title: Properties of a resiniferatoxin-stimulated, calcium inhibited but phosphatidylserine-dependent kinase, which is distinct from protein kinase C isotypes alpha, beta 1, gamma, delta, epsilon and eta.
abstract: We have separated a resiniferatoxin-stimulated histone-kinase activity from human neutrophils, elicited mouse macrophages and murine alveolar macrophages by hydroxyapatite chromatography. The assay conditions for resiniferatoxin kinase were optimized as part of this study and in the presence of phosphatidylserine but absence of Ca2+ the Ka for histone IIIs phosphorylation by resiniferatoxin was calculated as 16 nM. Using a phosphate gradient of 20-500 mM, peaks of protein kinase C activity could be washed from the hydroxyapatite column in 300 nM phosphate and resiniferatoxin kinase recovered in 500 mM phosphate. At the optimum concentration of 160 nM, the ability of resiniferatoxin to induce enzyme activity was compared with a range of phorbol esters all at the same concentration. These related compounds failed to activate resiniferatoxin kinase although they have previously been shown to activate protein kinase C isotypes. Similarly sn-1,2,-dioleoylglycerol and the potent irritant capsaicin at 30 microM failed to activate the kinase. A Scatchard analysis of [3H] phorbol dibutyrate binding produced a linear plot (Kd 41.6 nM; Bmax 11.6 fmol unit-1) and binding was inhibited by resiniferatoxin and 12-O-tetradecanoylphorbol-13-acetate (TPA), with resiniferatoxin 700 times more potent than TPA in this respect. A radiolabelled resiniferatoxin binding assay was also used to demonstrate specific binding of [3H]resiniferatoxin which could be inhibited by unlabelled compound. Resiniferatoxin kinase activity was shown to be distinct from the protein kinase C isotypes alpha, beta 1, gamma, delta and epsilon by means of immunological analysis and from the eta isotype, because that isotype was not stimulated by resiniferatoxin but was stimulated by TPA when a pseudosubstrate was used. In addition the resiniferatoxin-stimulated activity was inhibited in-vitro by the addition of Ca2+ (Ki 0.1-0.5 nM free Ca2+). Further purification of resiniferatoxin kinase by Superose chromatography indicated a major activity fraction of about 70-90 kDa. Thus resiniferatoxin kinase, isolated from human and mouse inflammatory cells is distinct from the known isotypes of protein kinase C and is a major resiniferatoxin receptor.
authors.affiliation: Department of Pharmacognosy, School of Pharmacy, University of London, UK.
authors.forename: P
authors.initials: P
authors.lastname: Sharma
authors.forename: W J
authors.initials: WJ
authors.lastname: Ryves
authors.forename: P C
authors.initials: PC
authors.lastname: Gordge
authors.forename: A T
authors.initials: AT
authors.lastname: Evans
authors.forename: N
authors.initials: N
authors.lastname: Shaun
authors.forename: B
authors.initials: B
authors.lastname: Thomas
authors.forename: P J
authors.initials: PJ
authors.lastname: Parker
authors.forename: F J
authors.initials: FJ
authors.lastname: Evans
mesh_terms: D000818:Animals; D002118:Calcium / Q000494:pharmacology*; D004224:Diterpenes / Q000494:pharmacology*; D006801:Humans; D007527:Isoenzymes / Q000378:metabolism*; D051379:Mice; D008970:Molecular Weight; D015240:Phorbol 12,13-Dibutyrate / Q000378:metabolism; D010718:Phosphatidylserines / Q000494:pharmacology*; D006656:Protamine Kinase / Q000302:isolation & purification* / Q000378:metabolism; D011493:Protein Kinase C / Q000378:metabolism*
chemical_list: D004224:Diterpenes; D007527:Isoenzymes; D010718:Phosphatidylserines; D015240:Phorbol 12,13-Dibutyrate; C024353:resiniferatoxin; D006656:Protamine Kinase; D011493:Protein Kinase C; D002118:Calcium