Differentiation of Ca2+ pumps linked to plasma membrane and endoplasmic reticulum in the microsomal fraction from intestinal smooth muscle.
ATP promotes 45Ca uptake by the microsomal fraction from the longitudinal smooth muscle of guinea-pig ileum and this uptake is stimulated by oxalate. As the microsomal fraction is made up of various subcellular entities, we examined the localization of the Ca2+-transport activity by density gradient centrifugation, taking advantage of the selective effect of digitonin (at low concentration) on the density of plasmalemmal elements. When the 45Ca-uptake activity was measured in the absence of oxalate, its behavior in subfractionation experiments closely paralleled that of the plasmalemmal marker 5'-nucleotidase. In contrast, the additional Ca2+-transport activity elicited by oxalate behaved like NADH-cytochrome c reductase, a putative endoplasmic reticulum marker. The endoplasmic reticulum vesicles constituted only a small part of the membranes in the microsomal fraction, which explains that their Ca2+-storage capacity was not detectable in the absence of Ca2+-trapping agent. Low digitonin concentrations selectively increased the Ca2+ permeability of the plasmalemmal vesicles. The two Ca2+-transport activities were further differentiated by their distinct sensitivity of K+, vanadate and calmodulin. In this respect, the oxalate-insensitive and oxalate-stimulated Ca2+-transport systems resembled, respectively, the sarcolemmal and sarcoplasmic reticulum Ca2+ pumps in cardiac and skeletal muscle, in accordance with the subcellular locations established by density gradient centrifugation.
D000818:Animals; D001693:Biological Transport, Active; D002118:Calcium / Q000378:metabolism*; D000252:Calcium-Transporting ATPases / Q000378:metabolism; D002147:Calmodulin / Q000494:pharmacology; D002462:Cell Membrane / Q000378:metabolism*; D004721:Endoplasmic Reticulum / Q000378:metabolism*; D006168:Guinea Pigs; D007082:Ileum / Q000378:metabolism; D007700:Kinetics; D008861:Microsomes / Q000378:metabolism*; D009130:Muscle, Smooth / Q000378:metabolism*; D010070:Oxalates / Q000494:pharmacology; D019815:Oxalic Acid; D011188:Potassium / Q000494:pharmacology; D014638:Vanadates; D014639:Vanadium / Q000494:pharmacology
D002147:Calmodulin; D010070:Oxalates; D014639:Vanadium; D014638:Vanadates; D019815:Oxalic Acid; D000252:Calcium-Transporting ATPases; D011188:Potassium; D002118:Calcium