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1、c15PrinciplesofMetabolicRegulation.qxd12/6/124:35PMPageS-173chapterPrinciplesofMetabolicRegulation151.MeasurementofIntracellularMetaboliteConcentrationsMeasuringtheconcentrationsofmetabolicintermediatesinalivingcellpresentsgreatexperimentaldifficulties—usuallyacellmustbedest
2、royedbeforemetaboliteconcentrationscanbemeasured.Yetenzymescatalyzemetabolicinterconversionsveryrapidly,soacommonproblemassociatedwiththesetypesofmeasurementsisthatthefindingsreflectnotthephysiologicalconcentrationsofmetabolitesbuttheequilibriumcon-centrations.Areliableexper
3、imentaltechniquerequiresallenzyme-catalyzedreactionstobeinstanta-neouslystoppedintheintacttissuesothatthemetabolicintermediatesdonotundergochange.Thisobjectiveisaccomplishedbyrapidlycompressingthetissuebetweenlargealuminumplatescooledwithliquidnitrogen(190C),aprocesscalled
4、freeze-clamping.Afterfreezing,whichstopsenzymeactioninstantly,thetissueispowderedandtheenzymesareinactivatedbyprecipitationwithperchlo-ricacid.Theprecipitateisremovedbycentrifugation,andtheclearsupernatantextractisanalyzedformetabolites.Tocalculateintracellularconcentrations
5、,theintracellularvolumeisdeterminedfromthetotalwatercontentofthetissueandameasurementoftheextracellularvolume.Theintracellularconcentrationsofthesubstratesandproductsofthephosphofructokinase-1reac-tioninisolatedrathearttissuearegiveninthetablebelow.MetaboliteConcentrationmM*
6、Fructose6-phosphate87.0Fructose1,6-bisphosphate22.0ATP11,400ADP1,320Source:FromWilliamson,J.R.(1965)GlycolyticcontrolmechanismsI:inhibitionofglycolysisbyacetateandpyruvateintheisolated,perfusedratheart.J.Biol.Chem.240,2308–2321.*Calculatedasmmol/mLofintracellularwater.(a)Cal
7、culateQ,[fructose1,6-bisphosphate][ADP]/[fructose6-phosphate][ATP],forthePFK-1reactionunderphysiologicalconditions.(b)GivenaGforthePFK-1reactionof14.2kJ/mol,calculatetheequilibriumconstantforthisreaction.(c)ComparethevaluesofQandKeq.Isthephysiologicalreactionnearorfarfr
8、omequilibrium?Explain.WhatdoesthisexperimentsuggestabouttheroleofPFK-1asare
