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ProteinExpressionandPuri cation34(2004)

improved

56–67

http://www.wenkuxiazai.net/locate/yprep

ImprovedexpressionandcharacterizationofCa2þ-ATPase

andphospholambaninHigh-Fivecellsq

JasonR.Waggoner,aJamieHu man,aBrianN.Gri th,a

LarryR.Jones,bandJamesE.Mahaneyc,*

a

DepartmentofBiochemistryandMolecularPharmacology,WestVirginiaUniversitySchoolofMedicine,Morgantown,WV26506-9142,USAb

DepartmentofMedicineandtheKrannertInstituteofCardiology,IndianaUniversitySchoolofMedicine,Indianapolis,IN46202,USA

c

EdwardViaVirginiaCollegeofOsteopathicMedicine,2265KraftDrive,Blacksburg,VA24060,USA

Received16August2003,andinrevisedform11November2003

Abstract

TheCa2þ-ATPaseaccountsforthemajorityofCa2þremovedfromthecytoplasmduringcardiacmusclerelaxation.TheCa2þ-ATPaseisregulatedbyphospholamban,a52aminoacidphosphoprotein,whichinhibitsCa2þ-ATPaseactivitybydecreasingtheapparenta nityoftheATPaseforCa2þ.TostudythephysicalmechanismofCa2þ-ATPaseregulationbyphospholambanusingspectroscopicandkineticexperiments,largeamountsofbothproteinsarerequired.Therefore,wedevelopedaCa2þ-ATPaseandphospholambanpreparationbasedonthebaculovirus-insectcellexpressionsystemusingHigh-FiveinsectcellstoproducelargeamountsofmicrosomalvesiclesthatcontaineitherCa2þ-ATPaseexpressedaloneorCa2þ-ATPaseco-expressedwithphospho-lamban.Theexpressedproteinswerecharacterizedusingimmuno uorescencespectroscopy,Ca2þ-ATPaseactivityassays,Ca2þuptakeande uxassays,andWesternblotting.Ourpuri cationmethodyields140mgofmicrosomalproteinperliterofinfection(1.7Â109cells),andtheCa2þ-ATPaseandphospholambanaccountfor16and1.4%,respectively,ofthetotalmicrosomalproteinbyweight,yieldingaphospholamban:Ca2þ-ATPaseratioof1.6:1,similartothatobservedinnativecardiacSRvesicles.TheenzymaticpropertiesoftheexpressedCa2þ-ATPasearealsosimilartothoseobservedinnativecardiacSRvesicles,andwhenco-expressedwithphospholamban,theCa2þ-ATPaseisfunctionallycoupledtophospholambansimilartothatobservedincardiacSRvesicles.

Ó2003ElsevierInc.Allrightsreserved.

Keywords:Ca-ATPase;Phospholamban;Baculovirus;High-Fivecells

TheCa2þ-ATPaseofcardiacsarcoplasmicreticulum(CSR)isa110kDaintegralmembraneproteinthatutilizesATPtodrivethetransportofCa2þionsintotheSRtopromotemusclerelaxation[1].IncardiacSR,thesarco(endo)plasmicreticulumCa2þ-ATPaseisoform(SERCA2a)isregulatedbyphospholamban,a52-aminoacidphosphoprotein,whichinhibitsCa2þ-ATPaseac-tivitybydecreasingtheapparenta nityoftheATPaseforCa2þ[2].Ca2þ-ATPaseinhibitionisrelievedbyphosphorylationofphospholambanatSer-16orThr-17,

ThisworkwassupportedinpartbygrantstoJ.R.W.(AHA0215055B),L.R.J.(NIHHL49428)andJ.E.M.(AHA0040094N).

*

Correspondingauthor.Fax:1-540-231-5252.

E-mailaddress:jmahaney@http://www.wenkuxiazai.net(J.E.Mahaney).1046-5928/$-seefrontmatterÓ2003ElsevierInc.Allrightsreserved.

improved

improved

doi:10.1016/j.pep.2003.11.005

q

resultinginamarkedincreaseinCa2þ-ATPaseactivity,anincreaseintherateofrelaxationofcardiacmuscle,andapositiveinotropice ect[3].

MuchattentionhasbeendevotedtounderstandingthephysicalandfunctionalinteractionbetweenCa2þ-ATPaseandphospholamban,becauseabnormalCa2þ-ATPaseregulationbyphospholambanhasbeenimplicatedasacausativefactorfordiastolicdysfunc-tion,cardiachypertrophy,andheartfailure[4].Mostrecently,Seidmanandcoworkers[5]haveshownthatanaturallyoccurringphospholambanmissensemutation(Arg!Cys)resultsinheartdiseaseinhumans.Like-wise,Kraniasandcoworkers[6]foundthathumanslackingphospholambandevelopedlethaldilatedcardiomyopathy,whichisincontrasttomice,wheredisruptionofthephospholambangeneorexpression

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