Effect of Protein Kinase C on the Plasma Membrane Calcium Pump in Purified Beta Cells

Abstract

The effect of protein kinase C activation on (Ca²⁺-Mg²⁺)-ATPase and ⁴⁵Ca²⁺ uptake in purified plasma membranes and membrane vesicles from beta cells was examined. PKC activation was achieved by incubating cells for 10 or 30 min in 100 nM or 1 μM of the phorbol ester 12-O-tetradecanoylphorbol 13-acetate (TPA) and evident by translocation of the α-isoform from the cytosolic to the membrane fraction. (Ca²⁺-Mg²⁺)-ATPase had a K_m for Ca²⁺ of 0.56 ± 0.17 μM and the V_max was 120 ± 12 nmol/min*mg protein in membranes from cells treated with TPA, while it was 0.66 ± 0.14 μM and 135 ± 19 nmol/min*mg protein, respectively, in its absence. In inside-out vesicles ⁴⁵Ca²⁺ uptake had a K_m for Ca²⁺ of 79 ± 19 nM and a V_max of 1.68 ± 0.43 nmol/min*mg protein in the presence of TPA. In the absence of TPA, the K_m was 71 ± 17 mM, and the V_max was 1.59 ± 0.39 nmol/min*mg mg protein, respectively. It is concluded that in beta cells PKC activation does not regulate (Ca²⁺-Mg²⁺)-ATPase activity or Ca²⁺ transport directly.