Cellpelletswerethawedonice,resUSPendedin:

*10mMTris-HCl(pH7.4)/150mMKCL/1mMEDTA/2mMDithiothreitol(DTT)atproteinconcentrationof3-6mg/ml,andsnap-frozenondryice.Afterthawingat37°C,theextractswerechilledoniceandsonicatedfor30sec.

AtypicalassayforestablishtheapparentVmaxandKmoftheSubstrateANDROST-4-ENE-3,17-DIONEofthenormal&mutantHSD17B3enzymeused:

*20-30microgramoftotalproteinin0.2mLof100mMTris-citrate(pH6)/2mMDTT.

*Steroidsubstratewasaddedindifferentconcent.(0.1-8microM),andNADPHwasaddedtoafinalcocent.of2mM.

*Reactionswereinitiatedbytheadditionofenzymeandwerecarriedoutfor25’-30’minat37°C.

AtypicalassaytoestablishtheapparentpHofthenormal&mutantHSD17B3enzymeused:

*SerialvariationofpH(range4.5pH-8.5pH),forthereactionBuffer:100mMTris-citrate/2mMDTT.

*Steroidsubstratewasaddedatfinalconcent.of5mM(where1mMwere14C-ANDROST-4-ENE-3,17-DIONEand4mMwerecoldsubstrate).

TransfectionofDNAinto293TcellsbyusingLIPOFECTAMINEPLUSReagentpackage(LIFETECHNOLOGIESCat.No.10964-013):Protocol

*Thedaybeforetransfection,splitandplatingthecells,sothethattheyare50%-60%confluentthedayoftransfection.Avoidantibioticsatthetimeoftransfectionandduring.

*Culturevessel:100mm

*plasmidDNA;4microg*plasmidb-gal;1microg

*PLUSreagent;20microliter

*LIPOFECTAMINEPLUSReagent;30microliter

*Incubateat37°Cat5%CO2for3h(seeLIFETECHNOLOGIESprotocol.)

*After3hinc.,increasevolumeofmediumtonormalvolume(seeLIFETECHNOLOGIESprotocol.)

*Thecellextractwereassayedforreportergeneactivity48hafterthestartoftransfection.

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