Steady State ATPase Assays Coupled Enzyme System相关交流-蚂蚁淘在线

Materials

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).

Foreachassay,addtocuvetteinthefollowingorder:

₂

Mixquicklywithaplasticprobe,thenaddthefollowing:

(Note:thevolumeofmotorandMTsusedwillvarywitheachassay.Clickhereforanexample.)

Mixquickly,putintospectrophometer,closechamberdoorandpush"start"button.

7.	Collectdatafor300seconds,thenremovecuvetteandwashwellwithDDW.Inverttodrypriortonextassay.Alternateuseof4cuvettes.

DeterminethechangeinOD₃₄₀perminuteaftersteadystatehasbeenreached,typicallyafterthefirst60seconds.ConverttoµMbydividingthechangeinOD₃₄₀/minby6.22x10^-3/µMforNADH.ThendividebythemotorconcentrationinµM.

Divideby60s/mintoconverttok_cat(s^-1).ThecontrolwithoutmotororMTsisthebackgroundandshouldbesubtractedfromallotherk_catvalues.ThecontrolswithMTsbutwithoutmotorgivethenucleotidehydrolysisbyMTsandshouldbesubtractedfromcorrespondingvalueswithmotorandthesameconcentrationofMTs.

Thek_catvalues(s^-1)canbeplottedvsMTconcentrationandthedatapointsfitwiththeMichaelis-MentonequationusingaprogramsuchasKaleidagraph.Theequationforthecurvefitwherey=k_catandx=[MTs]is:

y=m1*x/(m2+x)

m1=V_maxandm2=K_M,MTs

Theinitialvaluesofm1=1,m2=1canbeenteredtoapproximatethevaluesofm1andm2.

Notes

1.	Assaysaresensitivetosomebuffersandwillnotwork,forexample,inTris-HCl.AvoidusingphosphatebuffersforATPaseassays.

ThecoupledenzymeATPaseassayisbasedontheconversionofphosphoenolpyruvate(PEP)topyruvatebypyruvatekinase(PK)coupledtotheconversionofpyruvatetolactatebylactatedehydrogenase(LDH).ThelattersteprequiresNADHwhichisoxidizedtoNAD⁺.NADHabsorbsstronglyat340nmbutNAD⁺doesnot,enablingtheultilizationofNADHtobefollowedbymonitoringabsorbanceat340nm.ThedecreaseinOD₃₄₀canbeconvertedintoATPaseactivitywhere1moleculeofNADHoxidizedtoNAD⁺correspondstotheproductionof1moleculeofADPbythemotorATPase.

ModifiedfromHuang&Hackney(JBC269,16493-5011994)

IfyouusemethodsfromtheKinesinsite,weaskthatyoucitetheKinesinHomePageandauthors,ortheappropriatesourcepublicationinyourwork.

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