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ATP-dependent calcium transport and its correlation with Ca2+-ATPase activity in basolateral plasma membranes of rat duodenum
Authors:W.E.J.M. Ghijsen  M.D. De Jong  C.H. Van Os
Affiliation:Department of Physiology, University of Nijmegen, P.O. Box 9101, 6500 HB Nijmegen The Netherlands
Abstract:Isolated basolateral plasmamembrane vesicles from rat duodenum epithelial cells exhibit ATP-dependent calcium-accumulation and Ca2+-dependent ATPase activity. Calcium accumulation stimulated by ATP is prevented by the calcium ionophore A23187, inhibited 80% by 0.1 mM orthovanadate but is not effected by oligomycin. Calcium accumulation is not observed with the substrate β-γ-(CH2)-ATP, ADP and p-nitrophenyl phosphate. Kinetic studies reveal an apparent Km of 0.2 μM Ca2+ and a Vmax of 5.3 nmol Ca2+/min per mg protein for the ATP-dependent calcium-uptake system. Calmodulin and phenothiazines have no effect on calcium accumulation in freshly prepared membranes, but small effects are inducable after a wash with a 5 mM EGTA. The kinetic parameters of Ca2+-ATPase are: Km = 0.25 μM Ca2+ and Vmax = 19.2 nmol Pi/min per mg protein. Three techniques, osmotic shock, treatment with Triton X-100 or the channel-forming peptide alamethacin, reveal that about 40% of the vesicles are resealed. Assuming that half of the resealed vesicles have an inside-out orientation, the Vmax of ATP-dependent calcium uptake amounts to 25 nmol Ca2+/min per mg protein and of the Ca2+-ATPase to 23 nmol Pi/min per mg protein. The close correlation between kinetic parameters of Ca2+-ATPase and ATP-dependent calcium-transport strongly suggests that both systems are expressions of a Ca2+-pump located in duodenal basolateral plasma membranes.
Keywords:Kinetics  Plasma membrane  (Rat intestine)  EGTA  Hepes  4-(2-hydroxyethyl)-1-piperazineethane sulphonic acid  apparent half maximal saturation constant  maximal velocity
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