Divalent cation-induced changes in conformation of protein kinase C.
Using physical techniques, circular dichroism and intrinsic and extrinsic fluorescence, the binding of divalent cations to soluble protein kinase C and their effects on protein conformation were analyzed. The enzyme copurifies with a significant concentration of endogenous Ca2+ as measured by atomic absorption spectrophotometry, however, this Ca2+ was insufficient to support enzyme activity. Intrinsic tryptophan fluorescence quenching occurred upon addition to the soluble enzyme of the divalent cations, Zn2+, Mg2+, Ca2+ or Mn2+, which was irreversible and unaffected by monovalent cations (0.5 M NaCl). Far ultraviolet (200-250 nm) circular dichroism spectra provided estimations of secondary structure and demonstrated that the purified enzyme is rich in alpha-helices (42%) suggesting a rather rigid structure. At Ca2+ or Mg2+ concentrations similar to those used for fluorescence quenching, the enzyme undergoes a conformational transition (42-24% alpha-helix, 31-54% random structures) with no significant change in beta-sheet structures (22-26%). Maximal effects on 1 microM enzyme were obtained at 200 microM Ca2+ or 100 microM Mg2+, the divalent cation binding having a higher affinity for Mg2+ than for Ca2+. The Ca2(+)-induced transition was time-dependent, while Mg2+ effects were immediate. In addition, there was no observed energy transfer for protein kinase C with the fluorescent Ca2(+)-binding site probe, terbium(III). This study suggests that divalent cation-induced changes in soluble protein kinase C structure may be an important step in in vitro analyses that has not yet been detected by standard biochemical enzymatic assays.
Lester
D S
DS
Department of Membrane Research, Weizmann Institute of Science, Rehovot, Israel.
Brumfeld
V
V
D000818:Animals; D001921:Brain / Q000201:enzymology; D002413:Cations, Divalent / Q000378:metabolism / Q000494:pharmacology; D055598:Chemical Phenomena; D002627:Chemistry, Physical; D002942:Circular Dichroism; D066298:In Vitro Techniques; D008297:Male; D011487:Protein Conformation / Q000187:drug effects; D011493:Protein Kinase C / Q000737:chemistry* / Q000378:metabolism; D051381:Rats; D011919:Rats, Inbred Strains; D013050:Spectrometry, Fluorescence
D002413:Cations, Divalent; D011493:Protein Kinase C