Biochemical characterization of rat brain protein kinase C isozymes.
Biochemical characteristics of three rat brain protein kinase C isozymes, types I, II, and III, were compared with respect to their protein kinase and phorbol ester-binding activities. All three isozymes appeared to be alike in their phorbol ester-binding activities as evidenced by their similar Kd for phorbol 12,13-dibutyrate and requirements for Ca2+ and phospholipids. However, differences with respect to the effector-mediated stimulation of protein kinase activity were detectable among these isozymes. The type I enzyme could be stimulated by cardiolipin to a greater extent than those of the type II and III enzymes. In the presence of cardiolipin, the concentrations of dioleoylglycerol or phorbol 12,13-dibutyrate required for half-maximal activation (A1/2) of the type I enzyme were nearly an order of magnitude lower than those for the type II and III enzymes. In the presence of phosphatidylserine, differences in the A1/2 of dioleoylglycerol and phorbol 12,13-dibutyrate for the three isozymes of protein kinase C were less significant than those measured in the presence of cardiolipin. Nevertheless, the A1/2 of these two activators for the type I enzyme were lower than those for the type II and III enzymes. At high levels of phosphatidylserine (greater than 15 mol %), binding of phorbol 12,13-dibutyrate to the type I enzyme evoked a corresponding stimulation of the kinase activity, whereas binding of this phorbol ester to the type II and III enzymes produced a lesser degree of kinase stimulation. For all three isozymes, the concentrations of phosphatidylserine required for half-maximum [3H]phorbol 12,13-dibutyrate binding were almost an order of magnitude less than those for kinase stimulation. Consequently, neither isozyme exhibited a significant kinase activity at lower levels of phosphatidylserine (less than 5 mol %) and phorbol 12,13-dibutyrate (50 nM), a condition sufficient to promote near maximal phorbol ester binding. In addition to their different responses to the various activators, the three protein kinase C isozymes also have different Km values for protein substrates. The type I enzyme appeared to have lower Km values for histone IIIS, myelin basic protein, poly(lysine, serine) (3:1) polymer, and protamine than those for the type II and III enzymes. These results documented that the three protein kinase C isozymes were distinguishable in their biochemical properties. In particular, the type I enzyme, which is a brain-specific isozyme, is distinct from the type II and III enzymes, both have a widespread distribution among different tissues.
Huang
K P
KP
Section on Metabolic Regulation, National Institute of Child Health and Human Development, Bethesda, Maryland 20892.
Huang
F L
FL
Nakabayashi
H
H
Yoshida
Y
Y
D000818:Animals; D001921:Brain / Q000201:enzymology*; D002118:Calcium / Q000494:pharmacology; D004075:Diglycerides / Q000494:pharmacology; D007527:Isoenzymes / Q000378:metabolism*; D007700:Kinetics; D015240:Phorbol 12,13-Dibutyrate / Q000378:metabolism; D010703:Phorbol Esters / Q000494:pharmacology; D010743:Phospholipids / Q000494:pharmacology; D011485:Protein Binding; D011493:Protein Kinase C / Q000378:metabolism*; D051381:Rats
D004075:Diglycerides; D007527:Isoenzymes; D010703:Phorbol Esters; D010743:Phospholipids; D015240:Phorbol 12,13-Dibutyrate; D011493:Protein Kinase C; D002118:Calcium; C013965:diolein