Ehw. Pap et al., THE INTERACTION BETWEEN PROTEIN-KINASE-C AND LIPID COFACTORS STUDIED BY SIMULTANEOUS OBSERVATION OF LIPID AND PROTEIN FLUORESCENCE, The Journal of biological chemistry, 270(3), 1995, pp. 1254-1260
The interaction of protein kinase C (PKC) with lipids was probed by a
dual approach. Pyrene-labeled lipid analogues of diacylglycerol, phosp
hatidylserine (PS), phosphatidylinositol (PI), phosphatidylinositol 4-
phosphate (PIP), and phosphatidylcholine (PC) were used both as accept
ers of tryptophan excitation energy of PKC and as membrane probes for
intra- and intermolecular lipid chain collisions by measuring the rati
o of excimer-to-monomer fluorescence intensity (EM). Both in micelles
of polyoxyethylene 9-lauryl ether and in dioleoyl-PC vesicles, interac
tion of PKC with monopyrenyl PS (pyr-PS) in the absence of calcium res
ulted in a relatively slow decrease of the EM value. This effect on th
e lipid dynamics was accompanied by quenching of the tryptophan fluore
scence of PKC. Addition of calcium resulted in a rapid further decreas
e of the EM ratio of pyr-PS and in additional quenching of the tryptop
han fluorescence. When 4 mol % of pyr-PS was replaced by 0.5 mol % of
dipyrenyl-labeled diacylglycerol a decrease of the intramolecular exci
mer formation rate and tryptophan fluorescence could only be detected
in the presence of calcium and PS. Strong binding was also observed wi
th dipyrenyl-labeled PIP (dipyr-PIP), but not with the other dipyrenyl
-labeled lipids: PI, PS, or PC. In addition, the EM ratios of dipyr-PI
P were not affected by phorbol 12-myristate 13-acetate, indicating tha
t phorbol 12-myristate 13-acetate and dipyr-PIP can bind simultaneousl
y to PKC.