Activation of an islet cell plasma membrane (Ca2+ + Mg2+)-ATPase by calmodulin and Ca-EGTA.
Islet cell plasma membranes contain a calcium-stimulated and magnesium-dependent ATPase (Ca2+ + Mg2+)-ATPase) which requires calmodulin for maximum enzyme activity (Kotagal, N., Patke, C., Landt, M., McDonald, J., Colca, J., Lacy, P., and McDaniel, M. (1982) FEBS Lett. 137, 249-252). Investigations indicated that exogenously added calmodulin increases the velocity and decreases the Km for Ca2+ of the high affinity (Ca2+ + Mg2+)-ATPase. These studies routinely employed the chelator ethylene glycol bis(beta-aminoethyl ether)-N,N,N',N'-tetraacetic acid (EGTA) to maintain Ca2+ concentrations in the submicromolar range. During the course of these investigations, it was found unexpectedly that increasing the concentrations of EGTA (0.1-4 mM) and total calcium in the media, while maintaining constant free Ca2+ levels, increased the velocity of the high affinity (Ca2+ + Mg2+)-ATPase. The free calcium concentrations under these conditions were verified by a calcium-sensitive electrode. The (Ca2+ + Mg2+)-ATPase maximally activated by 2-4 mM EGTA was not further stimulated by calmodulin, whereas camodulin stimulation increased as the concentration of EGTA in the media was decreased. A similar enhancement by Ca-EGTA was observed on active calcium transport by the plasma membrane-enriched fraction. Moreover, Ca-EGTA had a negligible effect on both active calcium transport as well as Ca2+-stimulated ATPase activity by the islet cell endoplasmic reticulum, processes which are not stimulated by calmodulin. The results indicate that stimulation by Ca-EGTA may be used to differentiate calcium transport systems by these subcellular organelles. Furthermore, the concentration of EGTA routinely employed to maintain free Ca2+ levels may itself obscure effects of calmodulin and other physiological agents on calcium-dependent activities.
D000818:Animals; D017301:Ca(2+) Mg(2+)-ATPase; D002118:Calcium / Q000494:pharmacology*; D002135:Calcium-Binding Proteins / Q000494:pharmacology*; D000252:Calcium-Transporting ATPases / Q000378:metabolism*; D002147:Calmodulin / Q000494:pharmacology*; D002462:Cell Membrane / Q000201:enzymology; D004305:Dose-Response Relationship, Drug; D004533:Egtazic Acid / Q000494:pharmacology*; D004789:Enzyme Activation; D005026:Ethylene Glycols / Q000494:pharmacology*; D007515:Islets of Langerhans / Q000187:drug effects / Q000201:enzymology*; D008297:Male; D051381:Rats; D011919:Rats, Inbred Strains
D002135:Calcium-Binding Proteins; D002147:Calmodulin; D005026:Ethylene Glycols; D004533:Egtazic Acid; D017301:Ca(2+) Mg(2+)-ATPase; D000252:Calcium-Transporting ATPases; D002118:Calcium