Subcellular distribution of ATP-dependent calcium transport in rat duodenal epithelium

Abstract

Subcellular fractionation studies were performed to delineate plasma membrane and intracellular membrane populations which might be involved in intracellular Ca 2+-homeostasis of rat small intestinal epithelial cells. After a low-speed supernatant fraction had been suspended in 5% sorbitol and subjected to equilibrium centrifugation in a zonal rotor, the Golgi and endoplasmic reticulum markers, galactosyltransferase and NADPH-cytochrome −c reductase, were concentrated in a density region designated Window II. The basal-lateral membrane marker ( Na +− K +)- ATPase was concentrated in a higher-density region designated Window III. ATP-dependent Ca 2+ transport was equally distributed between the two windows. Several membrane populations could be resolved from each window with good recovery of Ca 2+-transport activity by a second density gradient centrifugation step. Second density gradient fractions were subjected to counter current partitioning in an aqueous polymer two-phase system. Basal-lateral membranes, characterized by an 11-fold enrichment of ( Na +− K +)- ATPase , contained ATP-dependent Ca 2+-transport activity with V max = 3.7 nmol/ mg per min and K m = 0.5 μ M . A major Golgi-derived population exhibited Ca 2+-transport activity with V max and K m values similar to those of the basal-lateral membranes. One membrane population, presumed to have been derived from the endoplasmic reticulum, contained Ca 2+-transport activity with V max = 4 nmol/ mg per min and K m = 0.5 μ M . In addition to demonstrating that ATP-dependent Ca 2+-transport activity has a complex distribution within enterocytes, this study raises the possibility that the basolateral plasma membranes might account for a relatively minor portion of the cell's Ca 2+-pumping ability.