Farnesol

October 21, 2014  |  By  | 


172 A.E.Sperry,S.E.Sen/InsectBiochemistryandMolecularBiology31(2001)171–178 mMNAD.Althoughtheseresultssuggestedthatfarnesol metabolismwasmediatedbyanicotinamide-dependent dehydrogenase,attemptstomoreclearlyidentifyNAD involvementmetwithdifculties.Incubationof1,5,9- [ 3 H]farnesol,followedbyradio-HPLCanalysisofthe polarmetabolites,indicatedthatneither[ 3 H]NADHnor [ 3 H]NADPHwaspresentincorporaallatahomogenates. However,theinabilitytodetectradiolabeledcofactordid notdisprovetheformationofNAD(H),sinceothersec- ondaryredoxprocessesmayhaveoccurredduringincu- bationwiththeinsectglandhomogenate. Inthispaper,wedescribethepartialcharacterization offarnesolmetabolisminlarvalcorporacardiaca– corporaallata(CC–CA)homogenatesof M.sexta . Despitethefactthatfarnesoloxidationinplants,ver- tebrates,andnon-JHproducinginsecttissueistypically catalyzedbynicotinamidedependentdehydrogenases, theformationoffarnesalininsectcorporaallataisan oxygen-dependentprocess.Additivestudiesindicatethat activitymayrequireametalcofactorandFAD,suggest- ingthatJHbiosynthesisinvolvestheintermediacyofone ormorealcoholoxidases. 2.Materialsandmethods 2.1.Chemicalsources Tween-80,nucleotidecofactors,yeastalcohol dehydrogenase(YADH),glucoseoxidase,catalase,and geranylgeraniolwereobtainedfromSigmaChemicalCo. (St.Louis,MO).Allothermaterialswerepurchased fromeitherAldrichChemicalCo.(Milwaukee,WI)or FisherScientic(Pittsburgh,PA),except1,2-[ 14 C]farne- sol,whichwasobtainedfromAmericanRadiolabeled Chemicals,Inc.(St.Louis,MO)andwaspuriedbysil- icagelcolumnchromatographyusinga5–10%ethyl acetateinhexanegradient. 2.2.Insectsandtissuesource Manducasexta larvaewererearedonanarticialdiet andmaintainedunderconstantphotoperiod(18L:6D), usingpreviouslydescribedprocedures(Belland Joachim,1976).Animalswerestagedpriortomolting atthe4thlarvalstadiumbyobservingheadcapsule slippageandweremadesynchronousbystarvationdur- ingthelastlarvalmolt.CC–CAcomplexesfromnewly emerged(0–12hold),5thstadiumlarvae(V/0),adevel- opmentalstagethatisknowntoproducesignicant quantitiesofJH(Bakeretal.,1987),wereremovedfol- lowingpreviouslyestablishedmicrodissectionpro- cedures(BhaskaranandJones,1980).Forallmetalstud- ies,corporaallatadissectionswereperformedinthe absenceof M.sexta saline(4mMNaCl,40mMKCl, 18mMMgCl 2 ,and3mMCaCl 2 ). 2.3.Enzymeassay Theconversionoffarnesoltofarnesalwithinthecor- pusallatumwasmonitoredaspreviouslydescribed(Sen andGarvin,1995a).Briey,CC–CAcomplexeswere removedfromV/0 M.sexta larvae,homogenizedfor3 minonice,in100mMTris–HClbuffer,pH7,andthe resultinghomogenatewascentrifugedat3,000 g for1 min.Thesupernatantwastransferredtoamicrocentri- fugetube,thevolumeadjusted(typicallyto0.25gland pairequiv/25 µ l),andTween-80wasaddedtogivea nalconcentrationof0.05%(w/v).Afterstandingonice for5min,thesolutionwascentrifugedat16,000 g for5 minandaliquotsofsupernatant(25–50 µ l)wereplaced into500 µ lsiliconizedplasticmicrocentrifugetubes (FisherScientic). Theconversionoffarnesoltofarnesalwasassayedby addingradioactivelylabeledsubstrate(1,2-[ 14 C]farnesol, specicactivity27.5mCi/mmol)tothepreparedenzyme solutiontogiveanalconcentrationof10 µ M.Thesol- utionwasincubatedat26 ° Cfor1–2h,thenquenched bytheadditionofacetonitrilecontainingfarnesol,farne- sal,andfarnesoicacidstandards.Thereactionmixture wasextractedwithCH 2 Cl 2 andtheconcentratedorganic extractwasredissolvedinaminimumamountofCH 2 Cl 2 andappliedtoaplastic-backednormalphaseTLCplate (40 × 80mm,Macherey-NagelPolygram ® SilG/UV254, BodmanIndustries,Aston,PA).Doubleelutionwith 10%ethylacetateinhexanecontaining5%triethylamine gavecleanseparationofstartingmaterialandproduct( R f farnesol = 0.5, R f farnesal = 0.85).Theextentoffarnesol oxidation(expressedas%conversion)wasdetermined bycuttingeachTLCplateintovesections,thenquan- tifyingtheamountoffarnesolandfarnesalpresentby liquidscintillationcounting(BeckmanLS5801,using ScintiVerseBD,FisherScientic). 2.4.Additivestudies Substratespecicity,co-factorrequirement,and inhibitorstudiesweredeterminedbyobservingtheeffect (i.e.,enhancement,noeffect,orinhibition,ascompared toappropriatecontrols)ofvariousadditivesonfarnesol metabolism.Enzyme(preparedasdescribedinSection 2.3)wasrstpreincubatedwithseveralconcentrations ofeachadditive(15–30minformostexperiments, exceptmetalstudies,whichwerepreincubated1–2h), thenassayedforactivity.Forallinhibitors,furtherstud- ieswereperformedtodeterminetheconcentrationof additivethatyieldeda50%lossofenzymeactivity. 2.5.K m apparentdetermination The K m apparentforfarnesoloxidationin M.sexta larvalCC–CAhomogenatewasobtainedbydouble reciprocalLineweaver–Burkplotoftheamountoffarne-