The properties of the protein kinase C (PKC)-phorbol ester interaction were highly dependent on assay methods and conditions. Binding to cation-exchange materials or adsorption to gel matrices resulted in PKC that was capable of binding phorbol 12,13-dibutyrate (PDBu). The extraneous interactions were eliminated by measuring phorbol ester binding with a gel filtration chromatography assay in the presence of bovine serum albumin (BSA). In the absence of calcium, free PKC did not bind PDBu or phospholipids. Calcium caused structural changes in PKC which enhanced its interaction with surfaces such as the gel chromatography matrix. While BSA prevented this interaction, it did not interfere with PKC association with acidic phospholipids. Interaction of PKC with phospholipid resulted in two forms of membrane-associated PKC The initial calcium-dependent and reversible form of membrane-associated PKC was capable of binding PDBu Both PKC and PDBu were released from this complex by calcium chelation. Sustained interaction with phospholipid vesicles resulted in a PKC-membrane complex that could not be dissociated by calcium chelation and appeared to result from insertion of PKC into the hydrocarbon portion of the phospholipid bilayer. Membrane insertion was observed at calcium concentrations of 2-500 μM and with membrane compositions of 10-50% acidic phospholipid. However, the extent of insertion was dependent on the binding conditions and was promoted by high phospholipid to PKC ratios, high calcium, the presence of phorbol esters high membrane charge, and long incubations. Once PKC was inserted into a phospholipid bilayer, it bound PDBu in the presence (Kd = <0.5 nM, kdiss = 10-1 s-1 at 4°C) and in the absence of Ca2+. Calcium enhanced the affinity of PKC-PDBu interaction and decreased the dissociation rate. These results showed that dramatic changes occurred in the in vitro properties of PKC upon the formation of the irreversible PKC-membrane complex. These properties may be related to cellular events that induce formation of the chelator-resistant form of membrane-bound PKC.
|Original language||English (US)|
|Number of pages||9|
|State||Published - 1989|