Abstract
The CaZ+ and phospholipid-dependent protein kinase (protein kinase C) is believed to couple extracellular messenger-induced polyphosphoinositide turnover with diverse cellular events, e.g. thrombin-induced aggregation of, and neurotransmitter release from, platelets (Berridge, 1984; Berridge & Irvine, 1984; Nishizuka, 1984). Recent reports indicate that protein kinase C may function in the negative feedback regulation of this induced polyphosphoinositide turnover (Leeb-Lundberg et al., 1985; Orellina et al., 1985; Watson & Lapetina, 1985). Walsh et al. (1984) reported the use of Ca2+ -dependent hydrophobic interaction chromatography (using phenylSepharose CL4B) in the purification of protein kinase C and a Ca2+ -binding protein of M , 21 000 (21K CaBP) from bovine brain. The 21K CaBP was subsequently purified to homogeneity using heat treatment and hydroxyapatite chromatography. However, bulk purification of this calciprotein necessitated an additional fractionation step due to contaminating proteins. DEAESephacel chromatography yielded not only the 21K CaBP but also two previously undescribed Ca2+ binding proteins of M , 18 400 and 17 000 (McDonald & Walsh, 1985~). The 17K CaBP was found to be a potent inhibitor of protein kinase C (McDonald & Walsh, (l985a,b). We now describe a modified purification procedure which facilitates the isolation of not only the 17K CaBP with increased yields but also a novel potent protein kinase C inhibitor, an M , 12000Ca2+ -binding protein (12K CaBP). Bovine brain 100 OOOg supernatant (obtained from 750 g of tissue) was fractionated on phenyl-Sepharose exactly as described b Walsh et al. (1984). We have found the yield of CaL-binding proteins to be higher using fresh rather than regenerated phenyl-Sepharose. Fractions eluted from this column by chelation of Ca2+ with EGTA were split into 21KCaBPand 17KCaBPrich pools which were heat-treated, dialysed and freezedried. After being redissolved in 20 mM-Tris/HCI (pH 7.5)/1 mM-EGTA/I mMdithiothreitol, both protein mixtures were fractionated separately on DEAE-Sephacel (McDonald & Walsh, 1985~). Detection of the eluted proteins by SDS/polyacrylamide-gradient slab-gel electrophoresis revealed the presence of not only the 21 K and 17KCaBPs, but also a 12KCaBP. The 17K and 12K CaBPs inhibited protein kinase C-catalysed phosphorylation of histone 111-S by an average of 75% at 2 . 9 ~ ~ 17K CaBP and 85% at 4.1 ~ M I ~ K CaBP. The inhibitory effects of both calciproteins were not due to interaction with substrates or cofactors nor to phosphatase activity, as neither CaBP dephosphorylated substrates which had been previously phosphorylated by protein kinase C, even after a prolonged incubation period. The inhibitory effect was shown to be a property of these proteins since trypsin digestion destroyed the inhibitory effects of both calciproteins (Fig. I ) .
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