Activation and inhibition of protein kinase C isozymes alpha and beta by Gd3+.
Gd3+ was evaluated as a probe for Ca2+ sites on protein kinase C (PKC) by studying its ability to replace Ca2+ in activation of PKC isozymes II (beta) and III (alpha) in the lipid systems phosphatidylserine/1,2-dioleoyl-sn-glycerol (PS/DO) and diheptanoylphosphatidylcholine (PC7)/DO. PKC beta was stimulated by Ca2+ or Gd3+ in PS/DO whereas activity in PC7/DO was independent of these metals. Thus, it is suggested that Gd3+ replaces Ca2+ at a site involving metal-lipid interactions. High concentrations of Ca2+ or Gd3+ inhibited activity in both lipid systems. Analysis of the Gd3+ inhibition in the PC7/DO system suggests that it is due to formation of GdATP, which competes at the MgATP site. Activity of PKC alpha was dependent on low concentrations of Ca2+ in both lipid systems. The ability of Gd3+ to substitute for Ca2+ could not be evaluated in the PS system due to the inability to completely remove contaminating Ca2+ without chelating buffers. Successful reduction of contaminating Ca2+ was achieved in the PC7 system but Gd3+ failed to substitute for Ca2+ in activating PKC alpha and only caused inhibition. This is consistent with binding of Gd3+ to a Ca2+ site at or near the active site of the enzyme rather than to a site on the lipid. These results indicate that interactions between PKC and Gd3+ are complex, involving occupation of more than one class of sites. Conditions for separately evaluating the individual sites can be manipulated by selection of isozyme and lipid system.
Maurer
M C
MC
Department of Chemistry, University of Virginia, Charlottesville 22908.
Grisham
C M
CM
Sando
J J
JJ
D000818:Animals; D000936:Antigen-Antibody Complex; D015153:Blotting, Western; D001921:Brain / Q000201:enzymology; D002118:Calcium / Q000494:pharmacology; D004789:Enzyme Activation; D005682:Gadolinium / Q000494:pharmacology*; D007527:Isoenzymes / Q000037:antagonists & inhibitors / Q000302:isolation & purification / Q000378:metabolism*; D007700:Kinetics; D011493:Protein Kinase C / Q000037:antagonists & inhibitors / Q000302:isolation & purification / Q000378:metabolism*; D051381:Rats
D000936:Antigen-Antibody Complex; D007527:Isoenzymes; D005682:Gadolinium; D011493:Protein Kinase C; D002118:Calcium