Proc. Nat. Acad. Sci. USAVol. 71, No. 4, pp. 1083-1087, April 1974

Calorimetric Studies of the Role of Magnesium Ions in YeastEnolase Catalysis

(metalloenzymes/proton equilibria/flow microcalorimetry/thermal titrations)

LARRY D. FALLER AND ALAN M. JOHNSON

Department of Chemistry, Wesleyan University, Middletown, Connecticut 06457

Communicated by Max Tishler, November 30, 1973

ABSTRACT The binding of magnesium ions and ofthe competitive inhibitor 3-phospho-D-glyceric acid toyeast enolase (2-phospho-D-glycerate hydrolyase, EC4.2.1.11) has been studied calorimetrically. Thermaltitration of the apoprotein with magnesium ions providesevidence that two magnesium ions bindc immeasurablytightly to the dimeric enzyme, either anticooperatively tointeracting sites or to two independent, nonidentical sites.Measurements of the saturation heat in buffers withdifferent enthalpies of protonation are consistent with therelease of two protons when the metal-binding sites arefilled at pH 7.5. The enthalpy of binding of the two mag-nesium ions, corrected for the release of two protons, is+11.7 kcal (+49.0 kJ) per mole of dimeric protein.Thermal titration of the magnesium-saturated enzyme

with 3-phosphoglyceric acid corroborates the conclusionof Spring and Wold [Biochemistry (1971) 10, 4655-46601that the enolase dimer possesses two equivalent and inde-pendent substrate-binding sites. The dissociation con-stant for the enzyme-inhibitor complex calculated fromthe thermal data is 2 mM. The thermal studies of 3-phosphoglyceric acid binding also confirm that metalions are required for substrate binding and that substratebinds at the two specific metal-binding sites on the apo-protein. Experiments in buffers with different enthalpiesof ionization provide evidence for proton uptake when 3-phosphoglyceric acid is bound.

Enolase (2-phospho-D-glycerate hydrolyase, EC 4.2.1.11)catalyzes the dehydration of 2-phospho-D-glycerate to phos-phoenolpyruvate in glycolysis. Divalent metal ions are re-quired for activity, and their function has been extensivelyinvestigated. In a recent review of the literature, Wold (1)concluded that enolase is a metal-activated metalloenzyme,in which metal ions play both structural and catalytic roles.The evidence that metal ions play a structural role in

enolase catalysis comes primarily from difference spectro-photometric and spectrofluorometric measurements and fromsedimentation equilibrium experiments. Yeast enolase dis-sociates into two, apparently identical subunits (2, 3). Thebinding of divalent metal ions has two important structuralconsequences. First, it reduces the dimer dissociation constantby as much as two orders of magnitude (2). Second, it causesultraviolet absorption and fluorescence emission changes inthe dimer that have been interpreted as a change to a cat-alytically active conformation of the enzyme (4, 5).

Nuclear magnetic resonance experiments provide theprincipal evidence for direct involvement of divalent metalions in the active site of enolase. The effect of substrate on

the relaxation rates of solvent protons has been interpretedas evidence for a ternary enzyme-metal-substrate complex,although the possibility of a substrate-induced conformationalchange affecting the environment of the paramagnetic Mn2+ion in the protein could not be excluded (6). Recently theeffect of the enolase-Mn2+ complex on the relaxation rates ofthe protons and phosphorous atom of a phosphoenolpyruvateanalogue has been measured (7). It was concluded that phos-phoenolpyruvate binds in the second coordination sphere ofthe enzyme-complexed Mn2+ ion. The proposed role of themetal ion in the hydration reaction is to increase the nucleo-philicity of the slowly exchanging, inner-sphere water mole-cule, facilitating hydroxyl attack on carbon-3 of the substrate.A total of four divalent metal ions have been shown to

bind specifically to the yeast enolase dimer by equilibriumdialysis (8). Two metal ions bind anticooperatively to theapoprotein. In the presence of substrate two additional metalions bind one to two orders of magnitude less tightly. Itappears that the order of binding to each subunit is metal,substrate, metal, and that both the structural and the cata-lytic functions that have been described are associated withthe first two metal ions bound per dimer.The first equivalent of Mg2+ bound stabilizes the dimer

and causes the ultraviolet difference spectrum between in-active and active enzyme (2, 5). Two experiments suggestthat occupation of the second metal-binding site may alsoinfluence the protein structure. First, the ultraviolet differencespectrum is caused by a single equivalent of Mn2+, but com-petitive binding experiments indicate that the preference ofMg2+ and Mn2+ for the first two metal-binding sites is re-versed (4, 8). Second, two equivalents of Zn2+ ion are requiredto cause the ultraviolet difference spectrum (Faller, to bepublished).

Titrations of yeast enolase with chromophoric competitiveinhibitors have been interpreted to mean that metal ions arerequired for substrate binding and that there are two equiv-alent substrate-binding sites per dimer (9). Since the nuclearmagnetic resonance measurements of the effect of the enolase-Mn2+ complex on the proton and phosphorous relaxationrates of a phosphoenolpyruvate analogue were made withless than one equivalent of metal ion present per dimer (7),substrate apparently binds at the two tight metal-bindingsites on the apoprotein.Flow microcalorimetry offers important advantages for

studying the role of divalent metal ions in enzymatic catalysis.No optical change is required, since the heat changes ac-companying metal ion and competitive inhibitor binding to

Abbreviations: Hepes, N-2-hydroxyethylpiperazine-N'-2-ethane-sulfonic acid; Pipes, piperazine-N,N'-bis(2-ethanesulfonic acid).

1083

1084 Biochemistry: Faller and Johnson

-15 ' ' '

-10_ daH

(kcal/mole)

-5

S0 2 3 1 5 10 IS5

Equivalents Mg2* Per Dimer

FIG. 1. Thermal titration of enolase with Mg(OAc)2 in pH 7.5Tris buffer, 0.05 M in acetate ion. 145 AM [Elo < 261 MM.T = 25.00C. The dashed line is the expected curve for bindingat a single site. The solid line is the theoretical curve calculatedfor anticooperative binding to two sites differing 50-fold in theiraffinity for the ligand. Details of the calculations are given in thetext.

the protein are measured directly. The high sensitivity of thetechnique permits accurate measurements on small amountsof recoverable enzyme (10). We report the initial results offlow microcalorimetric studies of the role of divalent metalions in yeast enolase catalysis. The order and stoichiometrywith which the first two metal ions and substrate have beenreported to bind are corroborated. It has been possible todistinguish between nonequivalent, possibly anticooperative,binding of the metal ions to the enolase dimer and the bindingof substrate molecules to two identical sites. Evidence ispresented that protons are released by the system when metalions bind and that protons are taken up when substrate binds.Neither of these proton equilibria has been previously re-

ported.

MATERIALS AND METHODS

Enolase was isolated from baker's yeast as described by West-head (11). The crude enzyme was purified by a modificationof the method used by Westhead and McLain (12) to separateenolase isozymes. Stepwise elution was followed by gradientelution from DE-32 microgranular cellulose (Whatman).The enolase used in the experiments reported had a specificactivity at 240 nm of 300 i 30 units. An optical factor of 0.89cm-1 mg-' ml (13) and a molecular weight of 88,000 (14)

TABLE 1. Enthalpy of binding of Mg2+ ions to enolase invarious buffers at pH 7.5 and 25°C

AH (kcal/mol)

Protonation Mg2 +Buffer* (ref.) Measured bindingtTris -11.3 (17) -11.4 +11.2Hepes -5.0 (18) +2.3 +12.3Pipes -2.7 (18) +6.2 +11.6

Mean + standard deviation = +11.7 i 0.6(+49.0 + 2.5 kJ/mole)

* Buffer value (15) 0.0214. Tris and Hepes, 0.05 M ionicstrength. Pipes, 0.06 M ionic strength.

t AH (binding) = AH (measured) -22 AH (protonation).

were used to convert absorbance readings at 280 nm to theconcentration of dimeric enzyme. Enolase containing lessthan 0.05 equivalents of divalent metal ions per dimer wasprepared by passage through a column containing AG 501-X8and AG50W-X8 ion-exchange resins (Bio * Rad).

Tris base was purchased from Sigma. Hepes (N-2-hydroxy-ethylpiperazine-N'-2-ethanesulfonic acid) and Pipes [pipera-zine-N,N'-bis(2-ethanesulfonic acid) ] were bought fromCalbiochem. All measurements were made in pH 7.5 bufferswith a buffering value (15) of 0.0214. The ionic strength ofthe Tris and Hepes buffers was 0.05 M, and that of the Pipesbuffer was 0.06 M. 3-Phospho-D-glyceric acid was obtainedfrom Calbiochem. Analyzed reagent grade magnesium acetatewas purchased from J. T. Baker.

Calorimetric measurements were made with a flow modifica-tion of the Beckman Model 190B microcalorimeter (16) at25.00 i 0.010C. Sturtevant (10) has estimated the usefulsensitivity of the instrument as 0.1 gcal/sec (0.42 ,IJ/sec).A conservative estimate of the precision attainable at the flowrate (0.736,gl/sec) and enzyme concentration (approximately0.2 mM in the driving syringe) used in the reported experi-ments is therefore 0.7 kcal/mole (3 kJ/mole). Appropriatecorrections were made for viscous heating and for the heatsof dilution of enzyme, metal ions, and inhibitor. Metal ionconcentrations were monitored with a Perkin-Elmer Model303 atomic absorption spectrophotometer.

RESULTS

Metal Binding. The result of thermally titrating enolasewith Mg2+ ions in pH 7.5 Tris buffer is shown in Fig. 1. Thirty-eight independent experiments at different enzyme andmetal ion concentrations are summarized in the figure. Thosecorresponding to the same number of equivalents of Mg2+ perdimer are shown averaged. The mean and standard deviationof eleven measurements of the saturation heat (>2 equivalentsof Mg2+) in Tris are -11.4 4± 0.6 kcal/mole (-47.7 + 2.5kJ/mole).The saturation heat was remeasured in two buffers with

heats of protonation significantly different from that of Tris.The results of those experiments are summarized in Table 1.Published values for the enthalpies of protonation at 250C ofTris (17) and of Hepes and Pipes (18) are tabulated in thesecond column. The heats evolved in each of the three bufferswhen saturating amounts of Mg2+ were mixed with apo-enzyme are reported in the third column. The value recordedfor Hepes is the average of three independent experiments,and the value recorded for Pipes is the average of two deter-minations.

Inhibitor Binding. 3-Phospho-D-glyceric acid competi-tively inhibits yeast enolase (19). The enzyme-Mg2+complex was thermally titrated with 3-phosphoglyceric acid.Fig. 2 is a Scatchard (20) plot of the results. The assumptionsmade in plotting the data in this form are described in theDiscussion. Measurements of the heat of competitive inhibitorbinding in the pH range where enolase is catalytically active(19) are complicated by the fact that the second pKa ofphosphoric acid is 7.21. Consequently, to avoid a contributionto the measured heat from ionization of the phosphoryl groupof 3-phosphoglyceric acid that is independent of inhibitorbinding to the protein, all solutions were carefully adjustedto pH 7.5 at the temperature of the calorimeter bath by use ofan expanded scale pH meter. Nevertheless, scatter in the

Proc. Nat. Acad. Sci. USA 71 (1974)

Role of Magnesium Ions in Enolase Catalysis 1085

2.0

.5

ZV

1.0 Co0

0.5 r

0 2 4 6 8 10

0-2 (Z)/C3-PGA:)

FIG. 2. Scatchard (20) plot for the thermal titration of theenolase-Mg2 + complex with the competitive inhibitor 3-phospho-D-glyceric acid (3-PGA). v is the fraction of the proteintitrated, assuming two identical and independent inhibitorbinding sites. The assumptions made in constructing the plotare discussed fully in the text. The best straight line shown drawnthrough the experimental points was obtained by the method ofleast squares. The bar indicates the scatter expected about theleast-square line from an uncertainty of 10% in the measuredenthalpy of binding. The measurements were made at 25.00Cin pH 7.5 Tris-acetate buffer, 0.05 M in acetate ion. 69 MAIM <

[E]o S 180 iMM. 0.13 mM < [3-phosphoglyceric acid]0 17.7mM. [Mg2 +] 0 = 1 mM.

measured heat of 3-phosphoglyceric acid dilution introducesadditional uncertainty into the enthalpies reported for 3-phosphoglyceric acid binding. The bar in Fig. 2 shows thescatter introduced into the Scatchard plot by a 10% un-

certainty in the enthalpy of binding.Fig. 3 shows the result of titrating enolase with Mg2+ and

then mixing it with an excess of 3-phosphoglyceric acid in thecalorimeter. Twenty-three independent experiments are

summarized in the figure. Those corresponding to the same

number of equivalents of Mg2+ per dimer are shown averaged.One millimolar EDTA was presented in the experimentsplotted as zero equivalents of Mg2+.The heat absorbed when 3-phosphoglyceric acid binds to

enolase has been measured in Tris, in Hepes, and in Pipesat the same enzyme, metal, and inhibitor concentrations. Theresults are reported in Table 2. Each recorded value is theaverage of three independent measurements.

DISCUSSION

Mletal Binding. Equilibrium binding studies indicate thattwo magnesium ions bind specifically to apoenolase (8) even

though the binding of only a single magnesium ion is observedin spectrophotometric and spectrofluorometric titrations (4,5). The thermal titration of enolase with Mg2+ confirms thatthere are two specific metal-binding sites per mole of dimer.The dashed line in Fig. 1 is the expected curve for binding at a

single site. It was constructed for an enzyme concentration of164 MM by averaging the heats evolved in titrations with more

than two equivalents of magnesium ion to obtain the satura-tion heat, and by evaluating the metal dissociation constantfrom the free Mg2+ ion concentration at which half the maxi-mum heat evolution was observed (26 MM). It is apparentthat the maximum decrease in enthalpy is approached tooabruptly to be explained by the binding of magnesium ions

15 |0

(kcal/mole)

5 X

0 2 3 4 5

Equivalents Mg2+ Per Dimer

FIG. 3. Enthalpy of binding of the competitive inhibitor 3-phospho-D-glyceric acid to enolase titrated with MIg2+. The dataare plotted for [E]o = 90 IAM and [3-phosphoglyceric acid]0 =

17.4 mALI. The solid line is the expected curve for binding of theinhibitor to two identical and independent sites with KI = 2 mM.The binding of Mg2+ ions was assumed to be immeasurably tight.Measurements were made at 25.00C in pH 7.5 Tris-acetate buffer,0.05 WI in acetate ion.

to a single site on the enzyme. Additional evidence that thereare two metal-binding sites is provided by the observationthat two equivalents of Zn2+ are required in the spectro-photometric titration of enolase (Faller, to be published)and by the stoichiometry and metal requirements for sub-strate binding discussed below.

Since only two equivalents of Mg2+ are required in thethermal titration of enolase (Fig. 1), the existence of twosites means that the binding to both sites must be immeasur-ably tight (dissociation constant < 10 AM). The dissociationconstants evaluated from equilibrium dialysis experiments are

9.6 and 470 MM (8), but those measurements were made athigh ionic strength (0.5 M). Spectrofluorometric titration atlow ionic strength gave more than an order of magnitudetighter binding for the first magnesium ion (5). Spectro-photometric titrations under the same experimental conditionsas used in this calorimetric study confirm that binding of thefirst Mg2+ ion and of both Zn2+ ions to apoenolase is im-measurably tight (Faller, to be published).The thermal titration curve in Fig. 1 is also incompatible

with two identical and independent binding sites, becausehalf of the total heat is evolved when less than one equivalent(0.66 equivalents) of Mg2+ has been added. The data are,

however, consistent either with anticooperative binding ofMg2+ ions to two interacting sites, or with binding to twononidentical, noninteracting sites.The solid line in Fig. 1 is the theoretical curve calculated for

anticooperative binding to two sites differing 50-fold in their

TABLE 2. Enthalpy of binding of 3-phosphoglyceric acid toenolase in various buffers at pH 7.5 and 250C

Buffer* AHt (keal/mol)

Tris +10.9Hepes +8.3Pipes +5.0

*Buffer value (15) 0.0214. Tris and Hepes, 0.05 M ionicstrength. Pipes, 0.06 M ionic strength.

t [ENo = 164 MM, [3-phosphoglyceric acid]o = 5.84 mM, and[Mg2+]O = 1 mM.

Proc. Nat. Acad. Sci. USA 71 (1974)

1086 Biochemistry: Faller and Johnson

affinity for the ligand (21). The factor fifty was estimated fromthe dissociation constant reported by Hanlon and Westhead(8) by assuming that the relative affinity of the two bindingsites for Mg2+ is unaffected by ionic strength. The curveshown was generated for dissociation constants from the twopresumed conformers of the protein of 2 and 100 nM. Theenthalpy change when the first equivalent of Mg2+ bindswas estimated as -8.8 kcal/mol (-36.8 kJ/mol) by linearlyextrapolating the initial portion of the experimental curveto one equivalent of Mg2+. The enthalpy change when twoequivalents of Mg2+ bind (-11.4 kcal/mol or -47.7 kJ/mol)is given by the saturation heat. An even better fit to theexperimental data could probably be obtained for slightlydifferent choices of the ratio of the two dissociation constantsand of the enthalpy of binding of the first magnesium ion,but the standard deviation of the experimental points fromthe theoretical curve shown (0.5 kcal/mol or 2.1 kJ/mol) iswithin our estimate of the precision of our measurements.

Satisfactory fit to the experimental data can also be ob-tained by assuming two nonidentical, noninteracting sites.The standard deviation of the experimental points from thetheoretical curve for binding to two independent sites differing50-fold in their affinity for Mg2+ ions (K1 = 2 nM and K2 =100 nM) is the same as the standard deviation from thecurve for anticooperative binding shown in Fig. 1 (0.5 kcal/mol or 2.1 kJ/mol). Nonequivalent binding sites could bederived from apparently identical subunits (3) by the forma-tion of an unsymmetrical dimer.Heat is absorbed when enolase is saturated with Mg2+

ions in Hepes and Pipes buffers (Table 1, column 3). It isapparent from the variation of the saturation heat in bufferswith different heats of ionization that metal binding is coupledto proton equilibria. If it is assumed that two protons arereleased by the system when the two metal-binding sites arefilled, then the enthalpy change attributable to binding of themetal ions (Table 1, column 4) is the difference between themeasured enthalpy change (column 3) and twice the heat ofprotonation of the buffer (column 2). As shown in column 4 ofTable 1, this difference in the three buffers studied is constant(+11.7 ±0.6 kcal/mole or +49.0 ± 2.5 kJ/mol) withinexperimental error, providing evidence for the release of twoprotons when the metal-binding sites on enolase are filled byMg2+ ions at pH 7.5. Since the observed stoichiometry couldresult from the partial ionization of more than two groups,we are investigating the release of protons as a function ofpH in an effort to learn the number and identity of the groupswhose acid dissociation constants are shifted by metal binding.

It is evident that the Mg2+ dissociation constants at lowionic strength must be evaluated before entropic contributionsto metal binding can be assessed. Two additional questionsare raised by the initial results of our calorimetric investiga-tion of Mgt+ binding to yeast enolase that must be answeredbefore the thermodynamics of metal binding to the proteincan be understood. First, is one proton released when eachMg2+ ion binds, or does the release of both protons coincidewith the conformational change triggered by binding of thefirst equivalent of magnesium ion? Second, what are thecontributions of metal binding and of the resultant conforma-tional change to the increase in enthalpy associated withbinding of the first equivalent of magnesium ion?

ITnhibitor Binding. Titrations of yeast enolase with chromo-

that there are two identical and independent substrate-bind-ing sites per dimer (9). Thermal titration of the enolase-Mg2+complex with 3-phosphoglyceric acid supports this interpreta-tion. Fig. 2 is a Scatchard (20) plot of the thermal titrationdata. In constructing the plot it was assumed that there aretwo substrate-binding sites per dimer, that the sites areidentical, and that they do not interact. In addition, it wasnecessary to know the enthalpy increase when all the sitesare filled with 3-phosphoglyceric acid. Linearity of the result-ing plot provides a test of the correctness of the assumptionsmade in constructing it. The number of binding sites cannotbe deduced from the titration with 3-phosphoglyceric acid.The experimental data can be fitted equally well by assuminga single site, or two equivalent, independent sites. The justi-fications for interpreting the data in terms of two sites are thework of Spring and Wold (9) and our experiments, describedin the next paragraph, in which enolase fractionally complexedwith Mg2+ was reacted with 3-phosphoglyceric acid. Since itwas impractical to use saturating amounts of 3-phospho-glyceric acid, the maximum enthalpy increase (+13.6 kcal/mol or +56.9 kJ/mol) was evaluated by an interative pro-cedure. For two identical and independent sites, the fractionof the protein titrated (v) is given by the measured enthalpychange divided by the enthalpy of 3-phosphoglyceric acidbinding per site (+6.8 kcal/mol or +28.5 kJ/mol). Thestraight line shown drawn through the experimental points inFig. 2 is the least-square line. The number of sites, given bythe ordinate intercept, is 1.98 i 0.09. The dissociation con-stant of the enzyme-inhibitor complex, calculated from theslope of the least-square line, is 1.93 ± 0.14 mM. The standarddeviation (0.5 kcal/mol) of the experimental points from thethermal binding curve calculated for a dissociation constantof 2 mM and an enthalpy of binding per site of +6.8 kcal/molis within the estimated precision of our measurements. Thedissociation constant evaluated from the thermal titrationcurve (2 mM) lies between the values of 0.5 (19) and 5 mM(8) previously published for the 3-phosphoglyceric acid-enolase complex.The results of titrating enolase with Mg2+ and then mixing

it with an excess of 3-phosphoglyceric acid in the calorimeterare shown in Fig. 3. The solid line is the calculated curve forbinding of the inhibitor to two identical, noninteracting siteswith a dissociation constant of 2 mM and an enthalpy ofbinding of +6.8 kcal/mol per site at an enzyme concentrationof 90 ,M and an inhibitor concentration of 17.4 mM. Mg2+binding was assumed to be immeasurably tight. The standarddeviation of the experimental points from the theoreticalcurve in Fig. 3 is 0.9 kcal/mol (3.8 kJ/mol). The consistencyof the results shown in Figs. 2 and 3 increases confidence inthe values of the 3-phosphoglyceric acid dissociation constantand the enthalpy of 3-phosphoglyceric acid binding deducedfrom the thermal titration of the magnesium enzyme with3-phosphoglyceric acid. More importantly, the results por-trayed in Fig. 3 corroborate the conclusion of Spring and Wold(9) that metal ions are required for substrate binding. Onemillimolar EDTA was added to insure that the enzyme wasmetal-free in the experiments plotted as zero equivalents ofMg2+. Since heat continues to be absorbed until two equiv-alents of Mg2+ are bound, the thermal data in Fig. 3 supportthe conclusion that there are two substrate-binding sites perdimer (9). Finally, since heat is absorbed when fewer thantwo equivalents of Mg2+ are present, the experiments sum-

Proc. Nat. Acad. Sci. USA 71 (1974)

phoric competitive inhibitors have been interpreted to mean

Role of Magnesium Ions in Enolase Catalysis 1087

marized in Fig. 3 provide evidence that substrate interactswith the first two metal ions bound by enolase. This conclusionis supported by the nuclear magnetic resonance experimentsof Mildvan and coworkers (7). Since only a fraction of an

equivalent of metal was present in their studies of the effectof the enolase-Mn2+ complex on the proton and phosphorousrelaxation rates of a phosphoenolpyruvate analogue, sub-strate must bind at the first and second metal-binding sites.The magnitude of the enthalpy increase when 3-phospho-

glyceric acid binds to metalloenolase is different in bufferswith different heats of ionization (Table 2). The same enolase,Mg2+, and 3-phosphoglyceric acid concentrations were usedin the experiments summarized in Table 2 to insure that thefraction of sites occupied in each buffer was the same. Quali-tatively similar results were obtained at a 20-fold higher3-phosphoglyceric acid concentration. The qualitative con-

clusion that protons are taken up when 3-phosphoglycericacid binds to the enolase-Mg2+ complex seems inescapable.Quantitative interpretation of the data is complicated by thefact that there are two sources of protons, the buffer and 3-phosphoglyceric acid. Neither the pKa of the phosphorylgroup of 3-phosphoglyceric acid nor its enthalpy of ionization,has been published. It may be that it is the phosphoryl group

of 3-phosphoglyceric acid that is protonated because of a shiftto higher pKa when the inhibitor binds to the protein.

We thank Professor Julian M. Sturtevant for the use of theflow microcalorimeters in his laboratory at Yale University.LDF is deeply grateful to Professor Edward W. Westhead of theUniversity of Massachusetts for his support and encouragement.The baker's yeast from which enolase was isolated was generouslysupplied by Anheuser-Busch, Inc. This research was supportedby National Science Foundation grant GB-36704.

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4247-4255.9. Spring, T. G. & Wold, F. (1971) Biochemistry 10, 4655-4660.

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Proc. Nat. Acad. Sci. USA 71 (1974)