Nucleotidemix

Motor(50-100µM;purity>95%)

0.5MTris-OAc,pH7.5

10mMEGTA

10mMMgCl2

DDW

SephadexG-50Mediumcolumn(0.8cminx20cm)

Columnbuffer=

50mMTris-OAc,pH7.51mMEGTA1mMMgCl2

Scintillationfluid

Scintillationvialsanddisposableinserts

1.	Preparereactionmixes(mutantandwild-type)bymixingthefollowing:
	__µL1µL10µL10µL10µL__µL______100µL	50-100µMmotorgammaoralpha-32PATPnucleotidemix0.5MTris-OAc,pH7.510mMEGTA10mMMgCl2DDW
	Adjustthevolumeofthemotortogive5-10µMinthemixandadjustthevolumeofDDWtogivethefinalvolumeof100µL.Centrifugethemotorbeforeaddingtothemix.Incubatethemixoniceovernight.Preparereactionmixesforbothmutantandwild-typemotorsonthesameday.

2.	ApplyreactionmixtoaSephadexG-50Mediumcolumn.

3.	Collecttwo1mLfractionsandlabelthemfr1andfr2,thencollect28x5-dropfractions(each=~250µL)andlabelthemfr3-fr30.

4.	Take50µLoffractions3-22andmixwith0.5mLofscintillationfluidinadisposableinsert.Putinsertintoascintillationvial,precountedtodeterminebackgroundoflessthan30cpm,thencountthevialonthe32Pchannel.

5.	Determineproteinconcentrationofeachfractionbyadding50µLofconcentratedBio-Radreagenttotheremainderoffractions3-12(~200µL).Prepareablankbyadding50µLofconcentratedBio-Radreagentto200µLofcolumnbuffer.ReadeachfractioninamicrocuvetteatOD595.CuvettecanberinsedoutwithDDWbetweeneachreADIng.

6.	PlotfractionnumberversuscpmandOD595.

1.	ForKmdetermination,theconcentration(µM)ofboundandunboundalpha-32PATPcanbedeterminedfromthestartingATPconcentration.

2.	Co-elutionofcpmwiththeproteinpeakisinterpretedasboundADP,andunboundcpmisATP+ADP+Pi.

ModifiedfromSongandEndow1998Nature396,587-590.

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