EFFECT OF PROTEIN-KINASE-C ON THE PLASMA-MEMBRANE CALCIUM-PUMP IN PURIFIED BETA-CELLS

The effect of protein kinase C activation on (Ca2+-Mg2+)-ATPase and Ca -45(2+) uptake in purified plasma membranes and membrane vesicles from beta cells was examined. PKC activation was achieved by incubating ce lls for 10 or 30 min in 100 nM or 1 mu M of the phorbol ester 12-O-tet radecanoylphorbol 13-acetate (TPA) and evident by translocation of the alpha-isoform from the cytosolic to the membrane fraction. (Ca2+-Mg2)-ATPase had a K-m for Ca2+ of 0.56 +/- 0.17 mu M and the V-max was 12 0 +/- 12 nmol/minmg protein in membranes from cells treated with TPA, while it was 0.66 +/- 0.14 mu M and 135 +/- 19 nmol/minmg protein, r espectively, in its absence. In inside-out vesicles Ca-45(2+) uptake h ad a K-m for Ca2+ of 79 +/- 19 nM and a V,, of 1.68 +/- 0.43 nmol/min mg protein in the presence of TPA. In the absence of TPA, the K, was 7 1 +/- 17 mM, and the Vmax was 1.59 +/- 0.39 nmol/minmg mg protein, re spectively. It is concluded that in beta cells PKC activation does not regulate (Ca2+-Mg2+)-ATPase activity or Ca2+ transport directly. (C) 1994 Academic Press, Inc.