When washed human platelets were disrupted by sonication in the presence of ethylene glycol bis(β-aminoethyl ether)-N,N,N′,N′-tetra-acetic acid, both the catalytic and [3H]phorbol-12,13-dibutyrate (PDBu)-binding activities of protein kinase C were recovered in the soluble fraction and were not separable from each other upon several column chromatographies. Platelet protein kinase C required diacylglycerol, Ca2+, and phospholipid for its activation and showed a molecular weight of about 87,000 as estimated by gel filtration analysis. However, when platelets were first incubated with 2 µm Ca2+-ionophore A23187 for 5 min at 37°C in the medium containing 3 mm CaCl2 and then disrupted under the same conditions, the catalytic and [3H]phorbol-12,13-dibutyrate-binding activities were separately recovered in the soluble and particulate fractions, respectively; moreover, the catalytic activity recovered in the soluble fraction became independent of diacylglycerol, Ca2+, and phospholipid, and showed a molecular weight of about 50,000 as estimated by gel filtration analysis. The kinetic properties of this Mr50,000 enzyme were similar to those of the catalytic fragment of rat brain protein kinase C described previously. In a cell-free system, digestion with trypsin of protein kinase C highly purified from rat brain caused the generation of a fragment which had no catalytic activity but showed full [3H]phorbol-12,13-dibutyrate-binding activity. The molecular weight of this fragment was estimated to be about 35,000 by sodium dodecyl sulfate-polyacryl-amide gel electrophoresis. These results indicate that protein kinase C consists of at least two functionally different domains, a hydrophobic phorbol ester- or diacylglycerol-binding and hydrophilic catalytic domains.
This investigation was supported in part by research grants from the Scientific Research Fund of Ministry of Education, Science, and Culture, Japan (1985), Investigation Committee on Abnormalities in Hormone Receptor Mechanisms, Public Health Bureau, the Ministry of Health and Welfare, Japan (1985), the Yamanouchi Foundation for Research on Metabolic Disorders (1985), Research Foundation for Cancer and Cardiovascular Diseases (1985), and Mitsukoshi Prize for Medicine (1985).