Guangxitoxin-1E(GxTx-1E) wasisolatedfromthevenomofChilobrachysjingzhao(Chineseearthtigertarantula).Guangxitoxin-1E wasshowntoblock Kv2.1/KCNB1,Kv2.2/KCNB2andKv4.3/KCND3channels withoutsignificanteffectonKv1.2/KCNA2,Kv1.3/KCNA3,Kv1.5/KCNA5,Kv3.2/KCNC2,Cav1.2/CACNA1C,Cav2.2/CACNA1B,Nav1.5/SCN5A,Nav1.7/SCN9AorNav1.8/SCN10Achannels. Guangxitoxin-1E inhibitsKv2.1withanIC50 valueof1nMandKv2.2withanIC50 valueof3nM.BlockofKv4.3occursat10-20foldhigherconcentrations.Guangxitoxin-1E actsasagatingmodifiersinceitshiftsthevoltage-dependenceofKv2.1K+ currentstowardsdepolarizedpotentials.Inpancreaticbeta-cells, Guangxitoxin-1E enhancesglucose-stimulatedinsulinsecretion bybroadeningthecellactionpotentialandenhancingcalciumoscillations.
RecentlyquotedDescription:
AAsequence: Glu-Gly-Glu-Cys4-Gly-Gly-Phe-Trp-Trp-Lys-Cys11-Gly-Ser-Gly-Lys-Pro-Ala-Cys18-Cys19-Pro-Lys-Tyr-Val-Cys24-Ser-Pro-Lys-Trp-Gly-Leu-Cys31-Asn-Phe-Pro-Met-Pro-OH
Presumeddisulfidebridgepattern:Cys4-Cys19,Cys11-Cys24,Cys18-Cys31
Length(aa): 36
Formula: C178H248N44O45S7
MolecularWeight: 3948.70Da
Appearance:Whitelyophilizedsolid
Solubility: waterorsalinebuffer
CASnumber: notavailable
Source: Synthetic
Purityrate: >95%
Reference:
Theroleofvoltage-gatedpotassiumchannelsKv2.1andKv2.2intheregulationofinsulinandsomatostatinreleasefrompancreaticislets
Thevoltage-gatedpotassiumchannelsKv2.1&Kv2.2arehighlyexpressedinpancreaticislets,yettheircontributiontoislethormonesecretionisnotfullyunderstood.HereweinvestigatetheroleofKv2channelsinpancreaticisletsusingacombinationofgenetic&pharmacologicapproaches.Pancreaticβ-cellsfromKv2.1(-/-)micepossessreducedKvcurrent&displaygreaterglucose-stimulatedinsulinsecretion(GSIS)relativetoWTβ-cells.InhibitionofKv2.xchannelswithselectivepeptidyl[guangxitoxin-1E(GxTX-1E)]orsmallmolecule(RY796)inhibitorsenhancesGSISinisolatedwild-type(WT)mouse&humanislets,butnotinisletsfromKv2.1(-/-)mice.However,inWTmiceneitherinhibitorimprovedglucosetoleranceinvivo.GxTX-1E&RY796enhancedsomatostatinreleaseinisolatedhuman&mouseislets&insituperfusedpancreatafromWT&Kv2.1(-/-)mice.Kv2.2silencinginmouseisletsbyadenovirus-smallhairpinRNA(shRNA)specificallyenhancedisletsomatostatin,butnotinsulin,secretion.Inmicelackingsomatostatinreceptor5,GxTX-1Estimulatedinsulinsecretion&improvedglucosetolerance.Collectively,thesedatashowthatKv2.1regulatesinsulinsecretioninβ-cells&Kv2.2modulatessomatostatinreleaseinδ-cells.DevelopmentofselectiveKv2.1inhibitorswithoutcrossinhibitionofKv2.2mayprovidenewavenuestopromoteGSISforthetreatmentoftype2diabetes.
LiXN., etal. (2013)Theroleofvoltage-gatedpotassiumchannelsKv2.1andKv2.2intheregulationofinsulinandsomatostatinreleasefrompancreaticislets. JPharmacolExpTher. PMID: 23161216
Regulationofvoltage-gatedK+channelsbyglucosemetabolisminpancreaticbeta-cells
Regulationofdelayedrectifier-typeK(+)channels(Kv-channels)byglucosewasstudiedinratpancreaticbeta-cells.TheKv-channelcurrentwasincreasedinamplitudesbyincreasingglucoseconcentrationfrom2.8to16.6mM,whileitwasdecreasedby2.8mMglucoseinareversIBLemanner(down-regulation)inbothperforated&conventionalwhole-cellmodes.ThecurrentwasdecreasedbyFCCP,intraPipette0mMATPorAMPPNP.Glyceraldehyde,pyruvicacid,2-ketoisocaproicacid,&10mMMgATPpreventedthedown-regulationinducedby2.8mMorlessglucose.TheresidualcurrentaftertreatmentwithKv2.1-specificblocker,guangxitoxin-1E,wasunchangedbyloweringorincreasingglucoseconcentration.WeconcludethatglucosemetabolismregulatesKv2.1channelsinratsbeta-cellsviaalteringMgATPlevels.
YoshidaM., etal. (2009)Regulationofvoltage-gatedK+channelsbyglucosemetabolisminpancreaticbeta-cells. FEBSLett. PMID: 19500583
AnautomatedelectrophysiologyserumshiftassayforK(V)channels
ThepresenceofseruminBIOLOGicalsamplesoftennegativelyimpactsthequalityofinvitroassays.However,assaystolerantofserumareusefulforassessingtheinvivoavailABIlityofasmallmoleculeforitstarget.Electrophysiologyassaysofionchannelsarenotoriouslysensitivetoserumbecauseoftheirrelianceontheinteractionoftheplasmamembranewitharecordingelectrode.Hereweinvestigatethetoleranceofanautomatedelectrophysiologyassayforavoltage-gatedpotassium(K(V))channeltoserum&purifiedplasmaproteins.Thedelayedrectifierchannel,K(V)2.1,stablyexpressedinChinesehamsterovarycellsproduceslarge,stablecurrentsontheIonWorksQuattroplatform(MDSAnalyticalTechnologies,Sunnyvale,CA),makingitanidealtestcase.K(V)2.1currentsrecordedonthisplatformarehighlyresistanttoserum,allowingrecordingsinashighas33%serum.UsingasetofcompoundsrelatedtotheK(V)channelblocker,4-phenyl-4-[3-(2-methoxyphenyl)-3-oxo-2-azaprop-1-yl]cyclohexanone,weshowthatshiftsincompoundpotencywithwholeserumorisolatedserumproteinscanbereliablymeasuredwiththisassay.Importantly,thisassayisalsorelativelyinsensitivetoplasma,allowingthecreationofabioassayforinhibitorsofK(V)2.1channelactivity.Hereweshowthatsuchabioassaycanquantifythelevelsofthegatingmodifier,guangxitoxin-1E,inplasmasamplesfrommicedosedwiththepeptide.Thisstudydemonstratestheutilityofusinganautomatedelectrophysiologyplatformformeasuringserumshifts&forbioassaysofionchannelmodulators.
RatliffKS., etal.(2008)AnautomatedelectrophysiologyserumshiftassayforK(V)channels. AssayDrugDevTechnol.PMID: 18471078
Gatingmodifierpeptidesasprobesofpancreaticbeta-cellphysiology
Pancreaticbeta-cellsdepolarizeinresponsetoglucose&firecalcium-dependentactionspotentialsthattriggerinsulinsecretion.Themajorcurrentresponsibleforactionpotentialrepolarizationinthesecellsisadelayedrectifier&Kv2.1subunitsarethoughtbeamajorcontributorofthedelayedrectifierchannels.Hence,blockersofKv2.1channelsmightprolongactionpotentials&enhancecalciuminflux&insulinsecretion.However,thelackofspecificsmallmoleculeKv2.1inhibitorshashinderedthetestingofthismechanism.Importantly,severalgatingmodifierpeptidesinhibitKv2.1channelsinarelativelyspecificfashion.Hanatoxin(HaTX)&guangxitoxin-1(GxTX-1)areexamplesthathavebeenusedtoprobetheroleofKv2.1channelsinbeta-cellphysiology.BothHaTX&GxTX-1stronglyinhibittheKvcurrentofbeta-cellsfromvariousspecies,arguingthatKv2.1subunitscontributesignificantlytothebeta-celldelayedrectifier.GxTX-1prolongsglucose-triggeredactionpotentials,enhancesglucose-dependentintracellularcalciumelevations&augmentsglucose-dependentinsulinsecretion.Takentogether,thesedatasuggestthatblockersofKv2.1channelsmaybeausefulapproachtothedesignofnoveltherapeuticagentsforthetreatmentoftype2diabetes.Thesestudieshighlighttheutilityofgatingmodifierpeptidesinthestudyofphysiologicalsystems.
HerringtonJ.,(2009)Gatingmodifierpeptidesasprobesofpancreaticbeta-cellphysiology. Toxicon. PMID: 17101164
SNAP-25(1-180)enhancesinsulinsecretionbyblockingKv2.1channelsinratpancreaticisletbeta-cells
Voltage-gatedoutwardK(+)currentsfrompancreaticisletbeta-cellsareknowntorepolarizetheactionpotentialduringaglucosestimulus,&consequentlytomodulateCa(2+)entry&insulinsecretion.ThevoltagegatedK(+)(Kv)channel,Kv2.1,whichisexpressedinratisletbeta-cells,mediatesover60%oftheKvoutwardK(+)currents.AnovelpeptidylinhibitorofKv2.1/Kv2.2channels,guangxitoxin(GxTX)-1,hasbeenshowntoenhanceglucose-stimulatedinsulinsecretion.Here,weshowthatSNAP-25(1-180)(S180),anN-terminalSNAP-25domain,butnotSNAP-25(1-206)(S206),inhibitsKvcurrent&enhancesglucose-dependentinsulinsecretionfromratpancreaticisletbeta-cells,&furThermore,thisenhancementwasinducedbytheblockadeoftheKv2.1current.ThisstudyindicatesthattheKv2.1channelisapotentialtargetfornoveltherapeuticagentdesignforthetreatmentoftype2diabetes.Thistargetmaypossessadvantagesovercurrently-usedtherapies,whichmodulateinsulinsecretioninaglucose-independentmanner.
ZhuangGQ., etal. (2009)SNAP-25(1-180)enhancesinsulinsecretionbyblockingKv2.1channelsinratpancreaticisletbeta-cells. BiochemBiophysResCommun. PMID: 19103161
Blockersofthedelayed-rectifierpotassiumcurrentinpancreaticbeta-cellsenhanceglucose-dependentinsulinsecretion
Delayed-rectifierK+currents(I(DR))inpancreaticbeta-cellsarethoughttocontributetoactionpotentialrepolarization&therebymodulateinsulinsecretion.Thevoltage-gatedK+channel,K(V)2.1,isexpressedinbeta-cells,&thebiophysicalcharacteristicsofheterologouslyexpressedchannelsaresimilartothoseofI(DR)inrodentbeta-cells.AnovelpeptidylinhibitorofK(V)2.1/K(V)2.2channels,guangxitoxin(GxTX)-1(half-maximalconcentrationapproximately1nmol/l),hasbeenpurified,characterized,&usedtoprobethecontributionofthesechannelstobeta-cellphysiology.Inmousebeta-cells,GxTX-1inhibits90%ofI(DR)&,asforK(V)2.1,shiftsthevoltagedependenceofchannelactivationtomoredepolarizedpotentials,acharacteristicofgating-modifierpeptides.GxTX-1broadensthebeta-cellactionpotential,enhancesglucose-stimulatedintracellularcalciumoscillations,enhancesinsulinsecretionfrommousepancreaticisletsinaglucose-dependentmanner.Thesedatapointtoamechanismforspecificenhancementofglucose-dependentinsulinsecretionbyapplyingblockersofthebeta-cellI(DR),whichmayprovideadvantagesovercurrentlyusedtherapiesforthetreatmentoftype2diabetes.
HerringtonJ., etal.(2006)Blockersofthedelayed-rectifierpotassiumcurrentinpancreaticbeta-cellsenhanceglucose-dependentinsulinsecretion. Diabetes. PMID: 16567526
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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。
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.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！
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！
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