Margatoxin(MgTx)isacomponentofthevenomofScorpioCentruroidesmargaritatus.Margatoxinpreferentiallyinhibitsvoltage-dependentpotassiumchannelsKv1.3withanIC50valuearound50pM(20foldmorepotentthanCharyBDotoxin)andirreversIBLyinhibitstheproliferationresponseofhumanT-cellsat20µMconcentration.MargatoxinisknowntobelesspotentonKv1.3expressedinXenopusOocytes(IC50around1nM).MargatoxinwasalsodescribedtobeapotentinhibitorofhumanvascularsmoothmusclecellmigrationwithanIC50of85pM.
FreesampleDescription:
AAsequence:Thr-Ile-Ile-Asn-Val-Lys-Cys7-Thr-Ser-Pro-Lys-Gln-Cys13-Leu-Pro-Pro-Cys17-Lys-Ala-Gln-Phe-Gly-Gln-Ser-Ala-Gly-Ala-Lys-Cys29-Met-Asn-Gly-Lys-Cys34-Lys-Cys36-Tyr-Pro-His-OH
(DisulfidebondsbetweenCys7-Cys29,Cys13-Cys34andCys17-Cys36)
Length(aa):39
Formula:C178H286N52O50S7
MolecularWeight:4179.03Da
Appearance:Whitelyophilizedsolid
Solubility:waterandsalinebuffer
CASnumber:[145808-47-5]Source:Synthetic
Purityrate:>97%
Reference:
PotentsuppressionofvascularsmoothmusclecellmigrationandhumanneointimalhyperplasiabyKV1.3channelblockers
AIM:
TheaimofthestudywastodeterminethepotentialforK(V)1potassiumchannelblockersasinhibitorsofhumanneoinitimalhyperplasia.
METHODSANDRESULTS:
Bloodvesselswereobtainedfrompatientsormiceandstudiedinculture.Reversetranscriptase-polymerasechainreactionandimmunocytochemistrywereusedtodetectgeneexpression.Whole-cellpatch-clamp,intracellularcalciummeasurement,cellmigrationassays,andorganculturewereusedtoassesschannelfunction.K(V)1.3wasuniqueamongtheK(V)1channelsinshowingpreservedandup-regulatedexpressionwhenthevascularsmoothmusclecellsswitchedtotheproliferatingphenotype.Therewasstrongexpressioninneointimalformations.Voltage-dependentpotassiumcurrentinproliferatingcellswassensitivetothreedifferentblockersofK(V)1.3channels.Calciumentrywasalsoinhibited.Allthreeblockersreducedvascularsmoothmusclecellmigrationandtheeffectswerenon-additive.Oneoftheblockers(margatoxin)washighlypotent,suppressingcellmigrationwithanIC(50)of85pM.Twooftheblockersweretestedinorgan-culturedhumanveinsamplesandbothinhibitedneointimalhyperplasia.
CONCLUSION:
K(V)1.3potassiumchannelsarefunctionalinproliferatingmouseandhumanvascularsmoothmusclecellsandhavepositiveeffectsoncellmigration.Blockersofthechannelsmaybeusefulasinhibitorsofneointimalhyperplasiaandotherunwantedvascularremodellingevents.
CheongA.,etal.(2011)PotentsuppressionofvascularsmoothmusclecellmigrationandhumanneointimalhyperplasiabyKV1.3channelblockers.CardiovascRes.PMID20884640
Kv1.3channelsinpostganglionicsympatheticneurons:expression,function,andmodulation
DocziMA.etal.(2008)Kv1.3channelsinpostganglionicsympatheticneurons:expression,function,andmodulation.AmJPhysiolRegulIntegrCompPhysiol.PMID18614767
PotentsuppressionofKv1.3potassiumchannelandIL-2secretionbydiphenylphosphineoxide-1inhumanTcells
ZhaoN.,etal.(2013)PotentsuppressionofKv1.3potassiumchannelandIL-2secretionbydiphenylphosphineoxide-1inhumanTcells.PLoSOne.PMID23717641
TheeffectsofKv1.3andIKCa1potassiumchannelinhibitiononcalciuminfluxofhumanperipheralTlymphocytesinrheumatoidarthritis
OBJECTIVE:
Thetransientincreaseofthecytoplasmicfreecalciumlevelplaysakeyroleintheprocessoflymphocyteactivation.Kv1.3andIKCa1potassiumchannelsareimportantregulatorsofthemaintenanceofcalciuminfluxduringlymphocyteactivationandpresentapossibletargetforselectiveimmunomodulation.
DESIGN:
Case-controlstudy.
SUBJECTSANDMETHODS:
Wetookperipheralbloodsamplesfrom10healthyindividualsand9recentlydiagnosedrheumatoidarthritis(RA)patientsreceivingnoanti-rheumatictreatment.WeevaluatedcalciuminfluxkineticsfollowingactivationinCD4,Th1,Th2andCD8cellsapplyinganovelflowcytometryapproach.WealsoassessedthesensitivityoftheabovesubsetstospecificinhibitionoftheKv1.3andIKCa1potassiumchannels.
RESULTS:
ThepeakofcalciuminfluxinlymphocytesisolatedfromRApatientsisreachedmorerapidly,indicatingthattheyrespondmorequicklytostimulationcomparedtocontrols.Inhealthyindividuals,theinhibitionoftheIKCa1channeldecreasedcalciuminfluxinTh2andCD4cellstoalowerextentthaninTh1andCD8cells.Onthecontrary,theinhibitionofKv1.3channelsresultedinalargerdecreaseofcalciumentryinTh2andCD4thaninTh1andCD8cells.NodifferencewasdetectedbetweenTh1andTh2orCD4andCD8cellsinthesensitivitytoIKCa1channelinhibitionamonglymphocytesofRApatients.However,specificinhibitionoftheKv1.3channelactsdifferentiallyoncalciuminfluxkineticsinRAlymphocytesubsets.Th2andparticularlyCD8cellsareinhibitedmoredominantlythanTh1andCD4cells.
CONCLUSION:
TheinhibitionofKv1.3channelsdoesnotseemtobespecificenoughinperipheralRAlymphocytes,sinceanti-inflammatoryTh2cellsarealsoaffectedtoanoteworthyextent.
ToldiG.,etal.(2013)TheeffectsofKv1.3andIKCa1potassiumchannelinhibitiononcalciuminfluxofhumanperipheralTlymphocytesinrheumatoidarthritis.ImmunoBIOLOGy.PMID22705192
OverexpressionofDelayedRectifierK(+)ChannelsPromotesInsituProliferationofLeukocytesinRatKidneyswithAdvancedChronicRenalFailure
Leukocytes,suchaslymphocytesandmacrophages,predominantlyexpressdelayedrectifierK(+)channels(Kv1.3),andthechannelsplaycrucialrolesintheactivationandproliferationofthecells.Sincelymphocytesareactivatedinpatientswithend-stagerenaldisease(ESRD),thechannelsexpressedinthosecellswouldcontributetotheprogressionofrenalfibrosisinadvanced-stagechronicrenalfailure(CRF).Inthepresentstudy,usingaratmodelwithadvancedCRFthatunderwent5/6nephrectomyfollowedbya14-weekrecoveryperiod,weexaminedthehistopathologicalfeaturesofthekidneysandtheleukocyteexpressionofKv1.3-channelsandcellcycleMarkers.Age-matchedsham-operatedratswereusedascontrols.InthecorticalinterstitiumofadvancedCRFratkidneys,leukocytesproliferatedinsituandoverexpressedKv1.3channelproteinintheircytoplasm.Treatmentwithmargatoxin,aselectiveKv1.3-channelinhibitor,significantlysuppressedthenumberofleukocytesandtheprogressionofrenalfibrosiswithasignificantdecreaseinthecorticalcellcyclemarkerexpression.ThisstudydemonstratedforthefirsttimethatthenumberofleukocyteswasdramaticallyincreasedinratkidneyswithadvancedCRF.TheoverexpressionofKv1.3channelsintheleukocyteswasthoughttocontributetotheprogressionofrenalfibrosisbystimulatingcellcyclingandpromotingcellularproliferation.
KazamaI.,etal.(2012)OverexpressionofDelayedRectifierK(+)ChannelsPromotesInsituProliferationofLeukocytesinRatKidneyswithAdvancedChronicRenalFailure.IntJNephrol.PMID22701172
CharacteristicsofACh-inducedhyperpolarizationandrelaxationinrabbitjugularvein
BACKGROUNDANDPURPOSE:
Therolesplayedbyendothelium-derivedNOandprostacyclinandbyendothelialcellhyperpolarizationinACh-inducedrelaxationhavebeenwellcharacterizedinarteries.However,themechanismsunderlyingACh-inducedrelaxationinveinsremaintobefullyclarified.
EXPERIMENTALAPPROACH:
ACh-inducedsmoothmusclecell(SMC)hyperpolarizationandrelaxationweremeasuredinendothelium-intactand-denudedpreparationsofrabbitjugularvein.
KEYRESULTS:
Inendothelium-intactpreparations,ACh(≤10⁻⁸M)marginallyincreasedtheintracellularconcentrationofCa²⁺([Ca²⁺](i))inendothelialcellsbutdidnotaltertheSMCmembranepotential.However,ACh(10⁻¹⁰-10⁻⁸M)inducedaconcentration-dependentrelaxationduringthecontractioninducedbyPGF(2α)andthisrelaxationwasblockedbytheNOsynthaseinhibitorN(ω)-nitro-l-arginine.ACh(10⁻⁸-10⁻⁶M)concentration-dependentlyincreasedendothelial[Ca²⁺](i)andinducedSMChyperpolarizationandrelaxation.TheseSMCresponseswereblockedinthecombinedpresenceofapamin[blockerofsmall-conductanceCa²⁺-activatedK⁺(SK(Ca),K(Ca)2.3)channel],TRAM34[blockerofintermediate-conductanceCa²⁺-activatedK⁺(IK(Ca),K(Ca)3.1)channel]andmargatoxin[blockerofsubfamilyofvoltage-gatedK⁺(K(V))channel,K(V)1].
CONCLUSIONSANDIMPLICATIONS:
Inrabbitjugularvein,NOplaysaprimaryroleinendothelium-dependentrelaxationatverylowconcentrationsofACh(10⁻¹⁰-10⁻⁸M).Athigherconcentrations,ACh(10⁻⁸-3×10⁻⁶M)inducesSMChyperpolarizationthroughactivationofendothelialIK(Ca),K(V)1and(possibly)SK(Ca)channelsandproducesrelaxation.TheseresultsimplythatAChregulatesrabbitjugularveintonusthroughactivationoftwoendothelium-dependentregulatorymechanisms.
ItohT.etal.(2012)CharacteristicsofACh-inducedhyperpolarizationandrelaxationinrabbitjugularvein.BrJPharmacol.PMID22595036
Charybdotoxinandmargatoxinactingonthehumanvoltage-gatedpotassiumchannelhKv1.3anditsH399Nmutant:anexperimentalandcomputationalcomparison
Theeffectofthepore-blockingpeptidescharybdotoxinandmargatoxin,bothscorpiontoxins,oncurrentsthroughhumanvoltage-gatedhK(v)1.3wild-typeandhK(v)1.3_H399Nmutantpotassiumchannelswascharacterizedbythewhole-cellpatchclamptechnique.Inthemutantchannels,bothtoxinshardlyblockedcurrentthroughthechannels,althoughtheydidpreventC-typeinactivationbyslowingdownthecurrentdecayduringdepolarization.Moleculardynamicssimulationssuggestedthatthefastcurrentdecayinthemutantchannelwasaconsequenceofaminoacidreorientationsbehindtheselectivityfilterandindicatedthattherigidity-flexibilityinthatregionplayedakeyroleinitsinteractionswithscorpiontoxins.Achannelwithaslightlymoreflexibleselectivityfilterregionexhibitsdistinctinteractionswithscorpiontoxins.Ourstudiessuggestthatthetoxin-channelinteractionsmightpartiallyrestorerigidityintheselectivityfilterandtherebypreventthestructuralrearrangementsassociatedwithC-typeinactivation.
NikoueeA.etal.(2012)Charybdotoxinandmargatoxinactingonthehumanvoltage-gatedpotassiumchannelhKv1.3anditsH399Nmutant:anexperimentalandcomputationalcomparison.JPhysChemB.PMID22490327
Voltage-dependentbiphasiceffectsofchloroquineondelayedrectifierK(+)-channelcurrentsinmurinethymocytes
LymphocytesareofrichindelayedrectifierK(+)-channels(Kv1.3)intheirplasmamembranes,andthechannelsplaycrucialrolesinthelymphocyteactivationandproliferation.Sincechloroquine,awidelyusedanti-malarialdrug,exertsimmunosuppressiveeffects,itwillaffectthechannelcurrentsinlymphocytes.Inthepresentstudy,employingthestandardpatch-clampwhole-cellrecordingtechnique,weexaminedtheeffectsofchloroquineonthechannelsexpressedinmurinethymocytes.Publishedpapersreportthatchloroquinewillinhibitvoltage-dependentK(+)-channelcurrentsbypluggingintotheopen-pore.Weobserved,indeed,thatchloroquinesuppressedthepulse-endcurrentsofKv1.3-channelsathighervoltagesteps.Surprisingly,however,wefoundthatthedrugenhancedthepeakcurrentsatbothhigherandlowervoltagesteps.SincechloroquineshowedsuchbiphasiceffectsonthethymocyteK(+)-channels,andsincethoseeffectswerevoltagedependent,weexaminedtheeffectsofchloroquineontheactivationandtheinactivationofthechannelcurrents.Wenotedthatchloroquineshiftedboththeactivationandtheinactivationcurvestowardthehyperpolarizingpotential,andthatthoseshiftsweremoreemphasizedatlowervoltagesteps.WeconcludethatchloroquinefacilitatesboththeactivationandtheinactivationofKv1.3-channelcurrentsinthymocytes,andthatthoseeffectsarevoltagedependent.
KazamaI.etal.(2012)Voltage-dependentbiphasiceffectsofchloroquineondelayedrectifierK(+)-channelcurrentsinmurinethymocytes.JPhysiolSci.PMID22328488
DifferentpotassiumchannelsareinvolvedinrelaxationofratrenalarteryinducedbyP1075
TheATP-sensitiveK(+)channelsopener(K(ATP)CO),P1075[N-cyano-N’-(1,1-dimethylpropyl)-N″-3-pyridylguanidine],hasbeenshowntocauserelaxationofvariousisolatedanimalandhumanbloodvesselsbyopeningofvascularsmoothmuscleATP-sensitiveK(+)(K(ATP))channels.Inadditiontothewell-knowneffectontheopeningofK(ATP)channels,ithasbeenreportedthatvasorelaxationinducedbysomeoftheK(ATP)COsincludessomeotherK(+)channelsubtypes.GiventhatthereisstillnoinformationonothertypesofK(+)channelspossiblyinvolvedinthemechanismofrelaxationinducedbyP1075,thisstudywasdesignedtoexaminetheeffectsofP1075ontheratrenalarterywithendotheliumandwithdenudedendotheliumandtodefinethecontributionofdifferentK(+)channelsubtypesintheP1075actiononthisbloodvessel.OurresultsshowthatP1075inducedaconcentration-dependentrelaxationofratrenalarteryringspre-contractedbyphenylephrine.Glibenclamide,aselectiveK(ATP)channelsinhibitor,partlyantagonizedtherelaxationofratrenalarteryinducedbyP1075.Tetraethylammonium(TEA),anon-selectiveinhibitorofCa(2+)-activatedK(+)channels,aswellasiberiotoxin,amostselectiveblockeroflarge-conductanceCa(2+)-activatedK(+)(BK(Ca))channels,didnotabolishtheeffectofP1075onratrenalartery.Incontrast,anon-selectiveblockerofvoltage-gatedK(+)(K(V))channels,4-aminopyridine(4-AP),aswellasmargatoxin,apotentinhibitorofK(V)1.3channels,causedpartialinhibitionoftheP1075-inducedrelaxationofratrenalartery.Inaddition,inthisstudy,P1075relaxedcontractionsinducedby20mMK(+),buthadnoeffectoncontractionsinducedby80mMK(+).OurresultsshowedthatP1075inducedstrongendothelium-independentrelaxationofratrenalartery.ItseemsthatK(ATP),4-AP-andmargatoxin-sensitiveK(+)channelslocatedinvascularsmoothmusclemediatedtherelaxationofratrenalarteryinducedbyP1075.
NovakovicA.,etal.(2012)DifferentpotassiumchannelsareinvolvedinrelaxationofratrenalarteryinducedbyP1075.BasicClinPharmacolToxicol.PMID22225832
Chemicalsynthesisandstructure-functionstudiesofmargatoxin,apotentinhibitorofvoltage-dependentpotassiumchannelinhumanTlymphocytes
The39aminoacidpeptide,margatoxin(MgTX),apotentinhibitorofthevoltage-activatedpotassiumchannel(Kv1.3)inhumanTlymphocytes,wassynthesizedbyasolidphasetechnique.FormationofthedisulfidebridgeswasrapidatpH8.2.Thefinalproductwaspurifiedtohomogeneityandwasphysicallyandbiologicallyindistinguishablefromthetoxinpreparedbiosynthetically.Thedisulfidebridgepairingwassimilartothatfoundpreviouslyfortherelatedtoxin-charybdotoxin(3):fromCys7toCys29,fromtestedforinhibitionof125Imargatoxinbindingtovoltage-activatedpotassiumchannels.TheresultsindicatethatthethreeC-terminalresiduesofMgTXareimportantfortheefficienttoxinbindingtoKv1.3.
Bednarek,M.A.,etal.(1994)Chemicalsynthesisandstructure-functionstudiesofmargatoxin,apotentinhibitorofvoltage-dependentpotassiumchannelinhumanTlymphocytes,BiochemBiophysResCommun.PMID: 8297371
Determinationofthethree-dimensionalstructureofmargatoxinby1H,13C,15Ntriple-resonancenuclearmagneticresonancespectroscopy
Thesolution structure ofthe39-residuepeptide margatoxin,ascorpiontoxinthatselectivelyblocksthevoltage-gatedpotassium-channelKv1.3,hasbeendeterminedbyNMR spectroscopy.Thetoxinwasisotopicallylabeledwith 13C and 15N andstudiedusingtwo-dimensionalhomonuclearandthree-andfour-dimensionalheteronuclearNMR spectroscopy.Thefinal structure wasdeterminedusing501constraints,comprising422NOEconstraints,60dihedralangleconstraints,9disulfideconstraints,and10hydrogenbondconstraints.StructureswereinitiallydeterminedwiththeprogramPEGASUSandsubsequentlyrefinedwithX-PLOR.Theaveragermsdeviationfromacalculatedaverage structure forthebackboneatomsofresidues3-38is0.40A.Ahelixispresentfromresidues11to20andincludestwoprolineresiduesatpositions15and16.Aloopatresidues21-24leadsintoatwo-strandantiparallelsheetfromresidues25to38withaturnatresidues30-33.Residues3-6runadjacenttothe33-38strandbutdonotformacanonicalbeta-strand.Thetwoadditionalresiduesof margatoxin,relativetotherelatedtoxinscharybdotoxinandiberiotoxin,insertinamannerthatextendsthebeta-sheetbyoneresidue.Otherwise,theglobal structure isverysimilartothatofthesetwoothertoxins.Thelongersheetmayhaveimplicationsforchannelselectivity.
Johnson,B.A.,etal.(1994)Determinationofthethree-dimensionalstructureofmargatoxinby1H,13C,15Ntriple-resonancenuclearmagneticresonancespectroscopy,Biochemistry.PMID: 7999764
Purification,characterization,andbiosynthesisofmargatoxin,acomponentofCentruroidesmargaritatusvenomthatselectivelyinhibitsvoltage-dependentpotassiumchannels
AnovelpeptidylinhibitorofK+channelshasbeenpurifiedtohomogeneityfromvenomofthenewworldscorpionCentruroidesmargaritatus.Theprimarystructureofthis39-amino-acidpeptide,whichwetermmargatoxin(MgTX),wasdeterminedbyaminoacidcompositionalanalysisandpeptidesequencing.MargatoxinpotentlyinhibitsbindingofrADIolabeledcharybdotoxin(ChTX)tovoltage-activatedchannelsinbrainsynapticplasmamembranes.LikeChTX,MgTXblocksthen-typecurrentofhumanT-lymphocytes(Kv1.3channel),butcomparedtoChTX,is20-foldmorepotent(half-blockatapproximately50pM),hasaslowerdissociationrate,andhasnoeffectoncalcium-activatedchannels.Todemonstratethatthesecharacteristicsareduesolelytothepurifiedtoxin,recombinantMgTXwasexpressedinEscherichiacoliaspartofafusionprotein.Aftercleavageandfolding,purifiedrecombinantMgTXdisplayedthesamepropertiesasnativepeptide.ReplacementoftheCOOH-terminalhistidineresidueofMgTXwithasparagineresultedinapeptidewitha10-foldreductioninpotency.Thiswasduetoafasterapparentdissociationrate,suggestingthattheCOOH-terminalaminoacidmayplayanimportantroleinthebindingofMgTXtotheKv1.3channel.MgTXdisplayssignificantsequencehomologywithpreviouslyidentifiedK+channelinhibitors(e.g.ChTX,iberiotoxin,noxiustoxin,andkaliotoxin).However,givenitspotencyanduniqueselectivity,MgTXrepresentsanespeciallyusefultoolwithwhichtostudythephysiologicroleofKv1.3channels.
Garcia-Calvo,etal.(1993)Purification,characterization,andbiosynthesisofmargatoxin,acomponentofCentruroidesmargaritatusvenomthatselectivelyinhibitsvoltage-dependentpotassiumchannels,JBiolChem. PMID: 8360176
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常用流动相加酸碱后PH的总结，希望大家能够提供一点自己测过的结果，谢谢先
1.直接用固体磷酸钠配制成50mM的磷酸钠溶液，再调pH到7.4；（我们试着用这个做了下，发现挂不上柱）
2.配置磷酸钠盐缓冲液：按NaH2PO4：Na2HPO4以19：81的摩尔比配制成pH7.4的缓冲液？（附一张百度出来的配方
）
3.如果是磷酸钠盐缓冲液，可以直接将50mM的NaH2PO4的水溶液用NaOH调成pH7.4吗？
再者，2和3这两个方法配制的磷酸钠盐缓冲液有什么区别？最终效果是一样的吗？如果不一样，有什么理论的知识支撑呢？个人感觉是分析化学中酸碱理论中的缓冲液那里的知识。求帮忙解答这些疑问。
另外，我还想问一下，pH对于Ni柱对His-tagged的蛋白的分离纯化影响大吗？是怎么影响的？谢谢大家了！
有了源数据之后把源数据按照大小排列，
选中源数据区域-->ALT+A1-->选中图标区右键-->更改图表类型-->散点图
因为是考察不同PH对药物的影响，样品又不好改变其PH值，这种情况怎么办？希望有经验的高手指教。
我的流动相是甲醇-水（90：10）
谢谢赐教！
请进子版按格式发贴，自行修改，谢谢。
由弱酸及其盐、弱碱及其盐组成的混合溶液，能在一定程度上抵消、减轻外加强酸或强碱对溶液酸碱度的影响，从而保持溶液的pH值相对稳定。这种溶液称为缓冲溶液。
:)
我在做一细菌不同酸碱度生长状况时,发现这些奇怪现象:pH=3的培养基灭菌(TSB液体培养基)灭菌后pH上升到到9.2!而原来pH=9.0的降到8.7(基本没多少变化),请问各位大侠,这是什么原因?
一般做不同酸碱度生长实验时,该如何才能防止pH在湿热灭菌后基本不变化?
是否可以理解为纯化水得PH范围为6.3-7.6？能否直接用pH计测量？谢谢!
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