Bdochem. J. (1976) 159, 719-728Printed in Great Britain

The Binding of Nucleotides and Bivalent Cations to the Calcium-and-Magnesium Ion-Dependent Adenosine Triphosphatase from Rabbit Muscle

Sarcoplasmic Reticulum

By DAVID W. YAT1ES and VICTOR C. DUANCE*Molecular EnzymologyLaboratory, Department ofBiochemistry,

University ofBristol, Bristol BS8 1TD, U.K.

(Received 24 June 1976)

The binding of MgATP to purified Ca2++Mg2+-dependent adenosine triphosphatasefrom rabbit muscle sarcoplasmic reticulum was studied by using a flow-dialysis method.Phosphoryl-enzyme formation and catalytic activity were also measured, and all threeprocesses demonstrated negative co-operativity, with half-saturation of all three para-meters at a MgATP concentration of4-50OM, and a Hill coefficient (h) of 0.8. The varia-tion of binding constant with pH was measured and showed tighter binding of MgATPwith increasing pH over the range 6.8-8.5. Binding parameters for ATP analogues werealso measured. The binding of Ca2+ in the presence and absence of ATP analogues gavehalf saturation at a Ca2+ concentration of 1.2-1.3uM. Hill plots of Ca2+-binding data gavea slope of 0.8. These results show that the binding of MgATP and Ca2+ can occur in arandom manner, with neither substrate influencing the affinity of the enzyme for theother.

The Ca2++Mg2-+dependent ATPaset from rabbitskeletal muscle sarcoplasmic reticulum is the enzymeresponsible for the active accumulation of COa+ bythe sarcoplasmic reticulum, causing muscle relaxa-tion. A crude enzyme preparation may be isolatedfrom homogenized rabbit muscle by differentialcejitrifugation; the resulting preparation contaiabout seven proteins and phospholipids. TheATPaseenzyme responsible for Ca2+ transport may be furtherpurified from the crude preparation by detergenttreatment and ammonium acetate fractionation, thepurified enzyme retaining about 100 molecules ofphospholipid for each enzyme molecule (MacLennanet al., 1972). The Ca2++Mg2+-dependent ATPasewill react with MgATP in the presence of Ca2+ toform a phosphoryl-enzyme complex (Yamamoto &Tonomura, 1968; Makinose, 1969; Martonosi,1969), and this complex is stable when quenched inacid.The present experiments have been performed to

obtain data on the dissociation constants of theenzyme for its substrates and to see how these com-

* Present address: Department of Animal Husbandry,University of Bristol, Langford, Bristol BS8 1TD, U.K.

t Abbreviations: ATPase, adenosine triphosphatase;EGTA, ethanedioxybis(ethylamine)tetra-acetic acid;AMP-P(NH)P, adenyl-5'-yl imidodiphosphate; AMP-PP(S), adenosine 5'-(3-thio)triphosphate; thio-ITP, 6-nercaptoinosine 5'-triphosphate; FTP, formycin 5'-triphosphate; TNP-ATP, trinitrophenyladenosine 5'-triphosphate.

Vol. 159

pare with the Km values of the enzyme for phos-phorylation and for steady-state ATPase activity.This information is required to elucidate themechanism of enzyme phosphorylation and dephos-phorylation. Experiments are described in which thebinding of Ca2+, ATP and variousATP analogues aremeasured by a rapid-flow dialysis method (Colowick& Womack, 1969). Phosphoryl.enzyme formationis also studied together with rates of steady-stateATPase activity and the relevance of these results isdiscussed with respect to possible enzyme mechan-isms.

Materials and Methods

Enzyme purificationThe Ca2++Mg2+-dependent ATPase was purified

by the method of MacLennan (1970) from leg andback muscles of New Zealand White Rabbits ob-tained from Hyline Rabbits Ltd., Marston, North-wich, Cheshire, U.K. The procedure designatedpreparation A by MacLennan (1970) was followed.The soluble purified enzyme so obtained was con-verted into insoluble enzyme by removal of thedeoxycholate by gel filtration. Samples (10ml) ofenzyme were passed through a column (2.5cmx30cm) ofSephadex G-25 previously equilibrated withbuffer containing 0.55M-sucrose, 0.045M-Tris/HCI,0.001 M-histidine and 0.58M-ammonium acetate,pH8.0. The cloudy white vesicular enzyme elutedfrom the column was concentrated by centrifugation

719

D. W. YATES AND V. C. DUANCE

at O00OOOg for 30min at 4°C in the 8 x 25ml head ofaMSE Superspeed 50 centrifuge. The pellet of enzymewas resuspended in the same buffer, by using a smallglass homogenizer, to a concentration of 15mg/mland stored at -20°C.Enzyme purity was determined by sodium dodecyl

sulphate/polyacrylamide-gel electrophoresis (Mac-Lennan, 1970). One major band of protein was pre-sent, with an apparent mol.wt. of 95000-105000.Traces (<2 %) of a minor component with a mol.wt.of 55000 were seen in some preparations, and thiscorresponds to the band identified by MacLennan(1970) as calsequestrin. Traces (<2%) of high-molecular-weight polymers (>200000mol.wt.) weresometimes seen.

Gels run at pH2.4 after incubation with Mg[y_32P]-ATP by the methods of Jordon & Raymond (1969)and Avruch & Fairbanks (1972) were radioauto-graphed on X-ray film (Kodirex KD 54T). The100000-mol.wt. band was the only component tocontain detectable 32P radioactivity.The enzyme preparation was taken to be over 95%

pure by these criteria, and a suspension of 1 mg ofprotein/ml as determined by the method of Lowryet al. (1951) was taken to be 10pM.

Adenylate kinase could possibly affect some of theresults ifpresent, and assays for this enzyme were per-formed as described below. No detectable activitycould be found (<0.002EC unit/mg).The Ca2++Mg2+-dependent ATPase preparation

was tested for Ca2+ accumulation in the presence ofoxalate, by using the conditions published by Weberetal. (1966). No C(2+ accumulation could be detected,and the preparation was assumed to be freely perme-able to Ca2+ when prepared in this manner.

Ca2+ depletion of reagentsCa2+-depleted enzyme was prepared by adding

buffered EGTA to a final concentration of 25mM tothe enzyme. Ca/EGTA and excess of EGTA werethen separated from the enzyme by gel filtration of1 ml samples on a column (30cmx I cm) of SephadexG-25 pre-equilibrated with Ca2+-depleted buffercontaining either 0.25M-sucrose, 0.05M-Tris/HCIand 0.001 M-histidine, pH8.0, or 0.1 M-Tris/HCI and0.001M-MgSO4, pH8.0.

All reagents except for KCl and MgSO4 weredepleted ofCa2+ by passage through Chelex-100 resincolumns (15cmx 1.5cm) (from Bio-Rad Labora-tories Ltd., Bromley, Kent, U.K.), in the K+ form.KCI and MgSO4 were depleted of Ca2+ by recrystal-lization from solutions containing 10mM-EGTA,followed by two recrystallizations from double-distilled water to lower EGTA contamination.Ca/EGTA buffers were used to obtain solutions

with a known and constant free Ca2+ ion concentra-tion.A 10mM-Ca/EGTA buffer was prepared by mixing

20mm solutions of EGTA and CaCl2 (diluted fromBDH standard 1 M-CaCl2 solution) in the appropriateratio to give a solution with the desired free Ca2+ con-centration and the pH adjusted to 7.4 with NaOHbefore diluting to volume with water. The ratio ofEGTA/CaCI2 required was determined by using thedissociation constant for Ca/EGTA of 1.2 x 1I0Mat pH7 given by Weber et al. (1966). For a 264uM-freecalcium solution the ratio of EGTA/CaCI2 was1: 0.96. Unless otherwise defined this solution isreferred to as Ca/EGTA buffer. The free Ca2+ con-centration can be varied by altering the EGTA/CaCl2ratio.

RaiochemicalsRadioisotopes were obtained from The Radio-

chemical Centre, Amersham, Bucks., U.K. [y-32p]_ATP was prepared from (32P]PI by isotope exchangeby the method of Glynn & Chappell (1964) and wasfurther purified on a Whatman DE-52 DEAE-cellulose anion-exchange column (C1- form) pre-equilibrated with 0.05 M-Tris/HCI, pH 8.0. The[y-32P]ATP was eluted with a linear KC1 gradient(0.05-0.5M). [y-32P]ATP was eluted at approx. 0.2M-KCI and was stored frozen at -20°C.

Other reagentsAT? (disodium salt), ADP (free acid), adenyl-5'-yl

imidodiphosphate [AMP-P(NH)P] and adenosine5'-(3-thio)triphosphate [AMP-PP(S)] (tetralithiumsalts), NADH, lactate dehydrogenase, pyruvatekinase and phosphoenolpyruvate were all obtainedfrom The Boehringer (London) Corporation Ltd.,Uxbridge Road, London, U.K. 6-Mercaptoinosine5'-triphosphate (thio-ITP), trinitrophenyladenosine5'-triphosphate (TNP-ATP) and formycin 5'-tri-phosphate (FTP) were kind gifts from Dr. J. F.Eccleston and Dr. D. R. Trentham, University ofBristol.Sodium deoxycholate (BDH reagent grade) was

recrystallized by the method of MacLennan (1970).The rare-earth elements gadolinium, europium andlanthanum were obtained from Koch-Light Labora-tories, Colnbrook, Bucks., U.K., as their chloridesalts. All other reagents were obtained from BDHChemicals Ltd. (Poole, Dorset, U.K.) and were ofAnalaR grade whenever available.

Protein determinationProtein concentrations were determined by the

method of Lowry et al. (1951), with bovine serumalbumin as the standard.

Enzyme activitiesThe Ca2++Mg2+-dependent ATPase catalytic

activity was measured either by pH-stat measurementor by linked-enzyme assay.pH-stat assay. Protons released on ATP hydrolysis

1976

720

SUBSTRATE BINDING BY Ca2++Mg2+-DEPENDENT ATP-ASE

were titrated with diluteNaOH by a Radiometer pH-stat assembly consisting of a type iTTi"llC titrator,type SBH la syringe burette and type SBR 2c penrecorder. Assays were performed at 20°C with anassay volume of 5ml. A 0.5ml burette containing5mM-NaOH allowed minimum rates of 0.02-0.031umol of ATP hydrolysed/min to be measured.The reaction vessel was flushed with N2 to minimizeinterference from absorption of atmospheric CO2.Linked enzyme-assay. The ADP formed by the

Ca2++Mg+-dependent ATPase was rephosphoryl-ated to ATP by using phosphoenolpyruvate withpyruvate kinase. Pyruvate was then converted intolactate with lactate dehydrogenase and NADH andthe rate ofNADH disappearance was measured in aspectrophotometer at 340nmn.

Reaction mixtures in general contained 20mM-Tris/HCl, 100mm-KCI, lOmM-Ca/EGTA giving afree Ca2+ concentration of 26pM, 0.5mM-MgCl2,0.1mM-phosphoenolpyruvate, 0.4mM-NADH,0.03mg of pyruvate kinase/ml and 0.03mg of lactatedehydrogenase/ml. A fivefold increase in pyruvatekinase concentration had no effect on the observedrate of NADH oxidation at all ATP concentrationsused. Pyruvate kinase and lactate dehydrogenase wereadded as suspensions in (NH4)2SO4.

Adenylate kinase assayAdenylate kinase was assayed in the presence of

ATPase by incubating enzyme with [2-3H]ADP,acid quenching, chromatographing the supernatantand counting the radioactivity of the [2-3H]AMPformed. In a typical assay, 3.4mg of enzyme was in-cubated in 1 ml of buffer containing 100mM-tri-ethanolamine/HCI, pH7.5, 3mM-[2-3H]ADP (specificradioactivity 3.33,uCi/,mol), 3nMM-MgCI2 and 30pM-CaC12. Samples (0.5ml) were removed and quenchedafter 0 and 5min, and the mono- and di-nucleotidesseparated by ion-exchange t.l.c. as described below.Radioactivity was measured by scintillation count-ing, after cutting out and eluting the spots.

Scintillation countingRadioactivity was measured by liquid-scintillation

counting in a Nuclear-Chicago Isocap 300 instru-ment. Samples were mixed with 15ml of scintillationliquid, composed of 3 litres of toluene, 2 litres of 2-methoxyethanol, 400g of naphthalene and 30g of2 -(4 - t-butylphenyl) -5 - (4 -biphenylyl) - 1-oxa-3,4-di-azole (butyl-PBD). This liquid would accept up to 5 %of aqueous sample without significant quenching of32p counts, but volumes of under 3% have been usedin the experiments described here. Radioactivity wasrecorded in two channels, and these were programmedfor each radioisotope used. Quenching was measuredeither from the ratio of counts in the two channels(sample-counts ratio) or from the distribution ofcounts from an external gamma source (9,uCi of

Vol. 159

133Ba), which produced a large number of countscompared with the sample (external-standard ratio).32p radioactivity was counted by using the 32P pro-gramme with sample-counts-ratio quench correction,45Ca radioactivity was counted by using the 14Cprogramme with sample-counts-ratio quench correc-tion, and 3H radioactivity was counted by using the3H programme with external-sample-ratio quenchcorrection. Quenching was normally insignificant for32P and 45Ca, but counts for 3H were calculatedfrom the channels ratio with the external source withthe aid of a quench curve prepared separately with aknown amount of 3H20 and increasing proportionsof water.

Phosphoryl-enzyme formationPurified Ca2++Mg2+-dependent ATPase was

mixed rapidly with Mg[y-32P]ATP and Ca/EGTAbuffer and the mixture quenched after 2s by squirtingan equal volume of trichloroacetic acid (0.612Mcontaining 2mM-ATP and 5mM-potassium phos-phate) into the reaction mixture from a syringe. Theprecipitated protein was separated by filtration on2.5cm-diam. Whatman glass-fibre discs (GF/D) andwashed with a further 30ml of trichloroacetic acid(0.306M) on a vacuum-filtration manifold. After beingsucked dry, the filter discs were placed in scintillationvials containing 15ml of scintillation fluid andcounted for radioactivity. Controls were carried outwith Mg[y-32P]ATP being added after quenching toestimate ATP contamination of the washed pellet.

Ligand bindingThe dissociation constants of MgATP, MgADP

and Ca2+ from the enzyme were measured by themethod described by Colowick & Womack (1969).A small flow-dialysis cell was used with a lowerchamber volume of 2.8ml and a membrane area of3cm2.Enzyme (1.8ml) and radioactive ligand in the

appropriate buffer (see Figure legends) were added tothe upper chamber and buffer alone was pumpedthrough the lower chamber at a flow rate of2.4ml/minby using anLKB Vario-Perpex pump. Both chamberswere stirred with magnetic stirring bars. A steady-state rate of dialysis was obtained 6min after addi-tions were made to the upper chamber.

Ion-exchange t.l.c.Nucleoside mono-, di- and tri-phosphates were

separated by ion-exchange t.l.c. on polyethylene-imine-cellulose thin-layer sheets (MN-Polygram Cel300 PEI/UV, from Camlab Ltd., Cambridge, U.K.).Streaks (20,ul) of samples were applied to the sheets(0cm x 5cm) and the chromatograms were deve-loped in 0.75M-KH2PO4, pH3.4, until the solventfront had travelled 8cm. This procedure is similarto that described by Goody & Eckstein (1971).

721

1). W. YATES AND V. C. DUANCE

Nucleotide spots were detected as dark areas agamsta fluorescent background when irradiated with short-wave u.v. light.

Results

Nucleotide bindingThe dissociation constant of MgATP from the

Ca2+tMg2+-dependent ATPase was determined bythe flow-dialysis method of Colowick & Womack(1969). It was essential that MgATP hydrolysis didnot occur during the time-course of the experiment;thus the binding was measured in the absence ofCa2+, all the solutions used were depleted of Ca+before use and the experiments were carried out in thepresence of 25mM-EGTA. This concentration ofEGTA complexed less than 1% of the added Mg2+at pH17.4. From the binding constants given bySchwarzenbach (1956), 98% of the ATP would becomplexed by Wg2+ ions at 54um-ATP and more than91 % at I mi-ATP. K+ ions have negligible bindingtoEGTA or ATP under these conditions (KATP3-is30mM at 1 mM-ATP). Typical results of such a deter-mination are shown in Fig. l(a), where the bindingcurve is shown with increasing MgATP concentra-tion. These data cannot be analysed as a simplerectangular hyperbola, as shown by the Scatchard(1949) plot in Fig, l(b), but replotting of the data as aHill plot (Brown & Hill, 1922-23) gives the resultshown in Fig. l(c), where points from three differentexperiments are plotted. A straight line is obtainedwith aHill coefficient (h) of0.79 and a half-saturating

MgATP concentration of SIMA. At the end of thebinding experiments the enzyme was removed fromthe dialysis cell, the protein precipitated with tri-chloroacetic acid (0.306M) and the denatured proteincentrifuged off in a bench centrifuge. The clear super-natant was neutralized with 4M-sodium acetate andthen analysed by tl.c. as described above. With theconditions stated above, less than 5% hydrolysis ofMgATP oCCurred as judged by [y32P]phosphateconcentrations in the enzyme supernatant.The binding of MgADP was also measured by the

rate-ofdialysis method, by using Mg[2-3H]ADP asthe radioactive ligand. The addition of non-radio-active MgAD)P were made with a Hamilton syringeto the upper chamber of the dialysis aell. The bindingcurveand Hill plot oftheresultsare shown inFigs.2(a)and 2(b). The results were corrected for quenching byusing a calibration curve obtained with 3H20 withthe scintillation counter set for 3H Counting in theextemal-standard-ratio mode. The Hill plot wasdrawn by using a bound maximum of 35MM obtainedfrom extrapolation of a Scatchard plot (not shown)to the bound axis. The plot gives a Hill coefficient of0.76, with half-maximum binding at a MgADP con-centration of 120um.

Phosphoryl-enzyme formationThe amount of phosphoryl-nz e formed at

different MgATP concentrations was measured by themethod given above, by quenching samples into tri-chloroacetic acid after 2s. The proportion of totalMgATP hydrolysed within this time period is negli-

4.0~~~~~~~~~~~~~~~~~~~~~'3 .0 40 ()

-2

S 2.0t 02.0 0 ,:i3~~~~~~~2 T.6

1.0 2

0 100 200 300 400t2o°1000 0 0.4 0.8 1.2 1.6 T.8 0.2 0.6 1.0 1.4 1.8

[MgATPJ (pM) Bound MgATP log[MgATP] (uM)Free MgATP

Fig. 1. BindingofMgATP to the Ca1++Mg2+-dependent ATPase determinedby the rate-of-dialysis method(a) Binding curve for MgATP to the Ca2++Mge+-dpendent ATPase detennined by the method of Colowick & Womnack(1969). To a solution of40pM-ATPase, lOOmM-KCl, 25mM-EGTA, l0mM-Tris/HCI, 1 mM-MgSO4 and 5pM_Mg&'-32PjATP(specific radioactivity of I.4mCi/pmol) at pH7.4 and 20QC was added non-radioactive MgATP, increasing the cncentrationfrom 5pm to 1 mm. The same mixturewithoutenzymeand MgATPwas pumpedthrough thelowerchamber.The radioactivity(c.p.m.) in each fraction was corrected for loss through dialysis. The radioactivity in the first fractions, before addition ofnon-radioactive MgATP, was subtracted from all subsequent fractions after correction for loss by dialysis, and results areplotted against the MgATP concentration. On average the increase in radioactivity over the baseline was approx. 50%.(b) The data in (a) were analysed as a Scatchard (1949) plot, [bound MgATPJ versus [bound MgATP]/[free MgATPJ. At agiven MgATP concentration, the ratio ofradioactivity/maximum radioactivity (determined at 1 mM-MgATP)representsthefraction ofthe MgATP unliganded. (c) A Hill plot of the data in (a). The maximum MgATP bound, [boundLm,,., was deter-mined by extrapolation ofa double-reciprocal plot to an infinite MgATP concentration. The half-saturating conoontrationof MgATP, i.e. when log([bound]i.m..- [boundD = 0.0, was 51 UM, with a Hill coefficient (slope) of 0.79. Points from twoadditional experiments have been added to this Hill plot.

1976

122

SUBSTRATE BINDING BY Ca2+M+Ng2+-DEPENDENT ATP-ASE 723

II' 'I ~~~~~~~~(b)3.0 (a) 0

O 2.0O

* '~ ~ ~~~~0

0 170 40 SQ0- 680 50 0 0.5 1.0 1.5 2.0 2.5[MgADP] (gMb) log[MgADP] (gm)

Fig. 2. Binding ofMgADP to the CQa++Mg2+-dependent ATPase determinedby the rate-of-dialysis method(a) A bindig curve for MgADP to the Ca2++Mg!-dependent ATPase determined by using the method of Colowick &Womack (1969) described in the Materials and Methods section. To a solution of40pM enzyme, lOOnM-KCl, lOmM-Tris/ttl,i1 MM-MgCI2, 10nM-Ca/ GTA giving a free [Caai of 26puM, and i*-Mg[2-H]ADP(specfic radioactivity 4.55mCi/

pmol), a(pIt7.4 aild J00C was added hon-radioactive MgADP, v fiti the-conceftaticn from Igm to I mM. Thsamemixture without enzyme and MgADP was pumped through the oWer chambr. The fAdioactivity in the fractions wascorreted for quencling and loss through dialysisas described inthe Materialsand Methods ection, and results are plottedagainstthe MgADP concntration. TW avrage max inmincresin uadioactivity over the baselin was 20-25%/. (b) AHill plot ofthe dstat io,() giving Kaw ,20 -nd Hill coefficient = 0.76.

0.8 .; 0

__0_ _4_-_ 4

I ~~- 2. 80 20 40 60 80 0.5 1.0 1.5

[MgATPI (aM) log[MgATP] (#M)Fig. 3.CFormtionofphosphoryl-enzymeby Ca2+Mg2+dependentATPaseasafunctionofMgATPconcentration

(a) The saturation curve was obtained by mixing 5pM enzyme with 0.5-48M-Mg[(-3PJATP (spific rdioactivity6.4jCi/gmol in a solution of lOOmM-KCI, 10mM-Tris/HCI and lOmu-Ca/EOTA giving a free 2+ concen;tration of33 pm at pH7A and 20°C. The reactions were terminated with trichioroacetic acid (O.306)2mM.ATP/Smm-Pt afterzero and 2s, and the concentration of phosphoryl-ezyme, [E-Pl wasma ease 'ibedin the Materials and Methodssection. (b) A Hill plot of the data 'in (a) giving KuY=37pM andaHl coeffic nt of 0.76.

0

0 .100 200 300 o 0.8 1.6 2.41/[MgATP](Gm1) log[MgATP] (.uM)

Fig. 4. Rate ofATP hydrolysis by Ca2++Mg2+-dependnt ATPase as afwniion ofATP coneentration monitored as NADHoxidation by the linked assay system

(a) A double-reciprocal4 plot of activity against MgATP concentration obtained by using reaction mixtures containinglOOmM-KCI, 1OmM-Ca/EGTA giving a free Ca+2 concentration of 26,pM, 20mM-Tris/HCI, O.5rMM-MgC12, 0.1 mM-phospho-enolpyruvate, 0.4mm-NADH, 0.1mg each of pyruvate kinase and lactate dehydrogenase, 0.3pM-Ca2++Mg2+-dependentATPase and MgATP concentrations ranging from 1.25,M to 1.22mM. Reactions were followed by measuring the dis-appearance of NADH extinction at 340mm at pH7.4 and 20°C. Two possible Km values werecalculated, l0 and444uM, thelatter from an expanded plot at the higher MgATP concentrations. (b) A Hill plot of the daia in (a), wher v is the ratemeasured at the various MgATP concentrations and V.ax. is the rate determined from plot (a) at an infinite MgATPconcentration. An apparent Km of 47.8pM and a Hil coefficient of 0.77 were calculated fromais.pjet.Vol.. 159

D. W. YATES AND V. C. DUANCE

gible. Fig. 3(a) shows a typical phosphorylationcurve. Analysis of these data by a double-reciprocalplot yields a non-linear graph but a Hill plot gives astraight line (Fig. 3b), with a Hill coefficient of 0.76and half-maximum phosphorylation at 374uM-MgATP. These values are close to those obtained forMgATP binding.

Ca2++Mg2+-dependent ATPase activityEnzyme catalytic activity was measured both by a

pH-stat assay and by using a MgATP-regeneratingsystem as described in the Materials and Methodssection. Although data from both methods gavesimilar results, those presented in Fig. 4(a) wereobtained by the linked-assay system, as this gavegreater accuracy with low MgATP concentrations.They are presented as a double-reciprocal plot andit is again evident that the data cannot be analysed asa simple rectangular hyperbola. Reprocessing theseresults as a Hill plot gives the result shown in Fig.4(b), where a straight line is obtained with a Hillcoefficient of 0.77 and a half-maximal rate at aMgATP concentration of 48AuM.The MgATP concentration giving half-maximal

activity (Ko.5) at different pH values was determinedby the pH-stat assay. The results are shown in Table1. Hill coefficients for assays at pH7.8 and 8.2 are oflimited accuracy, as all concentrations used wereabove the KO.5 value.

Ca2++Mg2+-dependent ATPase activity on ATPanaloguesCa2++Mg2+-dependent ATPase activity was as-

sayed in the presence of various chromophoric ATPanalogues by the pH-stat assay procedure to measurenucleotide hydrolysis. Those analogues that were notsubstrates were tested for inhibitory activity againstthe hydrolysis of MgATP. The results of these assaysare summarized in Table 2, which gives the values of

kcat. and Ki or K..5 for the analogues used. The datawere analysed as Lineweaver-Burk plots, because thelimited availability of these analogues allowed theconcentration to be varied over only a limited range.

Binding of cations to the Ca2++Mg2+-dependentATPaseThe binding ofCa2+ to the Ca2++Mg2+-dependent

ATPase was studied by the rate-of-dialysis methodof Colowick & Womack (1969) with 45Ca. Bindingwas measured both in the absence of nucleotide andin the presence of the MgATP analogues MgAMP-PP(S) and MgAMP-P(NH)P. MgATP could not beused because the catalytic rate of the enzyme was toogreat. MgAMP-P(NH)P was not hydrolysed by theenzyme, but MgAMP-PP(S) was hydrolysed slowly.To ensure that the MgAMP-PP(S) was not totallyhydrolysed during the course of the experiment, theenzyme was removed from the dialysis cell at the endof the experiment, precipitated with trichloroaceticacid and the solution clarified by centrifugation.

Table 1. Effect ofpH on the Ko.5 of the Ca2++Mg2+-dependent ATPasefor MgATP

Assays were performed by the pH-stat procedure withassay mixtures containing lO0mM-KCI, lOmM-Ca/EGTAgiving a free Caa2+ concentration of 33pm, and MgATPconcentrations varying from 0.075 to 0.575mM. Reactionswere started by the addition of0.4/M enzyme. Ko.5 valueswere obtained from Hill plots ofthe data at eachpH value.

pH Ko.5 (mM) Hill coefficient (h)

6.8 0.1317.1 0.1027.4 0.0477.8 0.0318.2 0.014

0.600.790.880.831.02

Table 2. Catalyticparameters ofATP analogues with the Ca2++Mg2+-dependent ATPaseAssays were performed by the pH-stat procedure with reaction mixtures containing lOOmM-KCl, lOmM-Ca/EGTA givinga free Ca2+ concentration of 26pM and various concentrations of substrates and inhibitors. Inhibition constants weredetermined by using MgATP as the substrate. Reactions were initiated by the addition of 0.5gM enzyme.

ATP analogue

MgATP, pH7.4MgATP, pH8.0ATP, pH7.4MgAMP-PP(S), pH7.4MgAMP-PP(S), pH8.0Mgthio-ITP, pH8.0MgFTP-P, pH8.0MgAMP-P(NH)P, pH7.4MgTNP-ATP, pH7.4

Ko.s (mM) K1 (DM) kcat. (s-1)0.0470.011

0.0650.0160.1660.154

8.5

0.2800.039

4.02.0

0.430.420.692.0

Concentrationrange used

(mM)

0.075-0.5750.050-0.5750.340-8.3400.156-0.9350.050-0.1900.050-0.1900.033-0.108

0.2700.100

1976

724

SUBSTRATE BINDING BY Ca2++Mg2+-DEPENDENT ATP-ASE

I300(a)~~~~~~~~b ~(c) $30(d)Iz 10 -( )1 -

co -+ 200 +' 200

~~cS~ ~ ~ ~ cS 'o~~5100 '0 1000

o 0 500 1000 1500 ° 0 500 1000 1500 0 8 16 24 E 0 8 16 24

[Ca2+] (pM) [Ca2+] (pM) [Bound Ca2+] [Bound Ca2O][Free Ca2+j [Free Ca2+]

Fig. 5. Ca2+ binding to the Ca2++Mg2+-dependentATPase measuredby the rate-of-dialysis method

Ca2+-binding curves were obtained by the rate-of-dialysis method of Colowick & Womack (1969) as described in theMaterials and Methods section, in solutions containing 12pM enzyme, lOOmM-KCl, lOmM-Tris/HCI, 5mM-MgSO4, 2pM-45CaCI2 (specific radioactivity 1 mCi/pmol), (a) without MgAMP-PP(S) and (b) with 5mM-MgAMP-PP(S) at pH7.4 and20°C. The Ca2+ concentration was increased to 1.5mM by the addition of portions of non-radioactive CaCI2. A similarsolution without enzyme and CaCl2 was pumped through the lower chamber. Scatchard plots of these data withoutMgAMP-PP(S) and with MgAMP-PP(S) are shown in (c) and (d) respectively. The maximum value for bound calcium,[bound]ma..,was taken from the intercept ofthe extrapolated line ofthe points for tight binding with the bound Ca2+-concen-tration axis ofthe Scatchard plots, at 24pM. Dissociation constants of 1.29 and 1.23pM and Hill coefficients of 0.82 and 0.83were determined from Hill plots (not shown).

6.0 '

4 4.0

i2.0 _- /

_ ~ ~~~~~~~~~/

0 200 400 600

1/[Cal+] (mm-')Fig. 6. Effect of Ca2+ on the steady-state phosphoryl-enzyme concentration during ATP hydrolysis by Ca2++

Mg2+-dependentATPaseA double-reciprocal plot of the results obtained when5pM enzyme was mixed with 5OpM-Mg[y-32P]ATP inlOOnM-KCI/lOnM-Tris/HCI and either lOmM-Ca/EGTAgiving free Ca2+ concentration of 1.8-26,pM, or for higherCa2+ concentrations CaC12, at pH7.4 and 20°C. The re-actions were quenched after 0 or 2s with trichloroaceticacid and the phosphoryl-enzyme concentrations measuredas described in the Materials and Methods section. Adissociation constant of 8.9pM was calculated from theintercept on the abscissa.

duced by hydrolysis would not bind to a significantdegree at these concentrations, and this is demon-strated by the good agreement between results in thepresence of MgAMP-PP(S) (slowly hydrolysed) andthose in the presence of MgAMP-P(NH)P (nothydrolysed). At the Mg2+ concentration used,nucleotides would be fully liganded with Mg2+ andless than 1% of Ca2+ added would be bound bynucleotide.The results of Ca2+-binding experiments in the

presence and absence of MgAMP-PP(S) are shownin Figs. 5(a) and 5(b). From Scatchard plots (Figs.Sc and 5d), two binding components are recognized,a tight-binding component with KD about 1 aM anda weaker component with KD about 200pM. Fromthe intercepts on the 'bound' axis, the tight-bindingcomponent represents 2mol of Ca2+/mol of enzyme,and the weaker component represents about 25molof Ca2+/mol of enzyme. The points for the tight-binding component can be replotted as a Hill plot,giving half-maximal Ca2+ binding at concentrationsof 1.29 and 1.23 flM without and with MgAMP-PP(S)respectively. The slopes of the Hill plots gave Hillcoefficients of 0.82 and 0.83. Similar results wereobtained by using MgAMP-P(NH)P as the nucleo-tide with half-maximal binding occurring at 3.3pM-Ca2+.

Samples of the supernatant were subjected to t.l.c.on polyethyleneimine-cellulose plates and inspectedat 254nm. The results showed less than 50% hydro-lysis of the MgAMP-PP(S) during the time-courseof the experiment. MgADP and thiophosphate pro-

Vol, 159

Phosphoryl-enzyme formation at different Ca2+concentrationsThe response of the steady-state phosphoryl-

enzyme concentration to different Ca2+ concentra-tions is shown in Fig. 6. To obtain these results theenzyme was incubated with 5OpM_Mg[y_32P]ATP andvarious concentrations of Ca2+ adjusted with Ca/

725

2 D. W. YATES AND V. C. DUANCE

6.0

4.0

- 2. 0

0

0 l/~~~I[Ca2+] (#M-1)Fig. 7. Effect of Ca2- on the steady-state catalytic activity

of the Ca2++Mg2+-dependent A7Pase

The enzyme activity was measured by the pH-stat methodby using Ca2+-depleted enzyme and reagents. Reactionmixtures contained lO0mM-KCI, 5mm-MgATP, 10mM-EGTA and various amounts of CaCi2 to give final freeCa2+ concentrations ranging from 0.1 to 100pM, and0.5pM enzyme at pH7.4 and 20'C. From the double-reciprocal plot, half-maximal activity was obtained at aCa2+ concentration of 4.4AM.

EGTA buffers. The reaction mixtures were quenchedafter 2s with trichloroacetic acid containing ATP andPi as described in the Materials and Methodsseion. The results are expressed as a double-recip-rocal plot, giving half-maximal phosphorylationwith a Ca2+ concentration of 8.9pM.-A Hill plot (results not shown) gave a similar Ko.5

for phosphorylation, with a Hill coefficient of 0.9. Ifphosphoryl-enzyme concentrations were measuredwith respect to time, they decreased as ATP washydrolysed.

Effect ofcation concentration on ATPase activityThe effect ofCa+ onATPase activity was measured

by monitoring the enzyme activity in the pH-stat andby using Ca2+-depleted enzyme and reagents. Theresults of these assays are shown in Fig. 7, wherethey are expressed as a double-reciprocal plot. Half-maximal MgATP hydrolysis occurs with a Ca2+concentration of 4.4gM. At Ca2+ concentrationsgreater than 354uM the ATPase is inhibited, as shownby the upward curve in the double-reciprocal plot.The chemical similarity between Ca2+ and the

rare-earth elements has led to their use as calciumanalogues in several enzymes, including a-amylase(Smolka et al., 1971), thennolysin (Colman et al.,1972) and trypsinogen (Damall & Bimbaum, 1970).Some experiments were performed to investigate theeffect of the rare-earth elements lanthanum, gado-

linium and europium- on the ATPase activity ofthe Ca2+-depleted Ca2++Mg2+-dependent ATPase.A pH-stat assay was used with reaction mixturescontaining lOOmM-KCI, 5mM-MgATP and 3.33-33.3piM-Ca2+ or rare earth at pH7.4 and 20°C.Reactions were started with O.5M (final concentra-tion) Ca2++Mg2+-dependent ATPase. The concen-trations of metals giving half-maximal rates were4.4,UM for Ca2+, 13/UM for gadolinium, 31A4M foreuropium and 15#uM for lanthanum. It is possiblethat a significant proportion of these cations arebound by ATP.

D

Nucleotide bindingThe binding of MgATP to the sarcoplasmic-

reticulum ATPase has been studied by flow dialysisand the results described in this paper show. thatMgATP binds, to the enzyme in the absence ofCa2+. The shape of the binding curve is not a simplerectangular hyperbola, but gives a curved Scatchardplot, as shown in Fig. 1. These data could be inter-preted as representing two binding constants forMgATP (Yamamoto & Tonomura, 1968), but analternative explanation would be weak negativeco-operative interaction between the protein mole-cules, and, ifthis is assumed, the data can be expressedas a Hill plot (Fig. 2), giving half-maximal bindingwith 44#M-MgATP and a Hill coefficient if 0.82.The enzyme has been shown to exist as a single poly-peptide chain with mol.wt. 100000 (Martonosi &Halpin, 1970) and the MgATP-binding data in Fig.1 indicate that one molecule of MgATP is bound perpolypeptide chain. It is therefore difficult to envisagetwo different binding sites for MgATP on two iden-tical polypeptide chains, unless there is interactionbetween adjacent molecules in the membrane. TheMgADP-binding data show a similar non-linearScatchard plot (Fig. 2), with an affinity for MgADPwhich is about one-third of that for MgATP.

Phosphorylation of the enzyme could be measuredby quenching the enzyme 2s after mixing with Mg-[y-3P]ATP in the presence of Ca2+. It has beenestablished by independent experiments (V. C.Duance & D. W. Yates, unpublished work) thatphosphoryl-enzyme concentrations remain constantfor at least 2s'after the initial rapid phosphorylation.The results in Figs. 3(a) and 3(b) show that the satura-tion curve for enzyme phosphorylation has a similarshape and half-saturation point to the binding curvefor MgATP, with half-maximal phosphorylation at37,M-MgATP. This suggests that the equilibriumfor the phosphoryl-transfer reaction:

Ca2 Ca2E-M&ATP; E.P

EMTMgADP1976

726

SUMSATkT BgtNING BY Ca2*+Mg2+-DEPENDENT ATP-ASE

mfust be netit to or less than uhity, with therate-limiting stop being subsequent to phosphoryl-ation.As a consequence of the equilibrium of the phos-

phoryl-transfer reaction being less than unity the/ Ca2

ternary compleX E-tMgATP is k major constituentof the steady-state complex. It then follows that theresponse of the stady-state catalytid activity to th-eMgAWT concentration should be similar to thatfound in the binding and phosphorylation experiAments (Fig. 2 and 3)j This was confirmed as shown inFigs. 4(a) and 4(b), half-maximal activity Wingattained mt 48 .MgATP1. Bsides the Sitnilatlty hithe MgATP oodenftfion tequlred tot halffMftnals&tuatibn, 6bvphbrylation nd catalytic actiVity,all three parameters showed similat iegativ eo-operatlve eftt with ttspeettto MgA1,- aS showh bythe slopst of the H1l1 plbt§, 0). 2, 0.76 anld 0.77 fbrMgATP bidi, phosphoriyatit,n and catalyticactivity respettively.The rate of phosphoryl-entMe formation is fast

compared with the hydrolysis of MgAT1 by theenzyme (3100s forthe observeddrate of phosphoryla-tion at high ATP cpmpared with 4s-1 for enzymecatalytic activity; V. C. biuanoe & D. W. Yates,unpublished work). As the equilibrium of the phos-phoryl-transfer reaction is near to unity, the reversereaction, phosphorylation ofMgADP by phosphoryl-enzyme, must also be fast, as suggested by results ofFroehlich & Taylor (1975) and V. C. Duance &D. W. Yates (unpublished work).As the half-maximal phosphorylation and ATPase

catalytic activity occur at the same MgATP concen-tration required for half saturation, with the formertwo being determined in the presence of Ca2+ and thelatter in its absence, it can be concluded that Ca2+does not influence the binding of MgATP.

Cutioft bi*dng to the ea2++Mg2+-dependent AtPaseFig. S shows the bindihg of CAI to the

Ca-++Mg2t-dependent AtPase in the presefce andab9bnce of a bound nUcledtide. In both cases thestOicheiobnetfy Of a2* binding ihdicates that 2Ca2+ioOs tar tightly bound pet enzyme molecule, inagreemtent with the published data of Hasselbachi(l064) and YaMada et at. (197b). MgAM-PP(S)was chosen as the analogud, as its hydrolysis tate ismuch slower than that of MgATP; thus an appreci-able aknunt remined at thd tnd of the exeimtnt.The resultS show that the pttsece of a nucleotide[AMP-PP(g) or AMP4PQ4HMP] does not Aftt thEaffinity of the enzye for C+ and that the enzymeshows negative co-operativity for Ca'4 bidding inboth cases. The degree of co-operativity is similarto that found by Meissner (1973) for Ca2+ binding.Although the presence of a bound nucleotide does

not affect the binding ofCa2+ to the enzyme; the bind-ing of C2+ to the phosphorylenzyme may be dif-ferent. The catailyti rate of hydrolysis of MgAMP-PP(S) has been shown to be sihilat at pH7.4 and 3.0(Table 2), di'ering from that of MgATP it that therate at pH7.4 is double that ireasuted at pt18.0. Apbssible explanation for this could be a thange ih therate-limiting step, as was found with the niysinATPase (Bagshaw et at., 1072), where the hydrolysisrate of the MgAMP-PP(S) is rate-limiting, If this isthe case with the Ca2++Mg2+-depandent ATPase,the steady-state concentration of phosphoryl-enzymewould be very low when hydrolysing MgAMP-PP(S);thus any change in affinity for Ca2+ between theenzyme and phosphoryl-enzyme would not be ob-served. The rather higher concentrations of Ca2+required for half-maximal phosphorylation of theenzyme may suggest a lower affinity of the phospho-ryl-enzyme for Ca2+ than the free enzyme, but moredata are required to test this suggestion.The data presented in this paper suggest that bind-

Ca2E + 2Ca2+ ,± E

E + MgATP ' E. MgATP + 2Ca2+

/Ca2

E-P\MgADP

Scheme 1. Reversibleformation ofphosphoryl-enzymefrom enzyme, MgATPandCa2+

Vol. 159

+ MgATP

,\\\ ,leCa2

MgATP

I1

172

728 D. W. YATES AND V. C. DUANCE

ing of Ca2+ and MgATP to the Ca2++Mg2+-depen-dent ATPase is a random process, as indicated byScheme 1, with affinities for Ca2+ and MgATP beingunaffected by the presence of liganded MgATP andCa2+ respectively. The data are consistent with theresulting ternary complex being able to react to givethe phosphoryl-enzyme in a reversible manner, withthe equilibrium being in favour of the ternary com-plex with MgATP and calcium.

We thank Professor H. Gutfreund for his help andencouragement and Dr. D. R. Trentham and Dr. J. Eccle-ston for helpful discussions and the gift ofATP analogues.This work was supported by a grant from the ScienceResearch Council.

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1976