Practical Approaches to Biological Inorganic Chemistry

315 Pages·2013·21.853 MB·English

by  Ricardo Louro and Robert Crichton (Auth.)

Practical Approaches
to Biological Inorganic
Chemistry
Edited by
Robert R. Crichton
Batiment Lavoisier
Universite´ Catholique de Louvain
Louvain-la-Neuve,Belgium
Ricardo O. Louro
ITQB, UniversidadeNova deLisboa
Oeiras,Portugal
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Preface
Shroudedinthemistsofscientificantiquity(thingsmovesoquicklythatevenadecadeortwoseemsalongtime),
inrealityalittlelessthan30yearsagoetheFederationofEuropeanBiochemicalSocieties,betterknownbyits
acronym FEBS, invited the Belgian Biochemical society to organise their annual Congress in Belgium. For the
first time in the history of these meetings (since the inaugural Congress, in London in 1964), two half day
symposiawereorganisedonthesubjectofmetalloproteins.Attheendofthesecondofthese,agroupofwhatin
thosedayswerecalledinorganicbiochemistsmettoenjoyadrinktogetherinthebaroftheSheratonHotel.The
outcomewasthattwoofthosepresent,oneofwhomisco-editorofthepresentvolume,togetherwithCeesVeeger
wereentrustedwiththetaskoforganisingaFEBSWorkshopCourseonInorganicBiochemistry.Thefirstofthese
washeldattheHotelEtapinLouvain-laNeuveattheendofApril,1985.Theoriginsofthisbookcanbetraced
back tothe long series ofAdvanced Courses which have followed that pioneering start.
At that very first Course, the pattern was established of organising lectures to introduce the subject and to
present a theoretical background to the methods which could be used to study metals in biological systems,
togetherwithpracticalsessionsinsmallergroups.Thefinallectureswerethendevotedtospecificexamples.Itis
interesting,andperhapsnottoosurprising,thatafteranintroductiontoligandfieldtheorybyBobWilliams,and
metal coordination in biology by Jan Reedijk, X-ray, EPR, NMR, Mo¨ssbauer and EXAFS spectroscopy of
metalloproteins were on the programme. The practicals included NMR, EPR and Mo¨ssbauer as well as Cees
Veeger’sfavourite,biochemicalanalysisofFeandSinFeeSproteins.TherewasaneveninglecturebyHelmut
Beinert (then on sabbatical in Konstanz) entitled ‘Limitations of Spectroscopic Studies on Metalloproteins and
ChemicalAnalysisofMetalsinProteins’.Whilethelecturerswereshuffledaroundfromyeartoyear,FredHagen,
AntonioXavier,AlfredTrautwein,andDaveGarnerrepresentedthecornerstoneofthespectroscopicpartofthe
courseoverthe early years.
Since then, over the period from 1985 until now we have organised some 20 courses, and trained over 800
students,mostofwhomweredoctoralorpost-doctoralstudentswhentheycame onthecourse.Itisasourceof
greatprideandsatisfactionthatmanyoftheformerstudentsstillenjoyactiveanddistinguishedcareersinthearea
of Biological Inorganic Chemistry, as we now call the subject. Even more rewarding are the number offormer
participantswhonowformthestaffofthecourse,notablytheotherco-editor,whohasalsotakenonthemantleof
co-organiser of the most recent courses. Indeed, with the exception of Rob Robson, who taught the Molecular
Biology lectures and practical for many years, the other authors contributing to this book, Frank Neese, Fred
Hagen,EckhardBill,MartinFeiters,ChristopheLegerandMargaridaArcherareallalumniofthe‘Louvain-la-
Neuve’course.
Our intention in editing this volume is that it can serve as a starting point for any student who wants to
study metals in biological systems. The presentations by the authors represent a distillation of what they have
taughtoveranumberofyearsintheadvancedcourse.Webeginwithanoverviewoftherolesofmetalionsin
biological systems, which we hope will serve as taster for the reader, who will find a much more detailed
account in the companion work to this volume (Crichton, 2012). Thereafter, after an introduction to that most
erudite of discipline (at least for non-inorganic chemists) ligand field theory, augmented by a good dose of
how molecular orbital theory can predict the properties of catalytic metal sites. This leads naturally into
a sequence which describes the physicochemical methods which can be used to study metals in biology,
concluding with an overview of the application of the powerful methods of modern genetics to
metalloproteins.
ix
x Preface
The considerations expressed by that pioneer of analytical precision Helmut Beinert in his 1985 evening
lectureinLouvain-la-Neuveareasrelevanttodayastheywerethen.Useasmanytechniquesaspossibletoanalyse
your sample e the more information from different approaches you have, the better we will understand your
protein.Donotwasteexpensiveandsensitivemethodsonshoddyimpuresamples,andconverselydonotemploy
primitivetechnicalmeanstoanalysehighlypurifiedsamples,whichhaverequiredenormousinvestmenttoobtain
them.Andaboveallrecognisethatthekeytometalloproteincharacterisationiscollaboration.Donotthinkyou
cansimplyphagocytiseatechniquefromthelaboratoryofacolleaguewhoknowsthemethodinsideouteitis
muchrichertocollaborate,incorporatinghisorherknow-howintoyourresearch.Andyouwillbethericherforit.
Bonnechance,goodluck,boasorteeandwelookforwardtogreetyouononeofthecourseswhichwill,we
hope,continueintothefuture.Hopefully,thislittleintroductorytextwillnotonlywhetyourappetite,buthelpyou
tofindyourway about the myriad practical methods which can be used to study metals inbiological systems.
Robert R. Crichton and Ricardo O. Louro
Louvain-la-Neuve, July, 2012
Chapter 1
An Overview of the Roles of Metals
in Biological Systems
Robert R. Crichton
BatimentLavoisier,Universite´CatholiquedeLouvain,Louvain-la-Neuve,Belgium
Chapter Outline
Introduction:WhichMetalsIonsandWhy? 1
SomePhysicochemicalConsiderationsonAlkaliMetals 3
NaDandKDeFunctionalIonicGradients 3
Mg2DePhosphateMetabolism 5
Ca2DandCellSignalling 6
ZinceLewisAcidandGeneRegulator 10
IronandCoppereDealingwithOxygen 12
NiandCoeEvolutionaryRelics 13
MneWaterSplittingandOxygenGeneration 16
MoandVeNitrogenFixation 18
INTRODUCTION: WHICH METALS IONS AND WHY?
Inthecompanionbooktothisone,‘BiologicalInorganicChemistry2ndedition’(Crichton,2011),weexplainin
greaterdetailwhylifeasweknowitwouldnotbepossiblewithjusttheelementsfoundinorganicchemistrye
namely carbon, oxygen, hydrogen, nitrogen, phosphorus and sulfur. We also need components of inorganic
chemistry as well, and in the course of evolution nature has selected a number of metal ions toconstruct living
organisms. Some of them, like sodium and potassium, calcium and magnesium, are present at quite large
concentrations,constitutingtheso-called‘bulkelements’,whereasothers,likecobalt,copper,ironandzinc,are
knownas‘trace elements’,with dietary requirements that are much lower than the bulkelements.
Justsixelementseoxygen,carbon,hydrogen,nitrogen,calciumandphosphorusemakeupalmost98.5%of
theelementalcompositionofthehumanbodybyweight.Andjust11elementsaccountfor99.9%ofthehuman
body (the five others are potassium, sulfur, sodium, magnesium and chlorine). However, between 22 and 30
elementsarerequiredbysome,ifnotall,livingorganisms,andofthesearequiteanumberaremetals.Inaddition
to the four metal ions mentioned above, we know that cobalt, copper, iron, manganese, molybdenum, nickel,
vanadiumandzincareessentialforhumans,whiletungstenreplacesmolybdenuminsomebacteria.Theessential
natureofchromiumfor humans remains enigmatic.
Justwhytheseelementsoutoftheentireperiodictable(Figure1.1)havebeenselectedwillbediscussedhere.
However,theirselectionwaspresumablybasednotonlyonsuitabilityforthefunctionsthattheyarecalleduponto
PracticalApproachestoBiologicalInorganicChemistry,1stEdition.http://dx.doi.org/10.1016/B978-0-444-56351-4.00002-6.
Copyright(cid:1)2013ElsevierB.V.Allrightsreserved. 1
2 PracticalApproachestoBiologicalInorganicChemistry
FIGURE1.1 Anabbreviatedperiodictableoftheelementsshowingthemetalionsdiscussedinthischapter.
playinwhat ispredominantlyanaqueousenvironment,butalsoontheir abundanceandtheir availabilityinthe
earth’scrustand itsoceans(which constitutethe major proportion of the earth’s surface).
The13metalionsthatwewilldiscussherefallnaturallyintofourgroupsbasedontheirchemicalproperties.In
þ þ þ (cid:2)
the first, we have the alkali metal ions Na and K . Together with H and Cl , they bind weakly to organic
ligands,havehighmobility,andarethereforeideallysuitedforgeneratingionicgradientsacrossmembranesand
þ þ
formaintainingosmoticbalance.Inmostmammaliancells,mostK isintracellular,andNa extracellular,with
þ
thisconcentrationdifferentialensuringcellularosmoticbalance,signaltransductionandneurotransmission.Na
þ
andK fluxesplayacrucialroleinthetransmissionofnervousimpulsesbothwithinthebrainandfromthebrain
tootherparts ofthe body.
Thesecondgroupismadeupbythealkalineearths,Mg2þandCa2þ.Withintermediatebindingstrengthsto
organicligands,theyare,atbestsemi-mobile,andplayimportantstructuralroles.TheroleofMg2þisintimately
associated with phosphate, and it is involved in many phosphoryl transfer reactions. Mg-ATP is important in
muscle contraction, and also functions in the stabilisation of nucleic acid structures, as well as in the catalytic
activity of ribozymes (catalytic RNA molecules). Mg2þ is also found in photosynthetic organisms as the metal
þ
centreinthelight-absorbingchlorophylls.Ca isacrucialsecondmessenger,signallingkeychangesincellular
metabolism, but is also important in muscle activation, in the activation of many proteases, both intra- and
extracellular,and as amajor component of arange of bio-minerals, including bone.
Zn2þ, which is arguably not a transition element,1 constitutes the third group on its own. It is moderate to
strongbinding,isofintermediatemobilityandisoftenfoundplayingastructuralrole,althoughitcanalsofulfil
averyimportantfunctionasaLewisacid.Structuralelements,calledzincfingers,playanimportantroleinthe
regulationof gene expression.
Theothereighttransitionmetalions,Co,Cu,Fe,Mn,Mo,Ni,VandWformthefinalgroup.Theybindtightly
to organic ligands and therefore have very low mobility. Since they can exist in various oxidation states, they
participate in innumerable redox reactions, and many of them are involved in oxygen chemistry. Fe and Cu are
constituentsofalargenumberofproteinsinvolvedinelectrontransferchains.Theyalsoplayanimportantrolein
oxygen-binding proteins involved in oxygen activation as well as in oxygen transport and storage. Co, together
with another essential transition metal, Ni, is particularly important in the metabolism of small molecules like
carbon monoxide, hydrogen and methane. Co is also involved in isomerisation and methyl transfer reactions.
A major role of Mn is in the catalytic cluster involved in the photosynthetic oxidation of water to dioxygen in
plants,and,fromamuchearlierperiodingeologicaltime,incyanobacteria. MoandWenzymes containapyr-
anopterindithiolate cofactor, while nitrogenase, the key enzyme of N fixation contains a molybde-
2
numeironesulfur cofactor, in which V can replace Mo when Mo is deficient. Other V enzymes include
1.IUPACdefinesatransitionmetalas“anelementwhoseatomhasanincompletedsub-shell,orwhichcangiverisetocationswithan
incompletedsub-shell.”
Chapter j 1 AnOverviewoftheRolesofMetalsinBiologicalSystems 3
haloperoxidases.TodatenoCr-bindingproteinshavebeenfound,addingtothelackofbiochemicalevidencefor
a biological roleof the enigmaticCr.
SOME PHYSICOCHEMICAL CONSIDERATIONS ON ALKALI METALS
Beforeconsidering,inmoredetail,therolesofthealkalimetals,NaþandKþ,andthealkalineearthmetals,Mg2þ
andCa2þ,itmaybeusefultoexaminesomeoftheirphysicochemicalproperties(Table1.1).Wecanobserve,for
þ þ
examplethatNa andK havequitesignificantlydifferentunhydratedionicradii,whereas,thehydratedradiiare
much more similar. It therefore comes as no surprise that the pumps and channels which carry them across
membranes,andwhichcaneasilydistinguishbetweenthem,aswewillseeshortly,transporttheunhydratedions.
Althoughnotindicatedinthetable,itisclearthatNaþisinvariablyhexa-coordinate,whereasKþandCa2þcan
þ þ
adjusttoaccommodate6,7or8ligands.Asweindicatedabove,bothNa andK arecharacterisedbyveryhigh
solvent exchange rates (around 109/s), consistent with their high mobility and their role in generating ionic
gradientsacrossmembranes.Incontrast,themobilityofMg2þissomefourordersofmagnitudeslower,consistent
with its essentially structural and catalytic. Perhaps surprisingly, Ca2þ has a much higher mobility (3(cid:3)108/s),
which explainswhy it isinvolvedin cell signalling via rapid changes on Ca2þfluxes.
TheselectivebindingofCa2þbybiologicalligandscomparedtoMg2þcanbeexplainedbythedifferencein
theirionicradius,aswepointedoutabove.Also,forthesmallerMg2þion,thecentralfieldofthecationdominates
itscoordination sphere, whereasforCa2þ,the secondandpossiblyeventhe third, coordinationspheres havean
importantinfluenceresultinginirregularcoordinationgeometry.ThisallowsCa2þ,unlikeMg2þtobindtoalarge
numberof centres at once.
ThehighchargedensityonMg2þasaconsequenceofitssmallionicradiusensuresthatitisanexcellentLewis
acid in reactions notably involving phosphoryl transfers and hydrolysis of phosphoesters. Typically, Mg2þ
functionsasaLewisacid,eitherbyactivatingaboundnucleophiletoamorereactiveanionicform(e.g.waterto
hydroxide anion), or by stabilising an intermediate. The invariably hexacoordinate Mg2þ often participates in
structureswherethemetalisboundtofourorfiveligandsfromtheproteinandaphosphorylatedsubstrate.This
leaves one or two coordination positions vacant for occupation by water molecules, which can be positioned in
a particulargeometry bythe Mg2þ toparticipate inthe catalytic mechanism ofthe enzyme.
NAD AND KD e FUNCTIONAL IONIC GRADIENTS
How,wemightask,dothepumpsandchannelsresponsiblefortransportacrossmembranesdistinguishbetween
þ þ
Na andK ions?Studiesoverthelast50yearsorsoofsyntheticandnaturallyoccurringsmallmoleculeswhich
bindionshaveestablishedthebasicrulesofionselectivity.Twomajorfactorsappeartobeofcapitalimportance,
TABLE1.1 PropertiesofCommonBiologicalCations
Ionic Hydrated Ionic Hydrated Exchange Transport
Cation radius(A˚) radius(A˚) volume(A˚3) volume(A˚3) rate(sec(cid:2)1) number
Naþ 0.95 2.75 3.6 88.3 8(cid:3)108 7e13
Kþ 1.38 2.32 11.0 52.5 109 4e6
Mg2þ 0.65 4.76 1.2 453 105 12e14
Ca2þ 0.99 2.95 4.1 108 3(cid:3)108 8e12
(FromMaguireandCowan,2002).
4 PracticalApproachestoBiologicalInorganicChemistry
namely the molecular composition and the stereochemistry (essentially the size) of the binding site. Synthetic
moleculeshavebeencreatedwhichselectivitybindLiþ(radius0.60A˚),Naþ(0.95A˚),Kþ(1.35A˚)andRbþ(radius
1.48A˚)bysimplyadjustingthecavitysizetomatchtheion(Dietrich,1985).Nowthatwehavethecrystalstructures
of membrane transport proteins, we can begin to understand how ion selectivity is accomplished (MacKinnon,
þ þ
2004; GouaxandMacKinnon, 2005). TheNa -selectivebindingsites inthe Na -dependantleucine transporter
þ þ
LeuTandtheK -selectivebindingsitesintheK channelhavebeendetermined,providingadirectcomparisonof
þ þ þ þ þ þ
selectivityforNa andK .TheNa andK ionsarecompletelydehydrated,boththeNa andtheK sitescontain
þ þ
oxygenligands,butbyfarthemostimportantfactordistinguishingNa andK sitesisthesizeofthecavityformed
bythebindingsite,whichagreeswellwiththerulesalreadylearnedfromhost/guestchemistry.Whatdetermines
alkalimetalcationselectivity,similartothatobservedinionbindingbysmallmolecules,isthattheproteinselects
þ þ
foraparticularion,Na orK ,byprovidinganoxygen-linedbindingsiteoftheappropriatecavitysize.
þ þ
MammaliancellsmaintainahighintracellularK (around140mM)andlowintracellularNa (around12mM)
þ þ
throughtheactionoftheNa ,K -ATPasepresentintheplasmamembrane.Theoverallreactioncatalysedis:
3Naþ(in) þ2Kþ(out) þATP þH O 53Naþ (out)þ 2Kþ (in) þADP þP
2 i
Theextrusionofthreepositivechargesforeverytwowhichenterthecell,resultsinatransmembranepotentialof
50e70mV,whichhasenormousphysiologicalsignificance,controllingcellvolume,allowingneuronsandmuscle
cells to be electrically excitable, and driving the active transport of important metabolites such as sugars and
aminoacids.Morethanone-thirdofATPconsumptionbyrestingmammaliancellsisusedtomaintainthisintra-
cellularNaþ(cid:2)Kþ gradient (in nervecellsthiscan rise toup to70%).
This thermodynamically unfavourable exchange is achieved by ATP-mediated phosphorylation of the
þ þ
Na ,K -ATPase followed by dephosphorylation of the resulting aspartyl phosphate residue, which drives
conformationalchangesthatallowionaccesstothebindingsitesofthepumpfromonlyonesideofthemembrane
atatime.TheATPaseexistsintwodistinctconformations,E andE ,whichdifferintheircatalyticactivityand
1 2
þ þ þ
theirligandspecificity(Figure1.2).TheE form,whichhasahighaffinityforNa ,bindsNa ,andtheE .3Na
1 1
þ
formthenreactswithATPtoformthe“high-energy”aspartylphosphateternarycomplexE ~P.3Na .Inrelaxing
1
þ
to its “low-energy” conformation E -P, the bound Na is released outside the cell. The E -P, which has a high
2 2
þ þ þ
affinityforK ,binds2K ,andtheaspartylphosphategroupishydrolysedtogiveE .2K ,whichthenchanges
2
þ
conformation to the E form, releasing its 2K inside the cell. The structures of a number of P-type ATPases,
1
includingtheNaþ-Kþ-ATPaseandtheCa2þ(cid:2)ATPaseoftheSarcoplasmicreticulumhavebeendeterminedand
are showninFigure1.3.
FIGURE1.2 AmodelfortheactivetransportofNaþandKþbytheNaþ-Kþ-ATPase.
Chapter j 1 AnOverviewoftheRolesofMetalsinBiologicalSystems 5
FIGURE 1.3 Overall structures and ion-binding site architectures of two P-type ATPases, rabbit sarcoplasmic reticulum Ca2þ-ATPase
(SERCA)andpigNaþ,Kþ-ATPase.TheupperpaneldepictsrabbitSERCA(E1ProteinDataBase[PDB]entry1T5S)andpigNaþ-Kþ-ATPase
(E2:Pi,PDBentry3KDP).N-,P-,andA-domainsarecolouredred,blueandyellow,respectively;theb-subunitandg-subunitofNaþ,Kþ-
ATPasewheatandcyan.Thelowerpaneldepictstheion-bindingsites,viewedapproximatelyperpendiculartothemembraneplanefromthe
extracytoplasmicside,intheE1state.Ionligandingresiduesareshownassticks,transmembranehelicesandcalciumionsinSERCAare
indicatedbynumbersandgreyspheres,respectively,andthesitessuperposedastransparentspheresontotheNaþ,Kþ-ATPasemodel.Putative
bindingsitesforthethirdsodiumionintheNaþ,Kþ-ATPaseareindicatedasgreyellipses.(FromBublitzetal.,2010.ReproducedCopyright
2010withpermissionfromElsevier).
MG2D e PHOSPHATE METABOLISM
TheintracellularconcentrationoffreeMg2þisabout5(cid:3)10(cid:2)3M,sothatalthoughMg2þ-bindingtoenzymesis
relatively weak (K not more than 105M(cid:2)1) and most Mg2þ-dependent enzymes have adequate local concen-
a
trationsofMg2þfortheiractivity.Mg2þisthemostabundantdivalentcationinthecytosolofmammaliancells,
bindsstronglytoATPandADP,andisthereforeextensivelyinvolvedinintermediarymetabolismandinnucleic
acidmetabolism.However,likeZn2þ,itisadifficultmetaliontostudy,sinceitisspectroscopicallysilent,withthe
consequence that manyspectroscopic studiesonMg2þ enzymes utilise Mn2þas areplacement metal ion.
6 PracticalApproachestoBiologicalInorganicChemistry
OfthefiveenzymesselectedintheEnzymeFunctionInitiative,recentlyestablishedtoaddressthechallenge
of assigning reliable functions to enzymes discovered in bacterial genome projects, but for which functions
have not yet been attributed (Gerlt et al., 2011), three of them are Mg2þ-dependent. We discuss two of them
briefly here.
Thehaloalkanoicaciddehalogenasesuperfamily(HADSF)(>32,000nonredundantmembers)catalyseadiverse
rangeofreactionsthatinvolvetheMg2þ-dependentformationofacovalentintermediatewithanactivesiteAsp.
Despitebeingnamedafteradehalogenase,thevastmajorityareinvolvedinphosphoryltransferreactions(Allenand
Dunaway-Mariano,2004,2009).WhileATPasesandphosphatasesarethemostprevalent,thehaloaciddehalogenase
(HAD)familycancarryoutmanydifferentmetabolicfunctions,includingmembranetransport,signaltransduction
andnucleic-acidrepair.Theirphysiologicalsubstratescoveranextensiverangeofbothsizeandshape,rangingfrom
phosphoglycolate, the smallest organophosphate substrate, to phosphoproteins, nucleic acids, phospholipids,
phosphorylateddisaccharides,sialicacidsandterpenes.
InHADenzymes,Aspmediatescarbon-grouptransfertowater(inthedehalogenases)andphosphoryl-group
transfer to a variety of acceptors. Thus, the HAD superfamily is unique in catalysing both phosphoryl-group
transfer (top) and carbon-group transfer (bottom) (Figure 1.4a). The roles of the four loops that comprise the
catalytic scaffold are shown in Figure 1.4b. The activity ‘switch’ is located on loop 4 of the catalytic scaffold
(yellow) which positionsone carboxylate residue tofunctionas ageneral base for the dehalogenases and either
two or three carboxylates to bind the Mg2þ cofactor essential for the phosphotransferases. CO represents the
backbonecarbonyloxygenofthemoietythatistworesiduesdownstreamfromtheloop1nucleophile(red).The
side-chainatthispositionisalsousedasanacid-basecatalystbyphosphataseandphosphomutaseHADmembers.
Loop2(green)andloop3(cyan)servetopositionthenucleophileandsubstratephosphorylmoiety.Figure1.4c
presents aribbondiagram ofthe fold supporting the catalyticscaffold ofphosphonatase.
ThemembersofanotherlargesuperfamilyofMg2þenzymes,theenolasesuperfamily(withmorethan6000
nonredundant members) catalyse diverse reactions, including b-eliminations (cycloisomerisation, dehydration
and deamination) and 1,1-proton transfers (epimerisation and racemisation). The three founder members of the
family are illustrated by mandelate racemase, muconate lactonising enzyme and enolase (Figure 1.5). They all
catalysereactionsinwhichthea-protonofthecarboxylatesubstrateisabstractedbytheenzyme,generatingan
enolateanionintermediate.Thisintermediate,whichisstabilisedbycoordinationtotheessentialMg2þionofthe
enzyme,isthen directed todifferent products inthe enzyme activesites.
CA2D AND CELL SIGNALLING
Calciumionsplayamajorroleasstructuralcomponentsofboneandteeth,butarealsocruciallyimportantincell
signalling.Topreventtheprecipitationofphosphorylatedorcarboxylatedcalciumcomplexes,manyofwhichare
insoluble,thecytosoliclevelsofCa2þinunexcitedcellsmustbekeptextremelylow,muchlowerthanthatinthe
extracellularfluidandinintracellularCa2þstores.Thisconcentrationgradientgivescellstheopportunitytouse
Ca2þasametabolictriggerethecytosolicCa2þconcentrationcanbeabruptlyincreasedforsignallingpurposes
bytransientlyopeningCa2þchannelsintheplasmamembraneorinanintracellularmembrane.Theseincreasesin
intracellular free Ca2þ concentration can regulate a wide range of cellular processes, including fertilisation,
muscle contraction,secretion, learning andmemory andultimately cell death, both apoptotic and necrotic.
Extracellular signals often act by causing a transient rise in cytosolic Ca2þ levels, which, in turn, activates
agreatvarietyofenzymesthroughtheactionofCa2þ-bindingproteinslikecalmodulin,aswewilldiscussindetail
below: this triggers such diverse processes as glycogen breakdown, glycolysis and muscle contraction. In the
phosphoinositide cascade (Figure 1.6), binding of the external signal (often referred to as the agonist2 when it
provokes a positive response) to the surface receptor R (step 1) activates phospholipase C, either through a G
2.Manydrugshavebeendevelopedeitherasagonistorantagoniststoreceptor-mediatedsignallingpathways,e.g.b-blockersblocktheaction
oftheendogenouscatecholaminesadrenaline(epinephrine)andnoradrenaline(norepinephrine)onb-adrenergicreceptors.