Reorientational properties of fluorescent analogues of the protein kinase C cofactors diacylglycerol and phorbol ester

The reorientational properties of the fluorescently labelled protein kinase C (PKC) cofactors diacylglycerol (DG) and phorbol ester (PMA) in vesicles and mixed micelles have been investigated using time-resolved polarised fluorescence. The sn-2 acyl chain of DG was replaced by diphenylhexatriene- (DPH) propionic acid, while a dansyl labelled analogue of phorbol ester was used. The extent of ordering of DPH-DG in vesicles turned out to be slightly different from that of the control choline lipid DPH-PC. Addition of PKC to vesicles containing 30 mole% brain PS considerably slowed down the DPH-DG anisotropy decay. This was not observed when DPH-DG was replaced by DPH-PC. Analysis of the fluorescence anisotropy decays of these DPH-lipids in micelles polyoxyethylene-9-laurylether mixed with 10 mole% of the essential phosphatidylserine allowed estimation of their lateral diffusion, orientation distribution and reorientational dynamics within the micelles. Addition of PKC resulted in a significantly slower decay of the fluorescence anisotropy of both DPH-DG and DPH-PC even in the absence of calcium, indicating a calcium independent complexation of PKC with the PS containing micelles. Addition of calcium resulted in a further reduction of the decay of anisotropy of DPH-DG but not of DPH-PC indicating that the Ca2+ dependent immobilisation is cofactor-specific. Similar specific interactions with PKC resulted in a slower decay of dansylated PMA when calcium and PS were present.