Tubulin(>5mg/mL)

100mMMg·GTP

4mMTaxolinDMSO

PM=	100mMPIPESpH6.82mMEGTA1mMMg2SO4

Motorprotein(>95%purity;15-20µM)

Cuvettes(200µLvolume,10mmpathlength)

0.5MTris-OAc,pH7.5

10mMMgCl2

10mMDTT

DDW

10mMMg·ATP

30mMPEP

6mMNADH(Makefreshin10mMTris-OAcpH7.5,4.26mg/mL=6mM)

PK/LDH(Sigma#P-0294,PK+LDHfromRabbitMusclein50%glycerol)

Procedure

1.	Assemblemicrotubulesforassays.
	E.g.,	58µLof50µMMTs=50µL5.8mg/mLtubulin+0.5µL100mMMg·GTP,incubatefor30-60minat37°C,thenadd7.5µL544µMTaxolinPM(1.02µL4mMTaxol+6.5µLPM)at37°C.

2.	DetermineproteinconcentrationusingtheBradfordassay.

3.	SetupspectrophotometertorecordOD340at10secondintervalsfor300seconds.Aratefactorof6.220x106cm2at340nm=molarextinctioncoefficientofNADHcanbeenteredintothedatacollectionprogram.

4.	Washandinvert4cuvettestodry.

5.	MakeTris-MgCl2-DTTmixforallassays.

Tris-MgCl2-DTTmix=(n=#ofassays+1)

nx20µL0.5MTris-OAc,pH7.520µL10mMMgCl220µL10mMDTT70µLDDW

Addtaxolto10µM(finalconcentrationinassay=6.5µM).

6.

Foreachassay,addtocuvetteinthefollowingorder: DDW=19.5µL-(volumeofmotor+MTs)130µLTris-MgCl2-DTT20µL10mMMg·ATP20µL30mMPEPinDDW7µL6mMNADH3.5µLPK/LDH Mixquicklywithaplasticprobe,thenaddthefollowing: ___µLmotor___µLMTs (Note:thevolumeofmotorandMTsusedwillvarywitheachassay.Clickhereforanexample.) Mixquickly,putintospectrophometer,closechamberdoorandpush"start"button. 7. Collectdatafor300seconds,thenremovecuvetteandwashwellwithDDW.Inverttodrypriortonextassay.Alternateuseof4cuvettes. 8. DeterminethechangeinOD340perminuteaftersteadystatehasbeenreached,typicallyafterthefirst60seconds.ConverttoµMbydividingthechangeinOD340/minby6.22x10-3/µMforNADH.ThendividebythemotorconcentrationinµM. Divideby60s/mintoconverttokcat(s-1).ThecontrolwithoutmotororMTsisthebackgroundandshouldbesubtractedfromallotherkcatvalues.ThecontrolswithMTsbutwithoutmotorgivethenucleotidehydrolysisbyMTsandshouldbesubtractedfromcorrespondingvalueswithmotorandthesameconcentrationofMTs. Thekcatvalues(s-1)canbeplottedvsMTconcentrationandthedatapointsfitwiththeMichaelis-MentonequationusingaprogramsuchasKaleidagraph.Theequationforthecurvefitwherey=kcatandx=[MTs]is: y=m1*x/(m2+x) m1=Vmaxandm2=KM,MTs Theinitialvaluesofm1=1,m2=1canbeenteredtoapproximatethevaluesofm1andm2. Notes 1. Assaysaresensitivetosomebuffersandwillnotwork,forexample,inTris-HCl.AvoidusingphosphatebuffersforATPaseassays. 2. ThecoupledenzymeATPaseassayisbasedontheconversionofphosphoenolpyruvate(PEP)topyruvatebypyruvatekinase(PK)coupledtotheconversionofpyruvatetolactatebylactatedehydrogenase(LDH).ThelattersteprequiresNADHwhichisoxidizedtoNAD+.NADHabsorbsstronglyat340nmbutNAD+doesnot,enablingtheultilizationofNADHtobefollowedbymonitoringabsorbanceat340nm.ThedecreaseinOD340canbeconvertedintoATPaseactivitywhere1moleculeofNADHoxidizedtoNAD+correspondstotheproductionof1moleculeofADPbythemotorATPase. ModifiedfromHuang&Hackney(JBC269,16493-5011994) IfyouusemethodsfromtheKinesinsite,weaskthatyoucitetheKinesinHomePageandauthors,ortheappropriatesourcepublicationinyourwork.