CHARACTERIZATION OF CALCIUM-DEPENDENT FORMS OF PROTEIN-KINASE-C IN ADULT-RAT VENTRICULAR MYOCYTES

The presence and subcellular localization of the Ca2+-dependent protei n kinase C (PKC) isoforms alpha and beta were investigated in freshly isolated adult rat cardiac ventricular myocytes. PKC activity was meas ured in cytosolic and particulate fractions prepared from control myoc ytes and those treated with either phorbol ester (phorbol 12-myristate 13-acetate, PMA) or a permeant synthetic diacylglycerol analog (1-ole oyl-2-acetylglycerol, OAG) in the absence or presence of an inhibitor of diacylglycerol kinase activity, compound R59022. Preliminary studie s detected no Ca2+/phpspholipid-dependent histone kinase activity in e ither subcellular fraction. To reproducibly observe Ca2+/phospholipid- dependent protein kinase activity, partial purification using a MonoQ HR 5/5 column and the presence of the peptide inhibitor of the cAMP-de pendent protein kinase were essential. MonoQ chromatography of cytosol ic and particulate fractions resulted in three peaks of Ca2+/phospholi pid-dependent protein kinase activity. In the cytosolic fraction a lar ge peak of activity eluted at 230-300 mM NaCl. Isoform-specific antise ra indicated both PKC alpha and PKC beta were present. In the particul ate fraction two peaks of Ca2+/phospholipid-dependent protein kinase a ctivity, both containing PKCa immunoreactivity, were observed. The lar ger peak eluted at 230-300 mM NaCl. In addition, a peak eluting at low er salt concentrations contained a Ca2+/phospholipid-independent histo ne kinase activity. This peak of kinase activity contained PKC alpha i mmunoreactive bands of 80- and 50-kDa. The 80-kDa band was the holoenz yme of PKC alpha whereas the band of lower molecular mass was likely a proteolytic fragment. In both cytosolic and particulate fractions, th e peak of kinase activity eluting at 230-300 mM NaCl contained PKC alp ha in the form of an 80-kDa doublet; this suggested the presence of au tophosphorylated PKC. Incubation of the myocytes with PMA, but not GAG , resulted in translocation of PKC from the cytosolic to the particula te fraction. Curiously, a transient decrease in PKC activity was obser ved in both subcellular fractions following treatment with either OAG or ethanol (1%). Results from this study show that freshly isolated ad ult rat cardiac ventricular myocytes contain both PKC alpha and PKC be ta, and that these isoforms translocate to the particulate fraction in response to treatment with PMA, but not OAG.