procedure modified in order to obtain an efficient extraction of extrinsic proteins. By using this method in combination with digitonin-treatment, an endoplasmic reticulum fraction contaminated with maximally 10 to 20% of plasma membranes was isolated, together with a plasma membrane fraction containing at most 30% endoplasmic reticulum. The endoplasmic reticulum and plasma membrane fractions differed in protein composition, reaction to digitonin, binding of wheat germ agglutinin, activities of marker enzymes and in the characteristics of the Ca2+ uptake. The Ca2+ uptake by the endoplasmic reticulum was much more stimulated by oxalate than the uptake by plasma membranes. Both fractions showed a (Ca2+ + Mg2+)-ATPase activity, but the largest amount of this enzyme was present in the plasma membranes. The study of the phosphorylated intermediates of the (Ca2+ + Mg2+)-ATPase by polyacrylamide gel electrophoresis revealed two phosphoproteins one of 130 kDa and one of 100 kDa (Wuytack, F., Raeymaekers, L., De Schutter, G. and Casteels, R. (1982) Biochim. Biophys. Acta 693, 45-52). The 130 kDa enzyme was predominant in the fraction enriched in plasma membrane whereas the distribution of the 100 kDa polypeptide correlated with the endoplasmic reticulum markers. The 130 kDa ATPase was the main 125I-calmodulin binding protein detected on nitrocellulose blots of proteins separated by gel electrophoresis. The (Ca2+ + Mg2+)-ATPase activity of the plasma membranes was higher than the (Na+ + K+)-ATPase activity, suggesting that the Ca2+ extrusion from these cells depends much more on the activity of the (Ca2+ + Mg2+)-ATPase than on Na+-Ca2+ exchange.
Raeymaekers
L
L
Wuytack
F
F
Casteels
R
R
D000818:Animals; D017301:Ca(2+) Mg(2+)-ATPase; D002118:Calcium / Q000378:metabolism; D000252:Calcium-Transporting ATPases / Q000378:metabolism*; D002458:Cell Fractionation; D002462:Cell Membrane / Q000201:enzymology* / Q000648:ultrastructure; D004721:Endoplasmic Reticulum / Q000201:enzymology* / Q000648:ultrastructure; D007700:Kinetics; D008854:Microscopy, Electron; D009130:Muscle, Smooth / Q000201:enzymology* / Q000648:ultrastructure; D011706:Pyloric Antrum / Q000201:enzymology*; D013552:Swine
D017301:Ca(2+) Mg(2+)-ATPase; D000252:Calcium-Transporting ATPases; D002118:Calcium