THE INTERACTION BETWEEN PROTEIN-KINASE-C AND LIPID COFACTORS STUDIED BY SIMULTANEOUS OBSERVATION OF LIPID AND PROTEIN FLUORESCENCE

Citation
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
Citations number
56
Categorie Soggetti
Biology
ISSN journal
00219258
Volume
270
Issue
3
Year of publication
1995
Pages
1254 - 1260
Database
ISI
SICI code
0021-9258(1995)270:3<1254:TIBPAL>2.0.ZU;2-Y
Abstract
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.