Lead activation of protein kinase C from rat brain. Determination of free calcium, lead, and zinc by 19F NMR.
Lead (Pb2+) has been reported to activate calcium/phospholipid-dependent protein kinase C (PKC) at subnanomolar concentrations (Markovac, J., and Goldstein, G. W. (1988) Nature 334, 732-734); however, others have failed to find any Pb(2+)-induced activation of PKC (Murakami, K., Feng, G., and Chen, S. G. (1993) J. Pharmacol. Exp. Ther. 264, 757-761). In neither of these studies was the actual free Pb2+ or Ca2+ concentration measured. In this study, 1,2-bis(2-amino-5-fluorophenoxy)ethane N,N,N',N'-tetraacetic acid (5F-BAPTA) was used to buffer Pb2+ and Ca2+ concentrations in the PKC reaction mixture. The specific free ion concentrations of Pb2+ and Ca2+, as well as Zn2+ and other divalent cations contained in the PKC reaction mixtures, were determined by 19F NMR spectroscopy. Using this approach to set and confirm the free Pb2+ and Ca2+ concentrations, we measured the Pb(2+)-dependent and the Ca(2+)-dependent activation of phosphotydylserine/diolein-dependent incorporation of 32P from ATP into histone and endogenous acid precipitable proteins in the 100,000 x g supernatant from homogenized rat brain cortex. We found that free Pb2+ activates PKC in the range from 10(-11) to 10(-8) M, Kact = 5.5 x 10(-11) M, while Ca2+ activates PKC in the range from 10(-8) to 10(-5) M, Kact = 2.56 x 10(-7) M. These findings clearly resolve the activation of PKC by subnanomolar concentrations of free Pb2+ from activation induced by Ca2+ or other divalent cations. Furthermore, it documents the utility of 5F-BAPTA as buffer and indicator when resolving the contributions of multiple divalent cations in biochemical processes.
Long
G J
GJ
Department of Pediatrics, Albert Einstein College of Medicine, Montefiore Medical Center, Bronx, New York 10467.
Rosen
J F
JF
Schanne
F A
FA
D000818:Animals; D002118:Calcium / Q000032:analysis / Q000494:pharmacology; D002474:Cell-Free System; D004789:Enzyme Activation / Q000187:drug effects; D007854:Lead / Q000032:analysis / Q000494:pharmacology*; D009682:Magnetic Resonance Spectroscopy / Q000379:methods; D008297:Male; D011493:Protein Kinase C / Q000378:metabolism*; D051381:Rats; D017207:Rats, Sprague-Dawley; D015032:Zinc / Q000032:analysis
D007854:Lead; D011493:Protein Kinase C; D015032:Zinc; D002118:Calcium