Abstract
A rabbit cytochrome P450 which catalyzes the epoxidation of arachidonic acid to two of the four possible regioisomeric epoxyeicosatrienoic acid metabolites was purified from renal cortex. A small amount of the unresolved omega/omega-1 hydroxylated eicosatetraenoic acid products were also produced. The enzyme had a specific content of 8.4 nmol of P450/mg of protein and exhibited a single band on sodium dodecyl sulfate-polyacrylamide gel electrophoresis after silver staining. Sequencing revealed a single NH2-terminal amino acid sequence with the first 20 residues identical to rabbit cytochrome P450 2C2. We suggest this enzyme be termed P450 2CAA (for arachidonic acid) until the complete sequence and substrate selectivity are established. Purified P450 2CAA was in the low spin state as evidenced by an absorption maximum at 415 nm; the reduced-carbonyl complex exhibited a maximum at 451 nm. The specific activity for metabolism of 7 microM arachidonic acid was 1.1 nmol of product formed/min/nmol of P450. About 75% of the metabolites were two of the four possible epoxyeicosatrienoic acids identified as the 11,12- and 14,15-epoxyeicosatrienoic acids by coelution with synthetic and commercial standards on reversed and normal-phase high pressure liquid chromatographic separations. The ratio of the 11,12- to 14,15-epoxyeicosatrienoic acids was 1.5:1. The purified enzyme exhibited no significant activity toward 7-ethoxyresorufin or progesterone, but demethylated aminopyrine and benzphetamine. Other fatty acids were also substrates for the enzyme. Oleic, linoleic, and lauric acids, all at about 10 microM, were metabolized at rates of 0.32, 0.72, and 0.73 nmol/min/nmol of P450, respectively. Monoclonal antibody that cross-reacts with P450 2C2 inhibited 63% of the microsomal epoxidation activity from renal cortex microsomes from phenobarbital-treated rabbits. The production of the epoxide metabolites of arachidonic acid suggests that P450 2CAA may have a significant role in arachidonic acid-mediated intra- and intercellular signalling pathways.
Highlights
In the present study we report the isolation of a cytochrome P450 enzyme from rabbit renal cortex microsomes that catalyzes the epoxidation of arachidonic acid to two of the four
This fraction was further purified by DEAE-Seph- The epoxygenase activity observed in fraction I1 did not arose chromatography as described by Ogita et al (1983) to give four separate 405-nm-absorbing peaks which catalyzed the formation of one major metabolite from arachidonic acid; the metabolite was identified by GC-MS as 20-HETE
A rabbitrenal P450 enzyme, termedP450 BCAA, that partment of Pharmaceutical Chemistry, University of California at catalyzes the epoxidation of arachidonic acid to two of the four possible regioisomeric epoxides was purified to electrophoretic homogeneity
Summary
Materials-Cholic acid, glucose 6-phosphate, glucose-6-phosphate 26 cm (510ml) Octyl-Sepharose CL-4B column equilibrated in buffer dctfTfmafIRNerrrnhceeoooareecihoorwdmdmmn.gylns.itHdineEAItSwwrUeoyC,noilledigwl[nNggtrlrmhiole-Neao.ta"rnesxaPanfoCgadr.fycdrnaos]roCtlniooemiaNdSgvihlmp(ceeetaoutaaNtisytslCcctettioipuelePcdtaelreeas--vlaaoC,c1bcXercnBiah0i.hndioIecda,Eiet,Icrosrwk)ayc2hwcc,-on(la-oehPNaaemlsdmaesmiarAltomeec.e.[ribrplh[DedAi-cl(t,cc'e-+aaHP4raI'r,4pi)aCnylanP(c.Ctcson]ageLht.l1]dnerdaiiAC,HandtldhfB2ldoorier-agae1zloinpnDeocre-miIea"iihobcdIcsCdl,ilIe,Nd,fea)aoa-r,Do)ucldluoTa,CeirnsTtmbdprioriMciSecoiTyws,tlMetla,i-e,laeotS-cidhsnr-aansiese2adonnnaplin,afulw-dldherou[3mBaogie4lr-Ecrmrli-ipo,yaec"opDhafscCchfDatoeTnerheo]csorduAprCeisopmdrmplhLPtloarhawww-giBoeuGcateeneaeriatisBrorierttldec-ee--s-,,yl-veAwvcgA1l11apoo1oi0iutm)ollDtlocwu.rubhuMxoliEmmumicyanNtfAhgnetefDasoaeessEonvinrnT0onl-dooifa.uSTedi6flsnt1leuea%i,eugo5ptbrm2atan0h0uePc0ceca.mfdto-4%hshfir1Mfev%ioaood0ri2gslfto.aep0PlcyAbtynTh%he4uciwCcoo.h5cefolshaef0rgTapensoartl0rofhhtclyeear,.miaecA4laidaauten%caenntrcitctheondibdooodcoylgulg,0hn0ndu0wfr..otrm.fa15(a3oeli0pf%iat%nrxmrnh.ht,a4yMeipyTcwt%ncl-hHaathegFaisErnroos0reec7gDdnlae.h.ai4w4actT0otcoc%t,Iea.liAoc)tl1,aosiolvt%nNc,hwtuenhihaem,tohteondTyaninnald.Icieainn1whtrvAt0weigdeoiwn.htrit1itlthgaathauaPohmcndesm1l-hMfdn1timNiwehdv0d0MsiieoDoogl.ow(u1nnhffTccEt%iiriedoocbtaTwDdhellucTuu/atTwatfmmcei1sfnAP-oeiir:1dxdnn5n--r, magnesium chloride, sodium citrate, and citric acid were from Fisher loaded at 1 ml/min ontoa 2.5 X 6 cm(30 ml) DEAE-Sepharose Scientific. m-Chloroperbenzoicacid was from Eastman Kodak. Greater than 80% of the obtained from Cayman Chemical Co. Synthesis of Arachidonic Acid Metabolites-A low specific activity Hydroxylapatite Chromatography-The nonbound fraction from mixture of 5,6-, 8,9-, 11,12- and 14,15-EETs was synthesized by the the CM-Sepharose step was loaded a t 1 ml/min onto a 2.5 X 6 cm nonselective epoxidation of arachidonic acid (0.2 pCi, 10 mg) with 1.5 (30 ml) hydroxylapatite column equilibrated with 10 mM phosphate equivalents of m-chloroperbenzoicacid in CHzC12 for 30 min a t room buffer, pH 7.5, containing 20% glycerol and 0.1% Tergitol Nonidet temperature (Chung and Scott, 1974). Two ml of elution buffer (1:lO dilution of Polybuffer 94 containing 20% glyceroland 0.2% Tergitol Nonidet P-10 and adjusted to pH 6.0 with acetic acid) was applied to thecolumn This was followed bythe dialyzed hydroxylapatite fraction and 120 ml of elution buffer a t a flow rate of0.2 ml/min. One was used for the determination of total radioactivity, and theother was spotted
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