GsMTx4(GsMTx-4,M-theraphotoxin-Gr1a) hasbeenisolatedfromthevenomofthespiderGrammostolarosea.Thiscationichydrophobicpolypeptideblocksselectivelythegatingofcationselectivechannelsandmechanosensitiveionchannelssuchas TRPC1 or TRPC6,withouthavinganyeffectonwhole-cellvoltage-sensitivecurrents.Thistoxinactsbyperturbingtheinterfacebetweenthechannelandthelipidbilayerwithoutnecessarilybeinginphysicalcontactwiththechannel. GsMTx4 alsodemonstratedtoactive TRPA1 channelat1µMconcentrationandtoinhibit Piezo1currents.
Recentlyquoted
Description:
AAsequence: Gly-Cys2-Leu-Glu-Phe-Trp-Trp-Lys-Cys9-Asn-Pro-Asn-Asp-Asp-Lys-Cys16-Cys17-Arg-Pro-Lys-Leu-Lys-Cys23-Ser-Lys-Leu-Phe-Lys-Leu-Cys30-Asn-Phe-Ser-Phe-NH2
Disulfidebonds: Cys2-Cys17,Cys9-Cys23 andCys16-Cys30
Length(AA): 34
Formula: C185H273N49O45S6
MolecularWeight: 4095.2Da
Appearance: Whitelyophilizedsolid
Solubility: waterandsalinebuffer
CASnumber:
Source: Synthetic
Purityrate: >97%
Reference:
Citations
- AndersonM,etal.(2013)OpposingeffectsofpodocinonthegatingofpodocyteTRPC6channelsevokedbymembranestretchordiacylglycerol. AmJPhysiolCellPhysiol. PubMedlink
SynergybetweenPiezo1andPiezo2channelsconfershigh-strainmechanosensitivitytoarticularcartilage
Diarthrodialjointsareessentialforloadbearingandlocomotion.Physiologically,articularcartilagesustainsmillionsofcyclesofmechanicalloADIng.Chondrocytes,thecellsincartilage,regulatetheirmetabolicactivitiesinresponsetomechanicalloading.Pathologicalmechanicalstresscanleadtomaladaptivecellularresponsesandsubsequentcartilagedegeneration.Wesoughttodeconstructchondrocytemechanotransductionbyidentifyingmechanosensitiveionchannelsfunctioningatinjuriouslevelsofstrain.Wedetectedrobustexpressionoftherecentlyidentifiedmechanosensitivechannels,PIEZO1andPIEZO2.CombineddirectedexpressionofPiezo1and-2sustainedpotentiatedmechanicallyinducedCa(2+)signalsandelectricalcurrentscomparedwithsingle-Piezoexpression.Inprimaryarticularchondrocytes,mechanicallyevokedCa(2+)transientsproducedbyatomicforcemicroscopywereinhibitedbyGsMTx4,aPIEZO-blockingpeptide,andbyPiezo1-orPiezo2-specificsiRNA.Wecomplementedthecellularapproachwithanexplant-cartilageinjurymodel.GsMTx4reducedchondrocytedeathaftermechanicalinjury,suggestingapossIBLetherapyforreducingcartilageinjuryandposttraumaticosteoarthritisbyattenuatingPiezo-mediatedcartilagemechanotransductionofinjuriousstrains.
LeeW,etal.(2015)SynergybetweenPiezo1andPiezo2channelsconfershigh-strainmechanosensitivitytoarticularcartilage.PNAS. PubMedlink
TheMechanosensitiveIonChannelPiezo1IsInhibitedbythePeptideGsMTx4
Cellscanrespondtomechanicalstressbygating mechanosensitive ionchannels (MSCs).Thecloningof Piezo1,aeukaryoticcationselectiveMSC,definesanewsystemforstudyingmechanicaltransductionatthecellularlevel.Because Piezo1 haselectrophysiologicalpropertiessimilartothoseofendogenouscationicMSCsthatareselectively inhibited bythe peptide GsMTx4,wetestedwhetherthe peptide targets Piezo1 activity.ExtracellularGsMTx4 atmicromolarconcentrationsreversibly inhibited ∼80%ofthemechanicallyinducedcurrentofoutside-outpatchesfromtransfectedHEK293cells.Theinhibitionwasvoltageinsensitive,&asseenwithendogenousMSCs,themirrorimagedenantiomer inhibited likethel.Therateconstantsforbinding&unbindingbasedon Piezo1 currentkineticsprovidedassociation&dissociationratesof7.0×10(5)M(-1)s(-1)&0.11s(-1),respectively,&aK(D)of∼155nM,similartovaluespreviouslyreportedforendogenousMSCs.Consistentwithpredictedgatingmodifierbehavior,GsMTx4 producedan∼30mmHgrightwardshiftinthepressure-gatingcurve&wasactiveonclosed channels.Incontrast,streptomycin,anonspecificinhibitorofcationicMSCs,showedtheuse-dependentinhibitioncharacteristicofopen channel block.The peptide didnotblockcurrentsofthemechanical channel TREK-1onoutside-outpatches.Whole-cell Piezo1 currentswerealsoreversibly inhibited by GsMTx4,&althoughtheoffratewasnearlyidenticaltothatofoutside-outpatches,differenceswereobservedfortheonrate.TheABIlityof GsMTx4 totargetthemechanosensitivityof Piezo1 supportstheuseofthis channel inhigh-throughputscreensforpharmacologicalagents&diagnosticassays.
Bae,C., etal. (2011)TheMechanosensitiveIonChannelPiezo1IsInhibitedbythePeptideGsMTx4.Biochemistry. PMID: 21696149
TRPA1IsDifferentiallyModulatedbytheAmphipathicMoleculesTrinitrophenolandChlorpromazine
KerstinHill,MichaelSchaefer(2007)TRPA1IsDifferentiallyModulatedbytheAmphipathicMoleculesTrinitrophenolandChlorpromazine. JBC. PMID: 17218316
Moleculardynamicssimulationsofastretch-activatedchannelinhibitorGsMTx4withlipidmembranes:twobindingmodesandeffectsoflipidstructure
NishizawaM,NishizawaK.(2007)Moleculardynamicssimulationsofastretch-activatedchannelinhibitorGsMTx4withlipidmembranes:twobindingmodesandeffectsoflipidstructure.BiophysJ. PMID: 17384064
Localizationofthevoltage-sensortoxinreceptoronKvAP
Ruta,V.,andMacKinnon,R.(2004)Localizationofthevoltage-sensortoxinreceptoronKvAP,Biochemistry. PMID: 15287735
CDNAsequenceandinvitrofoldingofGsMTx4,aspecificpeptideinhibitorofmechanosensitivechannels
ThepeptideGsMTx4fromthetarantulavenom(Grammostolaspatulata)inhibitsmechanosensitiveionchannels.Inthiswork,wereportthecDNAsequenceencodingGsMTx4.Thegeneistranslatedasaprecursorproteinof80aminoacids.Thefirst21aminoacidsareapredictedsignalsequence&theC-terminalresiduesareasignalforamidation.AnarginineresidueadjacenttotheN-terminalglycineofGsMTx4isthecleavagesiteforrelease.Theresultingpeptideis34aminoacidsinlengthwithaC-terminalphenylalanine¬aserine-alaninepreviouslyidentified[J.Gen.Physiol.115(2000)583].Wechemicallysynthesizedthispeptide&foldeditin0.1MTris,pH7.9withoxidized/reducedglutathione(1/10).Propertiesofthesyntheticpeptidewereidenticaltothewildtypeforhighperformanceliquidchromatography(HPLC),massspectrometry,CD,&NMR.WealsoclonedGsMTx4inathioredoxinfusionproteinsystemcontainingsixhistidines.Nickelaffinitycolumnsallowedrapidpurification&foldingoccurredinconditionsdescribedabovewith0.5MguanidiniumHClpresent.ThrombincleavageliberatedGsMTx4withthreeextraaminoacidsattheN-terminus.TheretentiontimeinHPLCanalysis&theCDspectrumwassimilartowildtype.Boththesynthetic&clonedpeptideswereactiveinthepatchclampassay.
Ostrow,K.L., etal. (2003)cDNAsequenceandinvitrofoldingofGsMTx4,aspecificpeptideinhibitorofmechanosensitivechannels. Toxicon. PMID: 14559077
Solutionstructureofpeptidetoxinsthatblockmechanosensitiveionchannels
Mechanosensitivechannels(MSCs)playkeyrolesinsensoryprocessing&havebeenimplicatedasprimarytransducersforavarietyofcellularresponsesrangingfromosmosensingtogeneexpression.ThispaperpresentsthefirststructuresofanykindknowntointeractspecificallywithMSCs.GsMTx-4&GsMtx-2areinhibitorcysteineknotpeptidesisolatedfromvenomofthetarantula,Grammostolaspatulata(Suchyna,T.M.,Johnson,J.H.,Hamer,K.,Leykam,J.F.,Gage,D.A.,Clemo,H.F.,Baumgarten,C.M.,&Sachs,F.(2000)J.Gen.Physiol.115,583-598).InhibitionofcationicMSCsbythehigheraffinityGsMtx-4(K(D)approximately500nm)reducedcellsizeinswollen&hypertrophicheartcells,swelling-activatedcurrentsinastrocytes,&stretch-inducedarrhythmiasintheheart.Despitetherelativelylowaffinity,nocross-reactivityhasbeenfoundwithotherchannels.Usingtwo-dimensionalNMRspectroscopy,wedeterminedthesolutionstructureofGsMTx-4&aloweraffinity(GsMTx-2;K(D)approximately6microm)peptidefromthesamevenom.Thedominantfeatureofthetwostructuresisahydrophobicpatch,utilizingmostofthearomaticresidues&surroundedwithchargedresidues.ThespatialarrangementofchargedresiduesthatareuniquetoGsMTx-4&GsMTx-2mayunderlietheselectivityofthesepeptides.
Oswald,R.E., etal. (2002)Solutionstructureofpeptidetoxinsthatblockmechanosensitiveionchannels. JBiolChem. PMID: 12082099
IdentificationofapeptidetoxinfromGrammostolaspatulataspidervenomthatblockscation-selectivestretch-activatedchannels
Wehaveidentifieda35aminoacidpeptidetoxinoftheinhibitorcysteineknotfamilythatblockscationicstretch-activatedionchannels.Thetoxin,denotedGsMTx-4,wasisolatedfromthevenomofthespiderGrammostolaspatulata&has<50%homologytootherneuroactivepeptides.ItwasisolatedbyfractionatingwholevenomusingreversephaseHPLC,&thenassayingfractionsonstretch-activatedchannels(SACs)inoutside-outpatchesfromadultratastrocytes.Althoughthechannelgatingkineticsweredifferentbetweencell-attached&outside-outpatches,thepropertiesassociatedwiththechannelpore,suchasselectivityforalkalications,conductance(approximately45pSat-100mV)&amildrectificationwereunaffectedbyoutside-outformation.GsMTx-4producedacompleteblockofSACsinoutside-outpatches&appearedspecificsinceithadnoeffectonwhole-cellvoltage-sensitivecurrents.Theequilibriumdissociationconstantofapproximately630nMwascalculatedfromtheratioofassociationanddissociationrateconstants.Inhypotonicallyswollenastrocytes,GsMTx-4producesapproximately40%reductioninswelling-activatedwhole-cellcurrent.Similarly,inisolatedventricularcellsfromarabbitdilatedcardiomyopathymodel,GsMTx-4producedanearcompleteblockofthevolume-sensitivecation-selectivecurrent,butdidnotaffecttheanioncurrent.Inthemyopathicheartcells,wheretheswell-inducedcurrentistonicallyactive,GsMTx-4alsoreducedthecellsize.Thisisthefirstreportofapeptidetoxinthatspecificallyblocksstretch-activatedcurrents.Thetoxinaffectonswelling-activatedwhole-cellcurrentsimplicatesSACsinvolumeregulation.
Suchyna,T.M., etal. (2000)IdentificationofapeptidetoxinfromGrammostolaspatulataspidervenomthatblockscation-selectivestretch-activatedchannels. JGenPhysiol. PMID: 10779316
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拼音名：Chunhuashui
英文名：PurifiedWater
【性状】本品为无色的澄清液体；无臭，无味。
【检查】酸碱度取本品10ml，加甲基红指示液2滴，不得显红色；另取10ml，加溴麝香草酚蓝指示液5滴，不得显蓝色。氯化物、流酸盐与钙盐取本品，分置三支试管中，每管各50ml。第一管中加硝酸5滴与硝酸银试液1ml，第二管中加氯化钡试液2ml，第三管中加草酸铵试液2ml，均不得发生浑浊。
硝酸盐取本品5ml置试管中，于冰浴中冷却，加10%氯化钾溶液0.4ml与0.1%二苯胺硫酸溶液0.1ml，摇匀，缓缓滴加硫酸5ml，摇匀，将试管子50℃水浴中放置15分钟，溶液产生的蓝色与标准硝酸盐溶液［取硝酸钾0.163g，加水溶解并稀释至100ml，摇匀，精密量取1ml，加水稀释成100ml，再精密量取10ml，加水稀释成100ml，摇匀，即得（每1ml相当于1pgNO3）0.3ml，加无硝酸盐的水4.7ml，用同一方法处理后的颜色比较，不得更深（0.000006%）。
亚硝酸盐取本品10ml，置纳氏管中，加对氨基苯磺酰胺的稀盐酸溶液（1→100）lml与盐酸菜乙H肢溶液（0．l＋100）1ml，产生的粉红色，与标准亚硝酸盐溶液〔取亚硝酸钠0.750g（按干燥品计算），加水溶解，稀释至100ml，摇匀，精密量取1ml，加水稀释成100ml，摇匀，再精密量取1ml，加水稀释成50ml，摇匀，即得（每1ml相当于1μgNO2）]0.2ml，加无亚硝酸盐的水9.8ml，用同一方法处理后的颜色比较，不得更深（0.000002%）。
氨取本品50ml，加碱性碘化汞钾试液2ml，放置15分钟；如显色，与氯化铵溶液（取氯化铵31.5mg，加无氨水适量使溶解并稀释成1000ml）1.5ml，加元氨水48ml与碱性碘化汞钾试液2ml制成的对照液比较，不得更深（0.00003%）。
二氧化碳取本品25ml，置50ml具塞量筒中，加氢氧化钙试液25ml，密塞振摇，放置，小时内不得发生浑浊。
易氧化物取本品100ml，加稀硫酸10ml，煮沸后，加高锰酸钾滴定液（0.02mol/L）0.10ml，再煮沸10分钟，粉红色不得完全消失。
不挥发物取本品100ml，置105℃恒重的蒸发皿中，在水浴上蒸干，并在105℃干燥至恒重，遗留残渣不得过1mg。
重金属取本品50ml，加水18.5ml，蒸发至20ml，放冷，加醋酸盐缓冲液（pH3.5）2ml与水适量使成25ml，加硫代乙酰胺试液2ml，摇匀，放置2分钟，与标准铅溶液1.5ml加水18.5ml用同一方法处理后的颜色比较，不得更深（0.00003%）。
微生物限度取本品，采用薄膜过滤法处理后，依法检查（附录ⅪJ），细菌、霉菌和酵母菌总数每1ml不得过100个。
【贮藏】密闭保存。
【化学成分】本品为蒸馏法、离子交换法、反渗透法或其他适宜的方法制得的供药用的水，不含任何附加剂。
【分子式与分子量】H2O18.02
【药理作用】溶剂、稀释剂
这里药典纯化水标准中并无PH值项目，请问对纯化水有PH值的要求吗，范围应在多少？请说明出处？
在纯化水检测中，检验酸碱度合格，但是发现PH在8左右。如果按以上标准检验合格，是否要考虑PH值？请知道的解答，谢谢！
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
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~
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。
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