Activation of an islet cell plasma membrane (Ca2+ + Mg2+)-ATPase by calmodulin and Ca-EGTA

• Published in: The Journal of biological chemistry Vol. 258; no. 8; pp. 4808 - 4813
• Main Authors: Kotagal, N, Colca, J R, McDaniel, M L
• Format: Journal Article
• Language: English
• Published: United States Elsevier Inc 25-04-1983; American Society for Biochemistry and Molecular Biology
• Subjects: Animals; Ca(2+) Mg(2+)-ATPase; Calcium - pharmacology; Calcium-Binding Proteins - pharmacology; Calcium-Transporting ATPases - metabolism; Calmodulin - pharmacology; Cell Membrane - enzymology; Dose-Response Relationship, Drug; Egtazic Acid - pharmacology; Enzyme Activation; Ethylene Glycols - pharmacology; Islets of Langerhans - drug effects; Islets of Langerhans - enzymology; Male; Rats; Rats, Inbred Strains
• ISSN: 0021-9258; 1083-351X
• DOI: 10.1016/S0021-9258(18)32497-9

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.