High-affinity Ca(2+)- and substrate-binding sites on protein kinase C alpha as determined by nuclear magnetic resonance spectroscopy.
Water proton nuclear magnetic resonance (NMR) relaxation rates were used to identify metal sites on protein kinase C (PKC) isozymes alpha and beta using paramagnetic Gd3+ as a probe. The paramagnetic effect of Gd3+ on water proton relaxation was enhanced with PKC isozymes alpha and beta in the presence of diheptanoylphosphatidylcholine/1,2-dioleoyl-sn-glycerol (PC7/DO). The data are consistent with a single class of metal-binding sites on PKC beta and two classes of sites on PKC alpha: a single high-affinity site with a KD for Gd3+ of 0.2 microM and a larger class of sites with a lower affinity for Gd3+. Titration with Ca2+ abolished the observed enhancement of water proton relaxation by the PKC alpha.Gd3+ complex, consistent with displacement of Gd3+ by Ca2+. Titrations of the PKC alpha.Gd3+ complex with Co(NH3)4ATP, a substitution-inert analogue of ATP, caused a substantial decrease in the observed water proton relaxation enhancement, consistent with formation of a ternary enzyme.metal.substrate complex with a KPKC alpha.Gd.[CoATP] of 30-100 nM. Titration of the metal enzyme complex with a model peptide substrate derived from the pseudosubstrate sequence of PKC alpha caused a similar decrease in enhancement at stoichiometric concentrations consistent with the formation of a PKC alpha.Gd3+.peptide complex with a KPKC alpha.Gd.[peptide] of less than or equal to 13 nM. Titrations of the fully formed PKC alpha.Gd3+.peptide complex with Co(NH3)4ATP caused a further decrease in enhancement consistent with formation of a quaternary complex.(ABSTRACT TRUNCATED AT 250 WORDS)
Maurer
M C
MC
Department of Chemistry, University of Virginia, Charlottesville 22901.
Sando
J J
JJ
Grisham
C M
CM
D000255:Adenosine Triphosphate / Q000031:analogs & derivatives / Q000494:pharmacology; D000595:Amino Acid Sequence; D000818:Animals; D001665:Binding Sites; D001921:Brain / Q000201:enzymology; D002118:Calcium / Q000378:metabolism*; D005682:Gadolinium / Q000378:metabolism / Q000494:pharmacology; D007527:Isoenzymes / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism*; D007700:Kinetics; D008081:Liposomes; D009682:Magnetic Resonance Spectroscopy / Q000379:methods; D008962:Models, Theoretical; D008969:Molecular Sequence Data; D010455:Peptides / Q000737:chemistry; D011493:Protein Kinase C / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism*; D051381:Rats; D012984:Software
D007527:Isoenzymes; D008081:Liposomes; D010455:Peptides; D000255:Adenosine Triphosphate; D005682:Gadolinium; D011493:Protein Kinase C; D002118:Calcium