Characteristics of a Ca2+-ATPase activity measured in islet homogenates.

Ca2+-ATPase activity was measured in rat islet homogenates, in a medium of low ionic strength containing a low concentration of Ca2+ and Mg2+ and devoid of K+. The enzyme activity was highly sensitive to inhibition by compound 48/80 (a calmodulin inhibitor), stimulated by 120 nM calmodulin and slightly affected by 10 mM NaN3. The addition of Mg2+ to the assay medium promotes the disappearance of apparent Ca2+-ATPase activity. Ouabain (0.1 mM) did not modify this ATPase activity. The enzyme showed two kinetic components for Ca2+ as well as for ATP: one with high apparent affinity and low maximum velocity and the other with low apparent affinity and high maximum velocity. Incubation of islet homogenates in this assay medium with [gamma-32P]ATP in the presence of proteolytic inhibitors, results in the appearance of a single labelled band of 130 kDa, identified by gel electrophoresis. The incorporation of 32P into this band was similar in the presence of either 2.8 or 50 microM Ca2+ and susceptible to hydroxylamine attack. The results indicate that, under the conditions described above, the Ca2+-ATPase activity evidenced in the islet homogenates had characteristics resembling those of the enzyme which catalyzes the outward Ca2+ transport. On the other hand, the method could provide a useful tool to test the effect of different agents which affect insulin secretion upon the islet plasma membrane Ca2+-ATPase activity.