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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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!
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。
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!
pH(1)=pKa+lg[c(CH₃COONa)/c(CH₃COOH)]=pKa=4.74
通HCl后,溶液是c(CH₃COOH)=0.2mol/L、c(NaCl)=0.1mol/L的混合溶液,溶液pH按照弱酸溶液pH的求法求.
c(H⁺)=√[Ka*c(CH₃COOH)]=√(10^-4.74*0.2)=0.00191(mol/L)(采用了近似公式)
pH(2)=-lg{c(H⁺)}=2.72
两个pH求得,那么pH的变化量也就可得了.pH的变化量=|pH(2)-pH(1)|=|2.72-4.74|=2.02
1)PH缓冲溶液作用原理和pH值
当往某些溶液中加入一定量的酸和碱时,有阻碍溶液pH变化的作用,称为缓冲作用,这样的溶液叫做缓冲溶液.弱酸及其盐的混合溶液(如HAc与NaAc),弱碱及其盐的混合溶液(如NH3·H2O与NH4Cl)等都是缓冲溶液.
由弱酸HA及其盐NaA所组成的缓冲溶液对酸的缓冲作用,是由于溶液中存在足够量的碱A-的缘故.当向这种溶液中加入一定量的强酸时,H离子基本上被A-离子消耗:
所以溶液的pH值几乎不变;当加入一定量强碱时,溶液中存在的弱酸HA消耗OH-离子而阻碍pH的变化.
2)PH缓冲溶液的缓冲能力
在缓冲溶液中加入少量强酸或强碱,其溶液pH值变化不大,但若加入酸,碱的量多时,缓冲溶液就失去了它的缓冲作用.这说明它的缓冲能力是有一定限度的.
缓冲溶液的缓冲能力与组成缓冲溶液的组分浓度有关.0.1mol·L-1HAc和0.1mol·L-1NaAc组成的缓冲溶液,比0.01mol·L-1HAc和0.01mol·L-1NaAc的缓冲溶液缓冲能力大.关于这一点通过计算便可证实.但缓冲溶液组分的浓度不能太大,否则,不能忽视离子间的作用.
组成缓冲溶液的两组分的比值不为1∶1时,缓冲作用减小,缓冲能力降低,当c(盐)/c(酸)为1∶1时△pH最小,缓冲能力大.不论对于酸或碱都有较大的缓冲作用.缓冲溶液的pH值可用下式计算:
此时缓冲能力大.缓冲组分的比值离1∶1愈远,缓冲能力愈小,甚至不能起缓冲作用.对于任何缓冲体系,存在有效缓冲范围,这个范围大致在pKaφ(或pKbφ)两侧各一个pH单位之内.
弱酸及其盐(弱酸及其共轭碱)体系pH=pKaφ±1
弱碱及其盐(弱碱及其共轭酸)体系pOH=pKbφ±1
例如HAc的pKaφ为4.76,所以用HAc和NaAc适宜于配制pH为3.76~5.76的缓冲溶液,在这个范围内有较大的缓冲作用.配制pH=4.76的缓冲溶液时缓冲能力最大,此时(c(HAc)/c(NaAc)=1.
3)PH缓冲溶液的配制和应用
为了配制一定pH的缓冲溶液,首先选定一个弱酸,它的pKaφ尽可能接近所需配制的缓冲溶液的pH值,然后计算酸与碱的浓度比,根据此浓度比便可配制所需缓冲溶液.
以上主要以弱酸及其盐组成的缓冲溶液为例说明它的作用原理、pH计算和配制方法.对于弱碱及其盐组成的缓冲溶液可采用相同的方法.
PH缓冲溶液在物质分离和成分分析等方面应用广泛,如鉴定Mg2离子时,可用下面的反应:
白色磷酸铵镁沉淀溶于酸,故反应需在碱性溶液中进行,但碱性太强,可能生成白色Mg(OH)2沉淀,所以反应的pH值需控制在一定范围内,因此利用NH3·H2O和NH4Cl组成的缓冲溶液,保持溶液的pH值条件下,进行上述反应.
:)
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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