Hydrolysis Cyclic 3':5'-Adenosine Monophosphate ...

1 , , ,June1974 Short-TermBindingandHydrolysisofCyclic3' :5'-AdenosineMonophosphatebyAggregatingD ictyosteliumCells(chemotaxis/receptors/c yclic-AMPphosphodiesterase/cellmembranes /slimemolds) ,74 Tabingen,GermanyCommunicatedbySaulRosema n,April3, , ,kinetics, (cAMP)actsasanattractantforaggregatingce llsoftheslimemold,Dictyosteliumdiscoideu m(1,2), (3).ExtracellularcAMPishydrolyzedinthiso rganismbothbyextracellularandcell-boundp hosphodiesterases,thelatterexhibitingits highestactivityatthestageofaggrega-tion- competence(4,5).CyclicGMP(cGMP)isagoodsu bstrateforthesephos-phodiesterasesandapo tentinhibitorofcAMPhydrolysisbytheseenzy mes(6);itschemotacticandotherbiologicala ctivities,however,are1000-foldlowerthant hoseofcAMP(2,3).Theseresultssuggestthatt hereceptorsitesaremorespecificthanthecat alyticsitesofphosphodiesterase,andprompt edstudiesofcAMPbindingunderconditionsofp hosphodiesteraseinhibitioninordertodetec treceptorsthat,inlivingcells, (7).

2 ,aswellasthedevelopmentalregulationofthe bindingactivity, (8)andwashedtoinducedifferentiationtoagg regation-competenceasdescribed(5).Wild-t ypestrainvi2 IMS,theUV-mutantaggr60-2,andthespontaneo usrevertantaggr60-3(rev)werecultivatedin shakersuspensionsof1X1010cellspermlofeit herEscherichiacoliB/rorSalmoneUaminnesot aR595andwashedthreetimesin17mMphosphateb uffer, ,atthetimeofexhaustionofbacteria(to)(5). cA) , ,withlabelednucleotides(['H]cAMP,27-30Ci /mmol;['H]cGMP, ;['H]5'-AMP, ;Amersham) (7).Ifnotstatedotherwise, ,beginningwith5seeafternucleotideadditio n,100plofthecellsuspensionswascentrifuge dfor30secinconcialtubesinanEppendorfMicr o-centrifuge, (7).Toshowthatnosignificantexchangeoflab eloccurredbe-tweencellsedimentandsuperna tantduringcentrifugation,100 Alofcellsuspensionwascentrifugedat5secaf teraddi-tionof10nM['HI] ;thesuper> , (w/v) ['H]cAMPassociatedwiththecellsedimenttot hatassociatedwiththesupernatant,asweltas the'percenthydrolysisinboth, ;tothesupernatant30,ulof55% ,5'-AMP,andadenosinewereseparatedbypaper chromatographyanddeterminedbyscintillaio ncounting(5).

3 ThehydrolyzedcAMP(75-95%)appearedas5-AMP , , ,with2X107ilvingcellsperml,50pMcAMP,and9 00unitsofinhibitorofextracel-lularphosph odiesteraseperml(5). by guest on May 14, 2021 by guest on May 14, 2021 (1974)CyclicAMPB indingbyDictyosteliumCells2425-i0u4, , *. ~~~~--{ (U)orthegrowth-phase(0)stagewereincubate dwith70nM[3H]5' ,10nM['H]cAMPwasaddedtoaggregation-compe tentcells(X).Within5sec,95%oftheaddednuc leotidewashydrolyzedto5'-AMP;therefore, ,nocontinuationof5'-AMPhydrolysiswasfoun dinthesupernatant(0). , ( ).ThismeansthatcAMPdissociatedfromthecel lsappearedinthemediumas5' ( ),whichshowsthatundertheexperimentalcond itions, ,sincethesupernatantofcellsuspen-sionsdi dnotdetectablyhydrolyze5'-AMP( ).Undertheexperimentalconditions, , ,there-fore,theapproximationmorevalid( ).}

4 Scatchardplots(10)ofbindingdata( )revealedapparentdissociationconstantsin therangeof1to2X10-7M,andapproximately5X1 05bindingsitespercell( ). ,nosignificantcAMPbindingwasdetected( ).Inaccordwithpreviousresults(5),cAMPhyd rolysisbygrowth-phasecellswasmuchslowert hanbyaggregation-competentcells,excludin gexcessivehydrolysisasanexplanationforth eabsenceofdetectablebinding( ). ) ~~~~~~~~01234X105cAMPBOUND(MOLECULESPERC ELL] .(A)10nM[3H] (0)and50(0).(B)[3H]cAMP-bindingcurvesand (C) (0,a), (0,0),samplesweretakenafter5,20,70,and12 0sec,andthemeansofthetwohighestbindingva lueswereplotted.(0,0)uncorrecteddata;(0, El)datacorrectedforunspecificnucleotideb indingoruptake, ['H]5'-AMPequivalenttothe[31H] ,theapparentbindingvalueswere63% '-AMPadded;atthehighestconcentration,26% ofthecAMPand6%ofthe5' 'percell, ;thedatawouldbeinaccord,however,withashi ftoftheapparentkdi8, ,althoughadiffer-enceof1-2hrbetweenthetw otimecoursesispossible( ]Downloaded by guest on May 14, 2021 2426 CellBiology:MalchowandGerisch>, (A)andofcAMP-bindingactivity(B).)

5 (0,0)Wild-typestrainM2(datafromtwoexperi ments);(0,El)mutantaggr50-2;(U,*)reverta ntaggr50-3(rev). (0,0,*)or70nM(0,o,0)['H]cAMPtosuspendedc ells,centrifugationat5secafternucleotide addition, ,takenasacriterionforaggregation-compete nce, , ).Inanonaggregatingmutant,aggr60-2(9),bo thbindingsitesandcell-boundphosphodieste raseremainedlow,whereas4i'arevertantofth ismutant,aggrQ60-(rev),bothincreasednorm ally( ).IncontrasttocAMP-bindingandphosphodies teraseac-tivity,therateof5'-AMPhydrolysi sbycell-boundphos-phatasewasnotsignifica ntlydifferentingrowth-phaseandaggregatio n-competentcells( ). ,thisimpliesbindingofthlechemotacticfact ortoareceptor,couplingofthisreactiontoap rocessattheinnersideofthemembrane, (5).Thequestionwasaskedwhetherthecatalyt icsiteofthephosphodiesteraseisitselfther eceptorsitesothatthecellscountcAMPmolecu lesstrictlybyhydrolyzingthem;orifthetwos itesaredifferent(5).

6 ThefindingthatcGMPextendstheperiodofcAMP bindingtothesamedegreeasitretardshydroly sis,favors,inconjunctionwiththekineticda tadiscussedbelow, ,atleast90% , , ,theintracellularcAMPlevelinaggregatingc ells(21) ,inaccordwiththerelativelyweakchemotacti cactionofcGMP(2).Theirselec-tivity,aswel lastheirmarkedincreaseduringthepre-aggre gationphase( ),distinguishesthesesitesfromsolublecAMP -bindingprotein(s)incellhomogenates(11). (20).Scatchardplotssuggestatotalofabout5 X10' ,whichweexpectisaweakcompetitiveinhibito rforcAMPbindingatthereceptor, ,thenumberofbindingsitesshouldnotbeinflu encedbycompetitiveinhibition, `-M(2,3). ,however,thatthecAMPis"channeled"fromthe receptortothephosphodiesterase, , ,atwhichconcentrationrangewefoundtheappa rentdissociationconstantforthereceptorto be1to2X10-'MinthepresenceofcGMP,theappar entKm,ofthecell-boundoOO~oO0000 /~~~~~~0_%.

7 ,,-O/oo000/00a~~~~~~~~~~~~/'b8~~~V-o-13 ~0-0a,0~~ , (1974)IIDownloaded by guest on May 14, 2021 ,intheaggregation-deficientmutantaggr50- s2,andintherevertantaggr50-3(rev)Wild-ty peStationaryaggr50-3(rev)phaseaggr50-2 StationaryphaseCell-surfacesitesor(aggre gation-Stationary(aggregation-extracellu larfactorGrowthphasecompetence)phasecomp etence) (5)andpresentpaperInhibitorofextracellul arphospho-diesterase-+-+(9)Contact-sites A-+_+(18)Contact-sitesB+++Nottested(18)G lycosphingolipid("antigenI")+++Nottested (19)phosphodiesterasewas5X10-7 MintheabsenceofcGMP(unpublished).Measure doverawiderrangeofconcentra-tions,thecel l-boundphosphodiesteraseexhibitedkinetic sre-semblingnegativecooperativity(12),si milartomammaliancAMPphosphodiesterases(1 3).

8 ItremainstobecalculatedifthecAMPconcentr ationnearthecellsurfacecanbesignifi-cant ly"buffered" , ( ). (14,15)andtotheglucagonreceptorsystemofl ivercells(16,17).cAMPreceptorsaswellasce ll-boundphosphodiesteraseareplasma-membr anecomponentsthatareregulatedduringthepr e-aggregationphase,similartoantibody-bin dingsitesre-ferredtoas"contact-sitesA."B lockingofthelatterbyuni-valentantibodyfr agmentseliminatestheend-to-endadhesionch aracteristicofaggregatingcells(18).Aninh ibitorofextra-cellularcAMPphosphodiester aseisalsoformedduringtheinterphasebetwee ngrowthandaggregation(9).Table1showsthat theactivityofallfourcomponentsiseitherab sentorlowinanonaggregatingUV-mutant, ,twoclassesofcell-surfacestructuresprese ntalreadyingrowth-phasecells,glyco-sphin golipids("antigenI")and"contact-sitesB," , , , ,Barkley, ,Hall, ,Konijn, ,Mason, ,O'Keefe,G.

9 ,&Wolfe, (1969) , , (1972)Advancesin CyclicNucleotideResearch1, , ,B.(1974) , , , (1972)Science175, ,D.,Nagele,B.,Schwarz, ,G.(1972) , ,D.,Fuchila, ,B.(1973) , , ,G.(1973) , , , (1970) , ,V.,Gerisch,G.,Muller, ,H.(1973) , ,G.(1949) , , ,Kwasniak, , (1973) , , ,G.(1973)Hoppe-Seyler' , , ,Thompson, ,Schneider, , (1972) ,69, , , (1971) , ,A.(1973) , , ,Krans, ,Birnbaumer, ,M.(1972) , ,M.(1973) , ,H.,Katz, ,G.(1973) , ,H.,Luderitz, ,G., , ,Rosenfeld, , (1973) , , , (1973) , , (1973) , (1974)Downloaded by guest on May 14, 2021