Muscarinictoxin7(MT7–m1-toxin1)hasbeenisolatedfromthevenomofthegreenmamba(DendroaspisAngusticeps).MT7potentlyblocksM1-subtypeofmuscarinicacetylcholinereceptorsatasubnanomolaraffintiy.MuscarinicacetylcholinereceptorsareG-proteincoupledreceptorsthatmediatethemetabotropiceffectsofacetylcholine.M1-typemuscarinicacetylcholinereceptorsarewellknowntherapeutictargetstoimprovecognitivefunctionsinpatientswithAlzheimerdisease.ContrarytomanyligandsthattargetmAChRs(pirenzepine,carbamoylcholinechloride,4-DAMPoratropine),MT7isthemostselectiveM1-subtypeantagoNISTasitisabout10,000timesmoreselectiveforM1-subtypethanothersubtypes.MT7toxinisanidealtooltoidentifythemuscarinicreceptorsubtypeexpressionintissuesforexample.MT7toxinbelongstothethreefingertoxinfamilysuchasrho-Da1a.
Recentlyquoted
Description:
AAsequence:LTC3VKSNSIWFPTSEDC17PDGQNLC24FKRWQYISPRMYDFTRGC42AATC46PKAEYRDVINC57C58GTDKC63NK
Disulfidebonds:Cys3-Cys24,Cys17-Cys42,Cys46-Cys57,Cys58-Cys63
Length(aa):65
Formula:C322H484N90O98S9
MolecularWeight: 7472.53Da
Appearance:Whitelyophilizedsolid
Solubility: waterandsalinebuffer
CASnumber: notavailable
Source:synthetic
Purityrate: >98%
Reference:
Structuraldeterminantsfortheinteractionsbetweenmuscarinictoxin7andmuscarinicacetylcholinereceptors
XuJ.,etal.(2015)Structuraldeterminantsfortheinteractionsbetweenmuscarinictoxin7andmuscarinicacetylcholinereceptors.JMolRecognit.PMID:25683330
Muscarinicacetylcholinereceptors(mAChRs)havefivesubtypesandplaycrucialrolesinvariousphysiologicalfunctionsandpathophysiologicalprocesses.PoorsubtypespecificityofmAChRmodulatorshasbeenanobstacletodiscovernewtherapeuticagents.Muscarinictoxin7(MT7)isanaturalpeptidetoxinwithhighselectivityfortheM1receptor.Withthreetofiveresiduessubstituted,M3,M4,andM5receptormutantscouldbindtoMT7atnanomolarconcentrationastheM1receptor.However,thestructuralmechanismsexplainingMT7-mAChRsbindingarestilllargelyunknown.Inthisstudy,weconstructed10complexmodelsofMT7andeachmAChRsubtypeoritsmutant,performedmoleculardynamicssimulations,andcalculatedthebindingenergiestoinvestigatethemechanisms.OurresultssuggestedthatthestructuraldeterminantsfortheinteractionsonmAChRswerecomposedofsomecriticalresidueslocatedseparatelyintheextracellularloopsofmAChRs,suchasGlu4.56,Leu4.60,Glu/Gln4.63,Tyr4.65,Glu/Asp6.67,andTrp7.35.ThesubtypespecificityofMT7wasattributedtothenon-conservedresiduesatpositions4.56and6.67.ThesestructuralmechanismscouldfacilitatethediscoveryofnovelmAChRmodulatorswithhighsubtypespecificityandenhancetheunderstandingoftheinteractionsbetweenligandsandG-protein-coupledreceptors.
MolecularconversionofmuscarinicAcetylcholineReceptorM5toMuscarinicToxin(MT7)bindingprotein
RondinelliS.,etal.(2011)MolecularconversionofmuscarinicAcetylcholineReceptorM5toMuscarinicToxin(MT7)bindingprotein.Toxins.PMID:22174976
Muscarinictoxin7(MT7)isamambavenompeptidethatbindsselectivelytotheM(1)muscarinicacetylcholinereceptor.Wehavepreviouslyshownthatthesecond(ECL2)andthird(ECL3)extracellularloopsoftheM(1)receptorarecriticallyinvolvedinbindingthepeptide.InthisstudyweusedamutagenesisapproachontheM(5)subtypeofthereceptorfamilytofindoutifthispossessesasimilarstructuralarchitectureintermsoftoxinbindingastheM(1)receptor.AnM(5)receptorconstruct(M(5)-E(175)Y(184)E(474)),mutatedattheformerlydecipheredcriticalresiduesonECL2and3,gainedtheABIlitytobindMT7,butwithratherlowaffinityasdeterminedinafunctionalassay(apparentK(i)=24nM;apparentK(i)forM(1)=0.5nM).Afterscreeningfordifferentdomainsandresidues,wefoundaspecificresidue(P(179)toLinM(5))inthemiddleportionofECL2thatwasnecessaryforhighaffinitybindingofMT7(M(5)-EL(179)YE,apparentK(i)=0.5nM).MutationofP(179)toAconfirmedarolefortheleucinesidechaininthebindingofMT7.TogethertheresultsrevealnewbindinginteractionsbetweenreceptorsandtheMT7peptideandstrengthenthehypothesisthatECL2sequenceisofutmostimportanceforMTbindingtomuscarinicreceptors.
DifferentinteractionbetweenMT7toxinandthehumanmuscarinicM1ReceptorinitsfreesanN-Methylscopolamineoccupiedstates.
Fruchart-GaillardC.,etal.(2008)DifferentinteractionbetweenMT7toxinandthehumanmuscarinicM1ReceptorinitsfreesanN-Methylscopolamineoccupiedstates. MolPharmacol.PMID:18784346
MuscarinicMT7toxinisahighlyselectiveandpotentantagonistoftheM(1)subtypeofmuscarinicreceptorandactsbybindingtoanallostericsite.ToidentifythemoleculardeterminantsbywhichMT7toxininteractswiththisreceptorinitsfreeandNMS-occupiedstates,theeffectontoxinpotencyofalaninesubstitutionwasevaluatedinequilibriumandkineticbindingexperimentsaswellasinfunctionalassays.ThedeterminationofthecrystallographicstructureofanMT7-derivative(MT7-diiodoTyr51)allowedtheselectionofcandidateresiduesthatareaccessIBLeandpresentonbothfacesofthethreetoxinloops.TheequilibriumbindingdataareconsistentwithnegativecooperativitybetweenN-methylscopolamine(NMS)andwild-typeormodifiedMT7andhighlightthecriticalroleofthetipofthecentralloopofthetoxin(Arg34,Met35Tyr36)initsinteractionwiththeunoccupiedreceptor.Examinationofthepotencyofwild-typeandmodifiedtoxinstoallostericallydecreasethedissociationrateof[(3)H]NMSallowedtheidentificationoftheMT7residuesinvolvedinitsinteractionwiththeNMS-occupiedreceptor.Incontrasttotheresultswiththeunoccupiedreceptor,themostimportantresidueforthisinteractionwasTyr36inloopII,assistedbyTrp10inloopIandArg52inloopIII.ThecriticalroleofthetipsoftheMT7loopswasalsoconfirmedinfunctionalexperiments.ThehighspecificityoftheMT7-M(1)receptorinteractionexploitsseveralMT7-specificresiduesandrevealsadifferentmodeofinteractionofthetoxinwiththefreeandNMS-occupiedstatesofthereceptor.
MuscarinicToxin7selectivityisdictatedbyextracellularreceptorloops
KukkonenA.,etal.(2004)MuscarinicToxin7selectivityisdictatedbyextracellularreceptorloops.JBC.PMID: 15452105
Muscarinictoxin7(MT7)isamambavenomproteinantagonistwithextremelyhighselectivityfortheM1muscarinicacetylcholinereceptor.TomapthesitesfortheinteractionofMT7withmuscarinicreceptorswehaveusedchimericM1:M3receptorsandsite-directedmutagenesisoftheM3andM4receptorsubtypes.TwoGluresiduesinM1,oneinextracellularloop2andoneinextracellularloop3,werefoundtobeimportantforthehighaffinitybindingofMT7.SubstitutionofthecorrespondingLysresiduesintheM3receptorwithGluconvertedtheM3mutanttoanMT7bindingreceptor,albeitwithloweraffinitycomparedwithM1.APhe–>Tyrsubstitutioninextracellularloop2ofM3togetherwiththe2GlumutationsgeneratedareceptorwithanincreasedMT7affinity(apparentKi=0.26nMinafunctionalassay)comparedwiththeM1receptor(apparentKi=1.31nM).TheimportanceoftheidentifiedaminoacidresidueswasconfirmedwithamutatedM4receptorconstructs.TheresultsindicatethatthehighselectivityofMT7fortheM1receptordependsonveryfewresidues,thusprovidinggoodProspectsforfuturedesignandsynthesisofmuscarinicreceptor-selectiveligands.
EffectsofmuscarinictoxinsMT2andMT7,fromgreenmambavenom,onm1,m3andm5muscarinicreceptorsexpressedinChineseHamsterOvarycells
BradleyKN.etal.(2003)EffectsofmuscarinictoxinsMT2andMT7,fromgreenmambavenom,onm1,m3andm5muscarinicreceptorsexpressedinChineseHamsterOvarycells.Toxicon.PMID:12565740
Severalsmallproteinscalledmuscarinictoxins(MTs)havebeenisolatedfromvenomofgreenmamba(Dendroaspisangusticeps).TheyhavepreviouslybeenshowninrADIoligandbindingstudiestohavehighselectivityandaffinityforindividualmuscarinicreceptorsubtypes,butlessisknownoftheirfunctionaleffects.ThisstudyhasexaminedtheactionsoftwooftheseMTs,MT2andMT7,usingchangesincytosolicCa(2+)([Ca(2+)](i))measuredusingthefluorescentindicatorfura-2inChineseHamsterOvary(CHO)cellsstablytransfectedwithindividualmuscarinicreceptorsubtypes,m1,m3andm5.MT2activatedthem1receptor:atconcentrationsabove100nMitcausedsignificantandconcentration-dependentincreasesin[Ca(2+)](i).From25to800nMMT2alsoproducedincreasesin[Ca(2+)](i)byactivatingm3receptors,althoughtheseincreasesin[Ca(2+)](i)werenotstrictlyconcentration-dependentwithonlyintermittentresponsesbeingrecorded(i.e.itwasnotalwayspossibletoobtainaresponsetotheagonistwitheachapplicationofthecompound).MT2(800-1600nM)alsocausedsignificantincreasesin[Ca(2+)](i)inCHOcellsexpressingthem5muscarinicreceptorsubtype.MT7(1microM)displayednoagonistactivityatanyofthemuscarinicreceptorsbutwasapotentnon-competitiveantagonist(at20nM)atthem1muscarinicreceptorsubtype.Ithadnoantagonistactivityatthem3orm5subtypes.TheseresultsindicatethatMT7isahighlyspecificantagonistatthem1muscarinicreceptorsubtypeassuggestedbyresultsfromradioligandbindingstudies.However,MT2islessselectiveforthem1muscarinicreceptorthanpreviouslydescribedasitalsoexhibitsagonistactivityatthem3andm5muscarinicreceptors,whichwasnotdetectedinradioligandbindingstudies.
InhibitionofacetylcholinemuscarinicM1receptorfunctionbytheM1-selectiveligandmuscarinictoxin7(MT-7)
OlianasMC.,etal.(2000)InhibitionofacetylcholinemuscarinicM1receptorfunctionbytheM1-selectiveligandmuscarinictoxin7(MT-7).BrJPharmacol.PMID:11015294
MT-7(1–30nM),apeptidetoxinisolatedfromthevenomofthegreenmambaDendroaspisangusticepsandpreviouslyfoundtobindselectivelytothemuscarinicM(1)receptor,inhibitedtheacetylcholine(ACh)-stimulated[(35)S]-guanosine-5′-O-(3-thio)triphosphate([(35)S]-GTPgammaS)bindingtomembranesofChinesehamsterovary(CHO)cellsstablyexpressingtheclonedhumanmuscarinicM(1)receptorsubtype.MT-7failedtoaffecttheACh-stimulated[(35)S]-GTPgammaSbindinginmembranesofCHOcellsexpressingeithertheM(2),M(3)orM(4)receptorsubtype.InN1E-115neuroblastomacellsendogenouslyexpressingtheM(1)andM(4)receptorsubtypes,MT-7(0.3–3.0nM)inhibitedthecarbachol(CCh)-stimulatedinositolphosphatesaccumulation,butfailedtoaffecttheCCh-inducedinhibitionofpituitaryadenylatecyclaseactivatingpolypeptide(PACAP)38-stimulatedcyclicAMPaccumulation.InbothCHO/M(1)andN1E-115cellstheMT-7inhibitionconsistedinadecreaseofthemaximalagonisteffectwithminimalchangesintheagonistEC(50)value.InCHO/M(1)cellmembranes,MT-7(0.05–25nM)reducedthespecificbindingof0.05,1.0and15nM[(3)H]-N-methylscopolamine([(3)H]-NMS)inaconcentration-dependentmanner,butfailedtocauseacompletedisplacementoftheradioligand.Moreover,MT-7(3nM)decreasedthedissociationrateof[(3)H]-NMSbyabout5fold.CHO/M(1)cellmembranespreincubatedwithMT-7(10nM)andwashedbycentrifugationandresUSPensiondidnotrecovercontrol[(3)H]-NMSbindingforatleast8hat30degreesC.ItisconcludedthatMT-7actsasaselectivenoncompetitiveantagonistofthemuscarinicM(1)receptorsbybindingstablytoanallostericsite.
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这就是说不用酸碱预处理吗？
Whatman的网站上没有DE52最大耐受压力，请问又经验的战友应该是多少？
Whatman的网站上：
DE32DryMicrogranularDEAECellulose
SimilarperformancecharacteristicsafterprecyclingasDE52.
DE52PreswollenMicrogranularDEAECellulose
ProbablythemostwidelyusedDEAEcelluloseintheworld;usedforbiopolymerswithlowtohighnegativecharges;exhibitsexcellentresolutionwithgoodflowrates.
附件是一本图书(MethodsinMolecularMedicine,)的章节,上面说:
WhatmanDEAE52comesalreadypreswollenandonlyneedstobetransferred
totherunningbuffer50mMTE8.
lAntibodiesUsingIonExchangeChromatography.pdf(87.06k)
1、弱酸和它的盐（如：HAc---NaAc）的水溶液组成；
2、弱碱和它的盐（如：NH3·H2O---NH4Cl）的水溶液组成；
3、多元弱酸的酸式盐及其对应的次级盐（如：NaH2PO4---Na2HPO4）的水溶液组成。
酸碱缓冲溶液的选型一般应根据具体情况进行选择。缓冲酸性可选用碱性缓冲液，缓冲酸性可采用碱性缓冲液。常用作缓冲溶液的酸类由弱酸及其共轭酸盐组合成的溶液具有缓冲作用。生化实验室常用的缓冲系主要有磷酸、柠檬酸、碳酸、醋酸、巴比妥酸、Tris（三羟甲基氨基甲烷）等系统，生化实验或研究工作中要慎重地选择缓冲体系，因为有时影响实验结果的因素并不是缓冲液的pH值，而是缓冲液中的某种离子。如硼酸盐、柠檬酸盐、磷酸盐和三羟甲基甲烷等缓冲剂都可能产生不需要的化学反应。
【酸碱缓冲溶液】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。这种溶液称为酸碱缓冲溶液。
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!
两个CEX方法A和B测定同一单抗，结果碱性峰比例差不多，酸性峰比例相差约7%，相应主峰也差了7%左右。
具体来说，A方法酸性峰高，主峰低，碱性峰稍微低点；B方法酸性峰低，主峰高，碱性峰稍微高点；另外也做了CIEF，结果呢和A方法更接近。
仔细比较起来，AB两个方法的峰性和数量差不多，就不知道为什么会有这么大的差异。两个方法一个用的WCX柱-磷酸缓冲液，一个用SCX柱-MES缓冲液
大家帮我分析下：
1.两个方法哪个方法更准确，是以酸性峰高的为准还是什么？为什么？
2.这显著差异是由方法造成，具体原因是什么？柱子？
3.CIEF的结果和A方法更接近，是不是可以由此证明A方法更好或者CIEF的方法更好（因为CIEF更快更方便）？
欢迎讨论~
纠正下，A方法用的是Tosoh的柱子，B方法用的是SCX柱。TOSOH的柱子是7um的填料，10cm长。SCX是10um的填料。我本人TOSOH的阳离子柱子用的很少，这次信手用用，结果发现差异很大
那我现在就考虑，在以后方法开发过程中，除了通过流动相pH和组成、梯度、柱子选择来获得样品主峰和酸碱性的最大分离，还要关注各峰比例。因为之前比较方法好坏都只看分离度，尤其是主峰和邻近峰的分离度，获得最大分离度，自然可以做到主峰尽可能纯，但从未认真比较过各峰比例。这是一个大疏忽吧！
另外，CIEF和CEX方法原理还是有点差异的，所以分的是不同的异质体，原液放行两个方法肯定是都要做的。问题就是在早期细胞株筛选和工艺开发阶段，哪个方法才是又快又准。CIEF（iCE280)一般15分钟一个样，比CEX快多了。如果CIEF测得主峰要低于CEX结果，是不是真的完全可以取代CEX呢？CEX分离出的峰远比CIEF的多！
欢迎大家继续讨论~
:)
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?
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