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1、THEJOURNALOFBIOLOGICALCHEMISTRYVol.244,No.3,IssueofFebruary10,pp.755-765,1969PrintedinU.S.A.PyrophosphateBindingtoandAdenosineTriphosphataseActivityofMyosinandItsProteolyticFragmentsIMPLICATIONSFORTHESUBSTRUCTUREOFMYOSIN*(Receivedforpublication,July1,1968)KATHLEENMINIHAN~'AUSS,$SHOTAROKITAGAWA,~A
2、NDJ.GERGELYFromtheDepartmentofMuscleResearch,InstituteofBiologicalandMedicalXciences,RetinaFoundation;DepartmentofBiologicalChemistry,HarvardMedicalSchool;andDepartmentofNeurology,MassachusettsGeneralHospital,Boston,Massachusetts02Il~LSUMMARYATPaseactivitywhichwas1.86timeshigherthanthatofThebindi
3、ngofpyrophosphatetomyosin,heavymero-heavymeromyosinandacalcium-activatedATPaseactivitymyosin,andSubfragment1wasstudiedbyanequilibriumwhichwas2.60timeshigher.dialysistechnique.Myosinbindsapproximately2(1.82)Afteralo-min.trypticdigestionofheavymeromyosin,moles/5Xlo5gofprotein,K=2.07X10”.Identical2.
4、5to45%ofthetotalnitrogenwasnonprecipitablewith6%resultswereobtainedwithmyosinpreparedbytheam-trichloraceticacid.DiscelectrophoresisofSubfragment1moniumsulfateprecipitationprocedurewithorwithoutisolatedbygelfiltrationonSephadexG-200containedtwomajorbandsandseveralminoronesintheleadingedgeofLiCland
5、thedilutionmethod.Redandwhiteskeletalmusclemyosinboundthesamenumberofmolesofpyro-thepeak,whilethetrailingedgecontainedonlyonemajorbandandafewminorbands.phosphate,andvariationoftheionicstrengthupto0.6didnotaffecteitherthebindingnumberorthebindingconstant.Thesimplestexplanationoftheseresultsisthatt
6、rypticNoselectiveblockingofanyofthesitescouldbeachieveddigestionofheavymeromyosinproducestwosubfragments,withN-ethyhnaleimideorp-chloromercuribenzoate.eachofwhichbindspyrophosphate,hydrolyzesATP,andHeavymeromyosinbound1.83molesofpyrophosphatehastheabilitytocombinewithactin.per3.6Xlo5g,andSubfragm
7、ent1withamolecularweightof9.74Xlo4to1.19Xlo5bound0.85mole/lo5g,K=3.67x106.Naturalactomyosinboundonly1moleofpyrophosphateThereisgeneralagreementonanumberofstructuralfeaturesper5xlo5gofmyosin.Theadditionofactintomyosinof
