J. Biol. Chem.-1975-Knowles-1809-13

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THE JOURNAL F Bro~oa~cnr.CHEMISTRYVol. 250, No. 5, Issue f March 10, PP. X309-1813, 975

Printed in U.S.A.

Acetyl Phosphatidylethanolamine in theReconstitution of Ion Pumps*

(Received or publication, July 25,1974)AILEEN F. KNOWLES, ANNE KANDRACH, AND EFRAIM RACKERFrom the Section of Biochemistry, Molecular and Cell Biology, Cornell University, Ithaca, New York 14850H. GOBIND KHORANAFrom the Departments of Biology and Chemistry, Massachusetts nstitute of Technology, Boston, Massachusetts02139

SUMMARYAcetyl phosphatidylethanolaminewas comparedwith phos-phatidylethanolamine in the reconstitution of several biolog-ical membrane activities with the following results.1. The proton pump reconstituted with the purple mem-brane of Halobacterium halobium and acetyl phosphatidyl-ethanolamine was quite active. However, some differencesin the kinetic properties, particularly in the decay rate, werenoted between vesicles reconstituted with phosphatidyleth-anolamine and acetyl phosphatidylethanolamine.2. Acetyl phosphatidylethanolamine could not replacephosphatidylethanolamine in the reconstitution of a Ca2+pump with ATPase isolated from sarcoplasmic reticulum.However, inclusion of suitable amounts of stearylamine or

oleylamine during reconstitution yielded acetyl phosphatidyl-ethanolamine vesicles with Ca2+ translocation activity com-parable to that of phosphatidylethanolamine vesicles.3. A mixture of acetyl phosphatidylethanolamine andstearylamine or oleylamine substituted for phosphatidyl-ethanolamine in the reconstitution of mitochondrial hydro-phobic proteins to form vesicles that catalyze 32Pi-ATP ex-change. Since phosphatidylcholine is also required in thissystem, these findings point to two functions of phosphatidyl-ethanolamine, one related to the specific properties of itsamino group, the other to a structural role of its small polarhead group. A hydrophobic alkylamine can fulfill the firstfunction, acetyl phosphatidylethanolamine the second.4. The importance of the charge was also observed in ex-periments with the reconstituted rutamycin-sensitive ATPaseof mitochondria. After depletion of phospholipids rom thehydrophobic proteins, ATPase activity and rutamycin sensi-tivity were restored only if a phospholipid as well as theappropriate charge were present.

* This work was supportedby National Institutes of HealthGrant CA-08964 and National Science Foundation Grant GB-30850 (E. R.) and National Institutes of Health Grant A-I 11479(G. H. K.).

Natural membranes contain complex mixtures of lipids.While relatively little definitive information is available regardingthe relationship between lipid structure and membrane function,at least two lines of investigation are desirable. One shouldfocus on the function of the fat ty acid side chains and the secondon the role of the polar head groups. Several studies have fo-cused on the former question. Nutritional studies in animalsas well as in microorganisms have revealed the importance of un-saturated fa tt y acids in maintaining fluidity of the membrane.Similarly, comparison of the composition of membranes of coldand warm blooded animals further support this conclusion (c f.Ref . 1). Of particular interest are the observations of the rela-tionship between the content of unsaturated fa tt y acids in yeastmitochondria and their capability to catalyze oxidative phos-phorylation (2). Further, it was also shown in an in vitro recon-stitution system of the mitochondrial 32Pi-ATP exchange (3) thatunsaturation in the fa tt y acid side chains was essential althoughthe saturated side chains could be varied widely. For the studyof the role of polar head groups in phospholipids, the successfulreconst.itution of defined membrane functions has provided apromising approach. For example, a reconstituted membranecatalyzing oxidative phosphorylation has a minimal requirementfor two phospholipids, phosphatidylcholine and phosphatidyl-ethanolamine (4).

The requirement for both phosphatidylcholine and phosphatid-ylethanolamine raised the question of the role of the distributionof charges on the one hand and of the structural role of the sizeof the head group in the assembly of the phospholipid bilayer onthe other hand.In this paper we describe the effect of a specif ic alteration of apolar head group achieved by acetylation of phosphatidyleth-anolamine on the properties of several reconstituted membranesthat are capable of catalyzing ion translocation and partial reac-tions of oxidative phosphorylation.

MATERIALS AND METHODSPhospholipids and other reagents required for the assay of thevarious ion transport systems were as described in previous refer-ences (3-8). Stearylamine and oleylamine were donated b y theAshland Chemical Co. Mgristylamine and palmitylamine werepurchased from K & K Laboratories, Inc.; dodeiylamine andnonylamine from Eastman Organic Chemicals. Dicetylphos-phate was a product of Sigma.

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1810Acetylation of Phosphatidylethanolamine-Three milliliters of

acetic anhydride were added to 750 pmoles of phosphatidyletha-nolamine purified from soybean phospholipid s (4) that were dis-solved in 9 ml of dry py ridine. After 10 min at 0, 30 ml of waterwere added and incubated further for 10 min at 0. The lipidswere then extracted three times w ith about 50 ml of ether and thecombined extracts were dried in vacua. The lipids were suspendedin chloroform-metha nol (4: 1) and passe d through a Dowex 50 (H+)column (1 X 10 cm) which had been previously washed with pyri-dine and chloroform-methanol. The phospholipids were then ap-plied to a Bio-Sil HA (Bio-Rad) column (1.5 X 40 cm) that hadbeen activated with hexane and washe d with chloroform (5).After w ashing with 100 ml of chloroform, acetyl phosphatidyl-ethanolamine was eluted with a linear gradient of methanol inchloroform (150 ml of chloroform in the mixing vessel and 150 mlof methan ol in the reservoir). The acetyl phosphatidylethanola-amine o btained by this procedure traveled as a single componenton thin layer chromatogram s with chloroform-methanol-water(65:24:4) with mobil ity higher tha n that of phosph atidylethan ol-amine . Trac es of pyridine in the preparation did not interferewith recon stitutions but coul d be removed by brief treatment ofthe sample with a small amount of Dowex 50 (H+). Unless spe-cifically stated otherwise, this preparation of acetyl phosphatidyl-ethanolamine was used in the reconstitution experiments.With synthetic dilaurylphosphatidylethanolamine, the sameacetylation procedure was used. However, fractionation on silic agel was omitted since, after passage through Dowex 50, the prep-aration revealed a single com ponent on thin layer chromatograms.

Assays-P roton translo cation (7, 9), aVZazf uptake (6, lo), andV-ATP exchange were measured as described in the references.Reconstitutions-Two methods of reconstitution were used withminor modifications specified in the legends of the tables and fig-ures: the cholate dialysis procedure (11) and the sonication method(8).

RESULTSAcetyl Phosphatidylethanolamine in Reconstitution of Proton

Pump with Bacterial Rhodopsin--The least demanding ion trans-locating system thus far reconstituted is the bacterial rhodopsinproton pump (7). It operates in vesicles reconstituted withsynthetic saturated phospholipids such as dimyristoylphos-phatidylcholine or dipalmitylphosphatidylcholine (9) which arecharacterized by known transition temperatures. As shownin Table I, the proton pump was operat ive with either phos-phatidylethanolamine or acety l phosphatidylethanolamine asthe only lipid in reconstitution. The initial rate of proton up-take was somewhat faster with the acetylated phospholipidparticularly at low pH values of the medium. The ! I/* ofdecay was considerably shorter with the result that the extentof proton uptake (which represents the equilibrium state of therate of uptake and back diffusion) was consistently lower. Thisbehavior is reminiscent of the response of reconstituted vesicles tovalinomycin (9). This ionophorc invariably accelerated the rateof proton uptake as well as of eff lux . Depending on which eff ectpredominated, the extent o f proton uptake was somet,imes in-creased, sometimes decreased. It can also be seen from Table Ithat at higher pH both the initial rate and the decay time ofphosphatidylethanolaminc vesicles were lowered, whereas withthe acetylated phospholipid the decay time was slightly increasedat a more alkaline pH. This explains why phosphatidylethanol-amine vesicles showed a marked drop in the extent of protonuptake at higher pH values, whereas the acetyl phosphatidyl-ethanolamine vesicles did not.

Acetyl Phosphatidylethanolamine in Reconstitution of CalciumPump-It was reported previous ly (6) that, a Ca2+ translocatingpump can be reconstituted by the cholate dialysis procedure withthe Ca2+-ATPase of sarcoplasmic reticulum (12) in the presenceof phospholipid mixtures or of phosphatidylethanolamine alone.It was observed later (13) that, when the reconstitution was per-

TABLE IAcetyl phosphatidylethanolamine in the reconstitution of bacterial

rhodopsin proton pumpTo 5 @moles of dry soybean phosphatidylethanolamine (PE) or

acetyl phosphatidylethanolamine (AC-PE), 0.2 ml of 0.15 M KC1and 20 ~1 of a suspension of purple membranes (6 mg per ml) wereadded. After adjus ting the pH to about 6, the mixtures weresonicated as described previously (7) until t,he suspension waseither clear or only slightly cloudy. Clarification occurred morequickly with the acetylated phospholipid. Initial rates of protonuptake, extent, and tl/z were measured with samples containing25 rg of bacterial rhodopsin as described previously (8).

Vesicles

PE

AC-PE

PH Initial rate Extent Decay, h , P-t5.306.607.345.506.707.47

tatoms H+/nin naloms H+ 569 11.1 1959 8.7 8.437 7.7 8.484 5.5 360 5.4 3.638 4.8 4.2

formed by sonication in the absence of detergents, both phospha-tidylethanolamine and phosphatidylcholine were required. Wetherefore returned to the cholate dialysis procedure to study re-constitution with acety l phosphatidylethanolamine. As can beseen from Fig. IA, vesicles reconstituted with Ca2+-ATPase andacety l phosphatidylethanolamine were completely inactive inCa2+ translocation. Act ,iv ity appeared when suitable amountsof stearylamine were also included during reconstitution withacety l phosphatidylethanolamine; however, excess of stearyl-amine was inhibit,ory. As shown in Fig. lB, ATPase act ivi ty ofthe reconstituted vesicles was also markedly depressed by acety lphosphatidylethanolamine and restored by stearylamine; how-ever, optimal reactivation of the ATPase act ivi ty and Ca*+ pumpact ivi ty did not coincide. It is of interest to note that incorpora-tion of stearylamine into phosphatidylethanolamine vesicles in-hibited the Ca*+ pump acti vity under conditions when the ATP-ase act ivi ty was either unaffected or only slightly enhanced.Olcylamine was much more effect ive than stearylamine in re-storing Ca*+ pump act ivi ty to acety l phosphatidylethanolaminevesic les. As shown in Fig. lA , at optimal concentrations ofolcylamine the rate of *%a*+ uptake was equal to that of phos-phatidylethanolamine vesicles reconstituted under identical con-ditions. The titration curve with oleylamine showed a sharppeak in comparison to that with stearylamine; excess oleylamineinhibited markedly. Again, maximal activation of the ATPaseact ivi ty took place in a range o f oleylamine which was inhibitoryto Ca*+ translocation. As shown in Table II , nonylamine was avery poor substitute for stearylamine. iit optimal concentra-tions (12 pmoles/ml) it activated only little C aZf translocationbut considerable ATPase act ivi ty. Dodecylamine was slightlybetter; myristylamine and palmitylamine restored at best about50% of the act ivi ty normally found with phosphatidylethanola-mine vesicles.

The inhibition of ATPase act ivi ty by acety l phosphatidyleth-anolamine was not dependent on reconstitution but could be ob-served on addition of the phospholipid to the enzyme (Fig. 2A).As shown in Fig. 2B, stearylaminc counteracted the inhibitionby acetylated lipid.

Acetyl Phosphatidylethanolamine in Reconstitution of 32Pi-ATPExchange and Rutamycin-sensitive A TPase of Mitochondrial Mem-brane-It was shown previously (3) that both phosphatidyl-

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5 IO 15 20p moles amine per ml

Acefyl- PE + stearyomine

5 IO 15 205. p moles omine per mlFIG. 1. Calcium uptake and ATPese act ivi ty of W+-ATPasevesicles reconstituted from phosphatidylethanolamine (PE),acety l phosphatidylethanolamine (AcetyZ-FE), and alkylamines.Phosphatidylethanolamine (12.5 amoles) or soybean acety l phos-phatidylethanolamine (12.5 /rmoles) were mixed with variousamounts of stearylamine or oleylamine. To the dry lipid mixturewere added 0.3 ml o f 0.4 M potassium phosphate, pH 7.4, and 0.06

ml of 10% potassium cholate, pH 8.0. The suspension was soni-cated until clear. To the clear lipid solution was added 0.2 mg ofCa*+-ATPase (12) and the fina l volume was made to 0.5 ml with 0.4M potassium phosphate, pH 7.4. The reconstitution was achievedby dialyzing against 200 volumes of the same buffer overnight.Calcium uptake and ATP hydrolysis were determined as described(10). A, calcium uptake act ivi ty; B, ATPase activity.choline and phosphatidylethanolamine are required for the re-constitution of an active 3Pi-ATP exchange with a mixture ofhydrophobic proteins from the mitochondrial membrane. Asshown in Table III , the exchange act ivi ty was abolished whenacety l phosphatidylethanolamine was used instead of phospha-tidylethanolamine. With optimal amounts of stearylamine dur-ing reconstitution, the act ivi ty was recovered, but excess amineinhibited. Oleylamine was less effect ive in this system and, withnonylamine, less than 20 v0 of the control act ivi ty with phospha-tidylethanolamine was recovered. Two control experiments aresignificant. Acetylated stearylamine was inactive and stearyla-mine with phosphatidylcholine alone was insuff icient for thereconstitution of active vesicles. As in the case of C& trans-port, myristylamine, dodecylamine, and palmitylamine gave onlypartial restoration of the Pi-ATP exchange activ ity .

We observed some time ago (14,15) that the hydrophobic frac-tion isolated from mitochondrial membranes strongly inhibitedthe ATPase ac tiv ity of added coupling facto r FL However, onaddition of phospholipids, an ATPase act ivi ty which was rutamy-tin-sensitive was restored.

As shown in Table IV, acety l phosphatidylethanolamine acti-

1811TABLE II

Restoration of calcium uptake activity in vesicles reconstituted withacetyl phosphatidylethanolamine by alkylamines

Reconstitutions and assays were performed as described in thelegend of Fig. 1. PE, phosphatidylethanolamine; AC-PE, acety lphosphatidylethanolamine.Phospholipid

Experiment 1PEAC-PEAC-PEAC-PEAC-PEExperiment 2PEAC-PEExperiment 3PEAC-PEAC-PE

Alkylamine

None 0 114 1.53None 0 0 0.09Stearylamine 10 109 0.8Oleylamine 10 161 1.01Nonylamine 12 3 0.54None 0 246 1.2Palmitylamine 12 99 1.0None 0 147 1.07Myristylamine 8 61.3 0.81Dodecylamine 12 36.7 0.76

ATPaseactivity

vated the ATPase of the hydrophobic protein complex. Addi-tion o f stearylamine decreased the rate of ATP hydro lysis and athigh concentration eliminated rutamycin sens itiv ity. Nega-tively charged dicetyl phosphate had no eff ect on the activatedATPase but eliminated rutamycin sens itiv ity. When stearyla-mine and dicetyl phosphate were added together, rutamycinsens itiv ity, which was absent with either alone, was restored.Similar eff ects were observed in the above sys tem with acety ldilaurylphosphatidylethanolamine. As shown in Fig. 3, a widerange of concentrations of the acetylated phospholipid activatedthe ATPase act ivi ty; however, rutamycin sens itiv ity was ob-served only at very low concentrations (10 to 20 PM), whereas atconcentrations above 100 PM little rutamycin sensitivity wasnoted. In the presence of stearylamine (Fig. 4), the ATPaseact ivi ty was suppressed but reactivation was observed with in-creasing amounts of dicetyl phosphate.

These experiments support the proposition that the charge atthe phospholipid surface plays an important role in the operationof the rutamycin-sensitive ATPase.

DISCUSSIONIt is apparent from the studies reported in this paper that ionpumps vary with respect to their phospholipid requirements.

The amino group of phosphatidylethanolamine is not requiredfor the proton pump reconstituted with bacterial rhodopsin butis required for the calcium pump and for the 32Pi-ATP exchangeof the mitochondrial membrane. The latter, a partial reaction ofoxidative phosphorylation and of the mitochondrial proton pump(11, 17)) is the most complex of the systems studied since a vesic-ular structure provided b y phosphatidylcholine together withamino groups provided by stearylamine are insuf ficient for theoperation of the pump. Pi-ATP exchange act ivi ty could notbe detected without addition of acety l phosphatidylethanolamine.We can tentatively conclude from this finding that the relativelysmall ethanolamine moiety is required for the proper packing ofthe polar head groups in one or both of the membrane surfaces.

Although from the above it would appear that it is possible toseparate the contribution of the structural component of the

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IA.2 0.4 0.6 08 IO 1.2 1.4 1.6p. moles acety l PE

$I 10.1 0.2 03 0.4 0.5p moles stearylamineFIQ. 2. Inhibition of CW-ATPase from sarcoplasmic reticulumby acety l phosphatidylethanolamine (Acetyl-PE) and reactivationby stearylamine. A, Cs?+-ATPase f rom sarcoplasmic reticulum (32pg) were incubated with various amounts of soybean acety l phos-phatidylethanolamine (a 20 mM stock solution of acety l phospha-tidylethanolamine in 5Om~ N-tris(hydroxymethyl)methylglyc ine(Tricine)-KOH, pH 8.0, was prepared by sonic dispersion) in avolume o f 0.2 ml. After preincubation for 5 min at room tempera-ture, the reaction was started by the addition of 1 ml of reactionmixture containing 0.1 M KCl, 50 mM Tris-Cl, pH 7.4,lO mM MgClt,10 m&f ATP, and 50 PM CaClz. The reaction was stopped after 4min at 37, and the inorganic phosphate released was determinedcalorimetrically. B, Cae+-ATPase (32 rg) was preincubated with0.8 rmole of soybean acety l phosphatidylethanolamine and theindicated amount o f stearylamine (a 50 mM stock solution ofstearylamine in 50 mM Tricine-KOH, pH 8.0, was prepared by

sonic dispersion). Preincubation and reaction conditions werethe same as described above.phospholipid from that of the amino groups, it is not as simpleto distinguish between the role of the amino groups as a donor ofa dissociable proton from their contribution to the over-all chargeof the lipid surface. A clue that the charge distribution is criticalwas provided by observations that the rutamycin sens itiv ity ofthe reconstituted mitochondrial ATPase exhibits a rather narrowoptimal range, apparently dependent on the charge which couldbe titrated back and forward by addition of negatively or posi-tively charged compounds.One of the most interesting and challenging problems in thebiogenesis of membranes is the mechanism of the asymmetric or-ganization of its components. The unidirectionality of ion

TABLE IIIEfect of acetyl phosphatidylethanolamine and alkylamines on Pi-

ATP exchangePhosphatidylcholine (PC) (1 pmole) and phosphatidylethanol-amine (PE) (1.6 pmoles) or soybean acety l phosphatidylethanol-amine (AC-PE) were dried together under Nz with the indicatedamounts of various amines, suspended in 0.2 ml of a buffer con-taining 5 mM N-tris(hydroxymethyl)methylglyc ine (Tricine)-

KOH, pH 8.2, 2 mM dithiothreitol, 0.1 mM EDTA, 25 mM sucrose,20 mM ammonium sulfate, and 4 mg of potassium-cholate andsonicated as described previous ly (4). The mixture was thencooled to 0 and 0.1 ml (1 mg) of hydrophobic proteins was added.After dialysis for 4 hours at 4 against 125 volumes of a solutioncontaining 10% methanol, 10 mM Tricine-KOH, pH 3.0, 0.2 mMEDTA, 0.1 mM ATP, 1 mM dithiothreitol, and 200 mM NaCl, thebuffer was changed and dialysis continued overnight. The vesi-cles (0.1 ml) were reconstituted with coupling facto rs and an-alyzed for Pi-ATP exchange as previous ly described (11).

Additions Amine

PE + PCAC-PE + PCAC-PE + PC + stearylamine 0.1250.250.51.01.52.00.51.01.51.01.02.01.0

AC-PE + PC + oleylamine

AC-PE + PC + nonylamineAC-PE + PC + acetyl stearylaminePC + stearylamine

[=P]ATP

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0.85016

TABLE IV

1435.36.6

246210030

1.46662

3.822

000

Eflect of acetyl phosphatidylethanolamine, stearylamine, and dicety lphosphate on rutamycin-sensitive ATPase

To 50 pg of the hydrophobic protein mixture isolated from bo-vine heart mitochondria (ll), soybean acetyl phosphatidyleth-anolamine, stearylamine, and dicety l phosphate in the amountsindicated in the table were added. ATPase assays were per-formed in a final volume of 1 ml in the presence of an ATP regen-erating system as described previous ly (16) with or without 4 pg ofrutamycin present.

AdditionsI ATPase activity

-Rut-amycin I Izy$i Inhibition

None. . 1.01 0.8875 nmoles of Ac-PE . 6.36 4.04+ 200 nmoles of stearylamine . . . . 3.54 2.03+ 400 nmoles of stearylamine 1.94 1.94+ 200 nmoles of dicetyl-P. 6.54 5.45+ 400 nmoles of dicetyl-P.. 6.0 5.72+ 400 nmoles o f dicetyl-P + 200nmoles of stearylamine. 5.09 2.59

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0 25 50 75 100n moles ocetyl dilouryl PE

FIG. 3. Ef fec t of acety l dilaurylphosphatidylethanolamine(acetyl dilauryl PE) on rutamycin-sensitive ATPase. Experimen-tal conditions were as described in the legend o f Table IV exceptthat acety l dilaurylphosphatidylethanolamine instead of soybeanacety l phosphatidylethanolamine was added in the amounts indi-cated.5.0 , 1

I I I I I50 100 150 200 300n moles dicetyl phosphate

FIG. 4. Activation of inhibited ATPase by dicetyl phosphate.Experimental conditions were as described in the legend of Fig. 3except that 37.5 nmol of acetyl dilaurylphosphatidylethanolamineand 20 nmol o f stearylamine were added to the ATPase complex to-gether with increasing amounts of dicety l phosphate as indicated.

pumps requires a highly specificand asymmetricassembly f theparticipating proteins. It does not appear from the limitedstudies eported n this paper that the aminogroupsof phospha-tidylethanolamine er seareessentialor the specificorganizationin the three systems ested.The problem of asymmetric organization is particularly puz-zling in view of the observations hat the reconstituted systemsmay either be representative of the orientation of the naturalmembrane s in the caseof the reconstituted Ca2+pump or beassembledn the oppositedirection as n the case f the reconsti-tuted rhodopsinproton pump. It seems ot unreasonablehere-fore to propose hat factors other than the general ipid composi-tion influence the orientation of the proteins and that specificorganizational components either proteins or lipids) participatein this important function.

1.

2.3.4.5.6.7.8.9.10.

11.12.13.14.15.16.17.

REFERENCESCHAPMAN, D., AND LESLIE, R. B. (1970) in Membranes of

Mitochondria and Chloroplasts (RACKER, E., ed) pp. 91-126,Van Nostrand Reinhold Co., New YorkHASLAM, J. M., SPITHILL, T. W., AND LINNANE, A. W. (1973)Biochem. J. 134, 947-957KAGAWA, Y., KANDRACH, A., AND RACKER, E. (1973) J. Biol.Chem. 240, 676-684RACKER, E., AND KANDRACH, A. (1973) J. Biol. Chem. 248,5841-5847RAGAN, C. I., AND RACKER, E. (1973) J. Biol. Chem. 246,2563-

2569RACKER, E. (1972) J. Biol. Chem. 247,8198-8200RACKER, E., AND STOECKENIUS, W. (1974) J. Biol. Chem. 249,662-663RACKER, E. (1973) Biochem. Biophys. Res. Commun. 66,224-230RACSER, E., AND HINKLE, P. (1974) J. Membr. Bio l. 17, 181-188KNOWLES, A. F., AND RACKER, E. (1975) J. Biol. Chem. 260,in pressKAGAWA, Y., AND RACKER, E. (1971) J. Biol. Chem. 246, 5477-5487MACLENNAN, D. H. (1970) J. Biol. Chem. 246,4508-4518RACKER, E., AND EYTAN, E. (1973) Biochem. Biophys. Res.Commun. 66, 174-178KAGAWA, Y., AND RACKER, E. (1966) J. Biol. Chem. 241, 2467-2474BULOS, B., AND RACKER, E. (1968) J. Biol. Chem. 243, 3901-3905PULLMAN, M. E., PENEFSKY, H. S., DATTA, A., AND RACKER,E. (1960) J. Biol. Chem. 236, 3322-3329MITCHELL, P. (1966) Biol. Rev. (Camb.) 41,445

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