Lipid dependence of surface conformations of protein kinase C.
The change of conformation of protein kinase C interacting with the surface of a mercury electrode directly from a solution or through a lipid monolayer was inferred from the number of cystine residues exposed and reduced on the electrode and from their reduction potentials. Soluble protein kinase C was estimated to have 5-6 disulfide bonds which could potentially react with the mercury electrode. Two major reduction peaks of cystine at different microenvironments within the protein molecule adsorbed to a mercury surface. They were observed in a.c. polarograms and cyclic voltamograms at two distinct potentials. The potential of these peaks became more negative as the pH of the solution increased, which was consistent with relaxation or decrease in alpha-helicity (ordered structure) of the protein as determined by circular dichroism (CD) estimations of secondary structure. The peak at the more positive potentials (-0.46 V relative to NAg/AgCl electrode at pH 7.4) tended to vanish upon cyclic reduction and reoxidation of the cystine, while the more negative peak (-0.62 V at pH 7.4) was enhanced. Addition of Mg2+ or Ca2+ had no significant effect on the potential but there was a reduction in their amplitude which appeared to affect the disappearance of these peaks upon pH adjustment. This suggests that the tertiary structure of the molecule is stabilized by Ca2+ and Mg2+, as substantiated by CD spectral analysis of secondary structures. Protein kinase C penetrated lipid monolayers to some extent. Addition of diacylglycerol or phorbol ester to the lipid monolayers facilitated this penetration. These compounds stabilized the protein surface conformation by destabilizing the monolayer at more positive potentials, resulting in an enhanced reduction peak at -0.42 V. This phenomenon was not significantly affected by Mg2+ or by Ca2+. The region of the protein kinase C (PKC) sequence which penetrated the monolayer contains cysteines and a primary amine(s), and may have homology to a region of phospholipase A2 which has been proposed as a phospholipid binding site for the two enzymes. Additionally, these polarographic studies suggest that PKC associates with and penetrates monolayers in a divalent cation-independent manner in agreement with our previous physical analyses of PKC interactions with lipid bilayers.