REORIENTATIONAL PROPERTIES OF FLUORESCENT ANALOGS OF THE PROTEIN-KINASE-C COFACTORS DIACYLGLYCEROL AND PHORBOL ESTER

The reorientational properties of the fluorescently labelled protein k inase C (PKC) cofactors diacylglycerol (DG) and phorbol ester (PMA) in vesicles and mixed micelles have been investigated using time-resolve d polarised fluorescence. The sn-2 acyl chain of DG was replaced by di phenylhexatriene- (DPH) propionic acid, while a dansyl labelled analog ue of phorbol ester was used. The extent of ordering of DPH-DG in vesi cles turned out to be slightly different from that of the control chol ine lipid DPH-PC. Addition of PKC to vesicles containing 30 mole% brai n PS considerably slowed down the DPH-DG anisotropy decay. This was no t observed when DPH-DG was replaced by DPH-PC. Analysis of the fluores cence anisotropy decays of these DPH-lipids in micelles polyoxyethylen e-9-laurylether mixed with 10 mole% of the essential phosphatidylserin e allowed estimation of their lateral diffusion, orientation distribut ion and reorientational dynamics within the micelles. Addition of PKC resulted in a significantly slower decay of the fluorescence anisotrop y of both DPH-DG and DPH-PC even in the absence of calcium, indicating a calcium independent complexation of PKC with the PS containing mice lles. 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+ de pendent immobilisation is cofactor-specific. Similar specific interact ions with PKC resulted in a slower decay of dansylated PMA when calciu m and PS were present.