title: Evidence that protein kinase C alpha has reduced affinity towards 1,2-dioctanoyl-sn-glycerol: the effects of lipid activators on phorbol ester binding and kinase activity.
abstract: The effect of 1,2-diacylglycerols on specific binding of [3H]phorbol 12,13-dibutyrate to cytosolic protein kinase C (PKC) was investigated in tissues reported to contain different proportions of PKC isoforms. In lung, frontal cerebral cortex and cerebellum cytosols (enriched in PKC alpha, beta and gamma, respectively) displacement of specific binding by phorbol 12,13-dibutyrate or diacylglycerols containing unsaturated acyl chains was of similar potency for each tissue. A range of 1,2-diacylglycerols containing saturated acyl chains exhibited varying affinities for [3H]phorbol 12,13-dibutyrate binding sites in each tissue; defining an optimal acyl chain length of around 14 carbons in each case. However, the affinities of saturated diglycerides were consistently lower in lung cytosol than in frontal cerebral cortex and cerebellum cytosols, with the greatest differences occurring at lower acyl chain lengths, especially with 1,2-dioctanoyl-sn-glycerol. Furthermore, a mixed micelle assay of PKC activity showed that 1,2-dioctanoyl-sn-glycerol displayed reduced potency at PKC alpha partially-purified from COS 7 cell cytosol compared to the mixture of PKC isoforms present in rat midbrain cytosol. Both low potency of 1,2-dioctanoyl-sn-glycerol as a displacer of [3H]phorbol 12,13 dibutyrate binding and the ability of arachidonic acid to act as an allosteric enhancer of binding, correlated with the proportional PKC alpha content of a range of tissues reported in the literature. In PKC enzyme activity assays, 1,2-dioctanoyl-sn-glycerol, but not phorbol 12,13-dibutyrate, was correspondingly a much poorer activator of PKC alpha from COS 7 cells than of the broad consensus of isoforms in rat midbrain. When alpha and beta isoforms were extensively-purified on DEAE-cellulose then hydroxyapatite, both the low affinity of 1,2-dioctanoyl-sn-glycerol for [3H]phorbol 12,13-dibutyrate binding sites and their allosteric regulation by arachidonic acid were confirmed to be characteristic of the alpha rather than the beta isoforms.
authors.affiliation: MRC Brain Metabolism Unit, University Department of Pharmacology, Edinburgh, UK.
authors.forename: D J
authors.initials: DJ
authors.lastname: MacEwan
authors.forename: R
authors.initials: R
authors.lastname: Mitchell
authors.forename: M S
authors.initials: MS
authors.lastname: Johnson
authors.forename: F J
authors.initials: FJ
authors.lastname: Thomson
authors.forename: E M
authors.initials: EM
authors.lastname: Lutz
authors.forename: R A
authors.initials: RA
authors.lastname: Clegg
authors.forename: K
authors.initials: K
authors.lastname: Connor
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D001095:Arachidonic Acids / Q000378:metabolism; D002531:Cerebellum / Q000201:enzymology; D002540:Cerebral Cortex / Q000201:enzymology; D003600:Cytosol / Q000201:enzymology; D004075:Diglycerides / Q000378:metabolism*; D007527:Isoenzymes / Q000276:immunology / Q000302:isolation & purification / Q000378:metabolism; D050356:Lipid Metabolism; D008168:Lung / Q000201:enzymology; D008297:Male; D008969:Molecular Sequence Data; D015240:Phorbol 12,13-Dibutyrate / Q000378:metabolism*; D010718:Phosphatidylserines / Q000737:chemistry; D011493:Protein Kinase C / Q000276:immunology / Q000302:isolation & purification / Q000378:metabolism*; D012333:RNA, Messenger / Q000096:biosynthesis; D051381:Rats; D017208:Rats, Wistar
chemical_list: D001095:Arachidonic Acids; D004075:Diglycerides; D007527:Isoenzymes; D010718:Phosphatidylserines; D012333:RNA, Messenger; C044295:1,2-dioctanoylglycerol; D015240:Phorbol 12,13-Dibutyrate; D011493:Protein Kinase C