THE Jounrh~ OP RIOLOGICAL CHEXISTRY Vol. 248, No. 20, Issue of October 25, PB. 6993~iOO0, 1973

Phted in U S.A.

Evidence for an Aspartyl Phosphate Residue at the Active Site

of Sodium and Potassium Ion Transport

Adenosine Triphosphatase*

(Received for publication, May 23, 1973)

ROBERT L. I’OST$ AND SEIOJI KUME$

Front the Department oj Physiology, Vanderbilt University Medical School, A’ashville, Tennessee 37232

SUMMARY

In order to characterize part of an active center of sodium and potassium ion transport adenosine triphosphatase, namely the active site of phosphorylation, the enzyme was phosphorylated with [32P]ATP and denatured with acid. The denatured [3SP]phosphoenzyme was digested to a limit [32P]phosphotripeptide with Pronase. The limit phospho- tripeptide was compared with the synthetic model com- pounds, [3?P]prolylphosphoaspartyllysine and [32P]prolylphos- phoglutamyllysine with respect to pH-hydrolysis profile, sensitivity to digestion by carboxypeptidase B, and iso- electric point. The active site phosphotripeptide closely resembled the synthetic phosphoaspartyl peptide and clearly differed from the synthetic phosphoglutamyl peptide in these respects. These similarities are strong, but not conclusive, evidence that the active site of this enzyme is the /3-carboxyl group of an aspartic acid residue. Previous work has shown that the limit phosphotripeptide contains an NH2-terminal serine or threonine residue and a COOH-terminal lysine residue. The enzyme source was guinea pig kidneys.

In order to characterize the active site before denaturation, the sensitivity of the phosphate group to hydroxylamine was tested. The enzyme was first treated with N-ethylmaleimide to inhibit acceleration of dephosphorylation due to a re- semblance between hydroxylamine and potassium ion. The treated enzyme was subsequently phosphorylated by [32P]adenosine triphosphate and the phosphoenzyme was chased with unlabeled adenosine triphosphate. During the chase, addition of hydroxylamine produced a dephosphoryla- tion which was 45-fold faster than that of the isolated phos- photripeptide under the same experimental conditions. It is concluded that the phosphoenzyme has sufficient sen- sitivity to hydroxylamine before denaturation to qualify as an acyl phosphate.

* This mark was supported by Grant 5ROl HL-01974 from the iXationa1 Heart and Lung Institute and by Grant 5POl AM-07462 from the Xational Institute of Arthritis and iCIetabolic Diseases, United States Public Health Service.

$ To whom correspondence should be addressed. Q Present address, First Department of Internal Medicine,

Facult,y of ;\ledicine, Universit,y of Tokyo, 7-3-l Hongo, Bunkyo- ku, Tokyo, Japan.

At one step in the reaction sequence of sodium plus potassium ion transport adenosine triphosphatase, (Na+ + EC+)-ATI’ase,’ the terminal phosphate group of ATP is transferred to a protein. Formation of this phosphoenzyme requires &I@+ and Na+; dephosphorylation is accelerated by I<+. At the active site of phosphorylation, the bond has been identified as an acyl phos- phate (carboxyl phosphate) on the basis of the chemical re- activity of the phosphate group in the denatured phospho- protein, specifically according to its pf1 hydrolysis profile, sensitivity to hydroxylnmine, acyl phosphntase or molybdate, and according to transfer OC the phosphate group to alcohols in acid solution (1, 2). Further characterization has been hindered by the small amount of material available and the lability of the phosphate bond.

Ordiuarily proteins have three types of cnrbosyl group avail- able for such a baud, namely, a y-glutarnyl group, a P-aspartyl group, or a COOH-terminal carboxyl group. Kahlenberg et al. (3) concluded “that the aryl phosph:ltc intermediate in the (Sa+ + I(+)-activated adenosine triphosphatase is an L-S&

tamyl-y-phosphate residue.” The conclusion n’as based on the following experiment n-ith the enzyme from guinea pig brain. Peptic [32P]phospllopeptidcs from the denatured phos- phoenzyme were treated with [2, 3-311]N-(n-propyl)-hydroxyl- amine with the intention ol replacing the unstable radioactive phosphate group with a more stable and equally radioactive hydroxamate group. Digestion of the treated peptidcs with Pronase and extensive purification yielded y-glutamyl hydrox- amate; the amount was 2c0 of that theoretically possible. Apart from uncertainty as to what sort of exchange reactions might occur during digestion by the mixture of enzymes in Pronase, it is difficult to see why the conclusion of Kahlenberg et al. (3) should not 1~ correct. However, the evidence pre- sented in this paper favors the conclusion that the active site phosphate group is attached t’o the P-carboxyl group of an aspartyl residue. In these new experiments the radioactive phosphate group, while still attached to the carboxyl group, remained the focus of attention. il limit [32P]phosphotripeptide

1 The abbreviations used are: (Xa+ + K+)-,4TPase, sodium olus potassium ion transport adenosine triphosphatase; Pr4, the limit phosphotripeptide of phosphorylated (Na+ + K+)-ATPase ob- tained by exhaustive digestion of the denatured enzyme with Pronase (or sequential treatment with pepsin, trypsin, and lcucine aminopeptidase) ; dansyl, 5dimethylaminonaphthalene-l-sul- fonyl.

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n-as isolated from the phosphoenzyme by digestion with Pronase and characterized sufficiently that analogous model [32P]phos- phoaspartyl and [321’]phosphoglutamyl tripeptides could be synthesized. The synthetic phosphopeptides differed from each other with respect to isoelectric point, sensitivity to digestion with carbosypcptidase B, and with respect to PI-I-hydrolysis profile. The corresponding properties of the limit phospho- peptide from the enzyme were T-cry similar to those of the syn- thetic aspartyl phosphopeptide and obviously different from those of the synthetic glutamyl phosphopcptide.

In the rest of this paper the nbbreyiation “Pr4” n-ill designate the limit [3”I’]pllosphotripeptidc producctl by digestion &h Pronase of the denatured phosphoenzyme of (;“\a+ + K+)- ATI’ase. Previous work has shown that I’r4 has a molecular weight about 400 (4) and contains one dicarbosylic and one dibasic amino acid (5). The basic amino acid is lysine, since it can be ncetylatcd before digestion \xith I’ronase (6) and E- dnnsyl-lysine has been isolated from dansylated I+4 (6). The lysine is positioned on the COOH-terminal side of the acidic residue binding the phosphate group according to the action of trypsill. Trypsin characteristically hydrolyzes pcptidcs on the COOTI-tx~rmi~ial side of a lysine or argininc resitluc. Digestion by trypsin of overlapping radioactive phosphopeptitles larger thaII I’r4 yields pcpticlcs which still contail the basic lysine (6). If the lysinc had been on the 1Z’I-IB-terminal side of the 321’- residue, it would have been cut off from it by the action of trypsin. This action of trypsin also excludes the possibility t,llat the phosphate is on a terminal carbosyl group. The S&tcrminal residue of Pr4 is serine or threonine since Pr4 is sensitive to oxidation Tvith periodate (6), which characteristically oxidizes vicinal hyclroxyl groups or \.icinal hyclrosyl and amino gI‘OllpS. The acidic residue is therefore central and is either a y-glutamyl phosphate or a P-aspartyl phosphate. That is,

Thr IV-l is ( ) SW -X(1’)-Lys n-here X is an acidic amino acid residue.

In this paper I’r4 was compared xyith synthetic Pro-Glu(P)- Lys and synthetic Pro-Asp(P)-Lgs. The use of a proline resitluc in place of a serine or threonine residue avoided potential obstruction by an additional blocking group during phosphoryla- tion of the blocked precursor synthetic tripeptides. It also provided synthetic phosphopeptides which could be hydrolyzed in the same reaction mixture with Pr4 and yet could be easily separated from l’r4 afterward by paper clectrophoresis at p1-T 7.

It is interesting that characterization in all the foregoing re- spects of the corresponding limit phosphotripeptide from the Ca?+-ATPase of sarcoplasmic reticulum of rabbit skeletal muscle has yielded results identical IT-it11 those found with the (Na+ + I<+)-ATPase of guinea pig kidney.2

METHODS

Synfhetic Model [32P]Pleosphopeptides-Preparation of benzyl- ox~carbonylprol~1-cu-glutamyl-~-benzylo~ycarbonyllysi~~e bcnzyl ester was a custom synthesis by International Chemical and Nuclear Corp., Irvine, Calif., and preparation of benzylosycar- bonylprol~l~a-ns~,nrt~yl-~-be~~z~lo?;~carbonyllysine benzyl ester lx-as a custom synthesis by Miles Laboratories, Elkhart, Jnd. The pllos~)llol?el?ticles TT-ere s@hesizcd from these blocked tripeptidcs by modifications of the method of Nishimura et al. (T). The folloxing procedure xvas used for the aspartyl tripcptitlc, n-hi& Teas more difficult to phosphorylate. The rrnction misturc contained 0.5 ml of pyridine, 0.0’7 ml of n-atcr, 0.02 ml of 14.7 ~1 1131’04, and 5 to 10 X 10G cpm of 32Pi at 23”.

2 Manuscript in preparation.

The reaction was started with 0.14 ml of 2 M tlicyclohesylcarbodi- imide (without insoluble residue) dissolved in mcthylene di- chloride. After 2 min of stirring, 200 Fmolcs of the blocked tripepticlc were added already dissolved in 0.4 ml of 1 :l (v/v) pyridinc-methylene chloride. Incubation was for 30 min at 23” with continuous stirring. The mixture was filtrrrtl through a pad of glass wool (Millipore Corp.) into 20 ml of ice-cold petroleum ether and the reaction vessels lucre kept on ice after this time. The original flask and the filter n-ere rinsed lvith 1 ml of a misture of pyridine and 3 RI H#OI 19: 1 (V/V). The rinsing solution n-as added to the organic solvent misturr, which n-as misecl n-cl1 and celltrifugcd briefly. It scpamted illto two phases; the upper one was discardccl. Cold 2 nr LiCl, 0.5 ml, was added and mised in. Then 20 ml of c~ltl petrolrum ether were added, mixed, and centrifuged as Marc. ‘She upper phase was discarded again. The loner phase \+a-: lyoph- ilizcd over P205 with a liquid nitrogen trap. The dry sample was dissolved in a cold mixture of 0.15 ml of glacial ncctic acicl, 1.0 ml of tliosanc, ailtl 3.0 ml of xvater. Thrc+tcnllis gram of 10yc pallatlium on charcoal (Matheson, (‘olcnlall atltl l+ll) I\-ad aclded and the mixture was hydrogenatctl at 23’ for 30 niin untlei hytlrogc~n at a prc~~rc of 3.5 kg per cm”. The chwoal n-as ro- movctl by filtratioli alid the yielcl was estiniatctl by a l~ydrosil- mate tclst (8) on an aliquot of 0.1 ml. Furtlic,r procc~lu~~ \verc at 0”. OIIC rnillilitcr of pyridille was atltl(~tl to the remaining sample alId the volume was brought to 10 ml with \vatc:r. The

phospllotrir)eI)title was put oIlto a column (1 x 15 cm) of 110~‘s XGI -X2 in the acetate form. It was elutctl wit.11 a lincxr gradi- ent formed by suitably mixing 20 ml of 0.03’ ( (v/v) a&ic acid and 0.27;) (V/V) pyridille in water as starting solution with 20 ml of 3.0%, (v/v) acetic acid and O.acyO (v/v) pyridinc as a finishing solutioii. The output from the column was monitored with a liquid flow Geigclr counter and the radioactive portion \ra col- lectecl in 5-ml samplrs to which 0.1 ml of diInctll\-lfol,maIni(le was addctl bcforc lyophilization over I’s05 with a liquid nitrogen trap. .Iftcr lyopliilization the samples n-crc tliisolvctl in 10 iii11 HCl and stored at -45”. The glutamyl l~llo~~~l~ot,ril~c~~titlr Teas prepared similarly cxept for simplifications. The substituted tripeptidc was soluble in the original reactioll nlixturr n11d was added as the dry solid before t,he dicyclol~r~~l~~~.rbodiin~itle. In place of pctrolrum ether after addition of the LiCl, colt1 acetone was usctl (also 20 ml). E‘urthcrmore, dioxano wa.s omitted from the hydrogenation mixture. The yield of the phosphotriprp- tides was 8 to 32y0 as estimated by the hyclrornmntc test. Elcc- trophoresis of each phosphopeptide on pnpcr showrtl 3 single major radioactive spot and a variable amount of l’i (6) (see later Fig. 6). Elution of the spot, acid hydrolysis, ant1 analysis on an amino acid analyzer and for radioactivity sho~vcd for each phosphotripeptide approximately equimolnr quantities of in- organic phosphate, proline, lysine, and aspnrtic or glutamic acid. Caution: in one case where an old preparation of tlicyclohesyl- carbodiimidc was used, there were found mult.iple spots on the paper after electrophoresis. This preparation was discnrdetl.

[3*P]Acetyl Phosphfe-The acetyl phosphate was prepared by incubation of 1 ~1 of 321’i in water with 2 ~1 of acetic anhydride in 10 ~1 of pyridine for 5 min at 0”. The rrnction was stopped by addition of 1.5 ~1 of SS7o (IV/IT) formic acid. The sample was dilutrd to about 1 ml lyith H&, lyophilizc(l as nborc, dis- solrcd in HIO, and stored at -45”.

Limit [32P]Pl~,ospl~ofripeptide Released jrom Dcnat~tred Pltos- phorglafed (Na+ + I<+)-)lTPnse by Digestion with Pronase- Crude (xa+ + Ii+)-ATPasc bound to guinea pig kicInc)- mem- brancs \I-as prepared, phosphorylated selcctir+- with [321’]ATP,

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and denatured with acid by the methods of Post and Sen (9, 10). The specific activity of the [Y’]ATP was about 400 cpm (pmole)-l and the specific activity of the enzyme preparation was about 2 pmoles of I’i relcnsed (mm)-’ (mg of protein-l under standard conditions. The entirc subsequent procedure was carried out at 0”. The tlvnatured membranes, about 48 mg of protein, were washed twice with 30 ml\r I-ICI by centrifugation and resuspen- sion. The denatured pellets were adjusted with 1.26 M

NII,HCOI to a pII between 7.0 and 7.4, as estimated with pH paper, and Tvvcre digested with Pronase (Calbiochem), 0.8 mg per mg of mcmbranc protein for 105 min at 0”. The digests were tlilutctl to 64 ml with O.O3y, (v/v) acetic acid and were ccntrifugc,tl at 34,000 X g for IO min. The supernatant vas applied to a column of Dowex AGl-X2 in the acetate form for further l)urification of the phosphopeptide by the same pro- cedure described for the synthetic phosphotripcptides.

Comparison of pH Ilydrolysis Pro/&-Since only picomole quantities of the radioactive natural tripeptide were available, and thcsc were heavily contaminated with nonradioactive pcptides, it, was not practical to estimate hydrolysis at several coircentr:rt,iorls of each buffer and to extrapolate to zero buffer conceirtr:~tiol~. To compare the rates of hydrolysis of a pair of compourltls, 11lcy were incubated together in a single reaction vessel ant1 \\ vrc separated afterward by paper clectrophoresis. By this approach, however imperfect the buffer system might be, a comparison of the relative rates of hydrolysis of two com- pounds slioultl still be valid. The basic purpose of these csperi- ments is comparison.

Lyophilized samples of the [32P]phospl~ol~eptitlcs or of [““I’]- acetyl phosphate vere dissolved in water. For a hydrolysis comparison, 15 11 of each such solution were mixed with 60 ~1 of a 0.1 M solution of the component listed first adjusted to the following pH values with the second component, unless other- wise stated: pII 0 = 1.5 M HClOd (not adjusted); pH I = 0.15 nr HC104 (not adjusted); pH 2 = 0.86 M formic acid (not ad- justed); pH values 3, 4, 5, and 6 = citric acid + NaOII; pH 7 = 2-(N-morpholino)propancsulfonic acid + N-ethylmor- pholine; pH 8 = (N-trishydroxymethyl)methyl-2-amino- propanesulfonic acid + (N-trishydrosymethyl)aminomethane; pH 9 = 0.1 fir 2-amino-2-methyl-1,3-propanediol adjusted with HCl; pH 10 = 0.1 JI KHC03 + 0.1 ~1 K&O3 adjusted Tvith KOH; pH 10 = p-alanine + diethylamine; ~1-1 11 = 0.5 RI triethylamine (not adjusted); ~1-1 12 = 0.1 &f piperidine (not adjusted); pH 13 = 0.15 hf KOH (not adjusted). Before in- cubation the mixtures were kept at 0”. The time of incubation at 37” was adjusted between 10 and 80 min so that more than 20% and less than 80% of the sample was hydrolyzed unless otherwise stated in the figure legend. ,411 buffers from pH 7 to 13 contained additionally 1 mM (1,2-cyclohexylene-dinitrilo)- tetraacctic acid, a chelator which reduced the rate of hydrolysis. After incubation the ~1-1 was measured with a small glass elec- trode (Beckman No. 39030) and this ~1-1 is plotted on the horizontal axis of the figures. Sometimes buffers initially at pH values 11 to 13 allowed the pH to fall 0.6 to 1.8 units before the end of incubation. After incubation the mixtures TT-ere cooled to 0” and the pH was adjusted to prevent distortion of the subsequent electrophoresis. Mixtures containing acetyl- phosphate were subjected to electrophoresis at pH 2 in 2% (v/v) formic acid. To 20 ~1 of the hydrolysis mixture at pH 3 and higher, I ~1 of glacial acetic acid was added. Mixtures containing the synthetic phosphopeptides were subjected to electrophorcsis at ~1-1 7 in a mixture of 0.1 M imidazole and 0.02 M citric acid. For adjustment of the pH a small amount of

solid imidazole was added to 20 ~1 of the hydrolysis mixtures at pH 6 and lower. After pH adjustment the 20.~1 samples were applied to moist Vhatman No. 3MM paper and clectrophoresis was for 2 hours at 80 volts per cm in a Locarte cold plate ap- paratus at 2”. =Ifter clectrophoresis the paper was dried and radioautographed. Radioactive spots corresponding to l’i, the phosphopeptides or to acetyl phosphate were cut out and counted in a thin window gas flow counter. The hydrolysis rate constant was estimated from the disappearance of the phosphopeptide in accordance with the assumption that hydrolysis was a first order reaction and with reference to zero time control samples. The control samples were treated identically t,xcept that no

buffer solution n-as added and they v-ere kept at 0” during the same time interval.

The buffers used above v-ere selected after a period of trial and error in which other buffers were found to give higher rate constants or to interfere with the electrophoresis. Formate ion at pH 4 and histidine at pH 6 were particularly active in promoting hydrolysis. Other rejected buffers include ethylene- diamine, fructose (pII 12), glycylglycine, maleic acid, and 2-(AT- morpholino)cthnncsulfonic acid. Diethylamine was a satis- factory substitute for tricthylamine. Boric acid was used at pH 9 without acceleration of hydrolysis but was less convenient than HCl for adjustment of the pII.

Paper I:‘lecfrophoresis-To estimate electrophoretic mobility as a function of 1’1-1, the following buffers were usctl on Whatman No. 31111 paper: pII 2 = 2cc (v/v) formic acid; pH 2.8 = 0.23 AI formic acid + 0.12 31 glycine; pI1 3.5 = 0.1 iv formic acid + 0.043 1RI Tris; pII 4.0 = 0.12 11 acetic acid + 0.02 31 Tris + 0.02 M KCl; pI-I 4.5 = 0.2 M acetic acid + 0.082 JI Tris; pH 5.0 = 0.075 hf acetic acid + 0.05 n!r Tris; ~1-1 6.0 = 0.02 11 succinic acid + 0.056 hf ‘l’ris; pH 8.0 = 0.1 31 acetic acid + 0.15 M Tris; and pH 8.5 = 0.1 M boric acid + 0.1 M Tris + 0.02 R;I KCI. The samples were applied to the cold moist paper lying in place on the apparatus. Electrophorcsis at 2” was for 2 hours at 80 volts per cm in a Locarte cold plate apparatus. The [32P]phosphopeptides were located by subsequent, radio- autography.

RESULTS

Since the bond between the phosphate group and the enzyme is usually considered to be an acyl phosphate bond, and since the pH hydrolysis profile of small molecules containing such bonds is sensitive to the architecture of the molecule in the neighborhood of the bond (11, 12), we undertook comparison of the pH hydrolysis profile of the limit phosphotripeptide from the active center of the enzyme with those of analogous syn- thetic phosphotripeptides.

Hydrolysis oj Limit Phosphotripeptide and ~~lcclylphosphate Simultaneously in Selected Buffer System-In order to find buffers n-hich gave the lowest rate of hydrolysis at each pI-I, they were tested first lvith acetyl phosphate. (The pH hydrolysis profile of fl-aspartyl phosphate is like that of acctyl phosphate (13). Glutamyl phosphate is extremely unstable (14).) It lvas found that inclusion of a chelator, (1,2-cycloliexylene-dinitrilo)tetra- acetic acid, reduced the rate of hydrolysis at moderately alkaline pH. Since the buffer system which produced the lowest rate of hydrolysis was considered the one least likely to distort effects produced by changes in the concentration of hydrogen ions, the chelator was incorporated in the chosen system. The system reproduced well the rate constants found by Koshland (15) for acetyl phosphate except at mildly alkaline pI1 where the chelator produced a lower rate of hydrolysis (Fig. 1). In the absence

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0.001~ ‘2 0 2 4 6 8 IO 12

PH

0.5 I

0.003 1 0 2 4 6 8 IO 12

FIG. 1 (lefl). Comparison of the pH hydrolysis profiles at 37’ of acetylphosphate (0, 0) and the limit phosphotripeptide of (Na+ + KC)-ATPase, Pr4 (A, A), estimated in a selected buffer system. The two substances were hydrolyzed in the same reaction vessel at each pH and were separated afterward by paper elec- trophoresis. The experimental procedure is given under “Meth- ods.” k is the phosphohydrolysis rate constant. In the case of the solid symbols, the extent of hydrolysis was greater than 20% and less than S070. In the case of the open symbols, hydrolysis was outside these limits, so that the results are probably less ac- curate. The solid line reproduces the data for acetylphosphate at 39” obtained by Koshland (15) with a different buffer system. The dashed line shows an arbitrary intercept on the vertical axis which is proportional to the concentration of hydroxyl ion.

FIG. 2 (center). Comparison of the pH hydrolysis profile at 37” of the limit phosphotripeptide of (Pl;a+ + K+)-ATPase, Pr4 (A., v), with that of Pro-Asp(P)-Lys (0, a) estimatedsimultaneously

of the chelator the chosen buffers gave rates which were the same as Koshland’s. Hydrolysis of the active site phospho- tripeptide, l’r4, was clearly more rapid than that of acetyl phos- phate bctwecn pH 5 and 10 (Fig. 1). In previous estimates of the pH hydrolysis profile of Pr4 or of the phosphoenzyme, there has been a minimum rate at about pH 2 to 3 and a rising shoulder in the curve with increasing pH. The more evenly U-shaped curve found here is probably due to the choice of buffers and the use of the chelator. The chelator was introduced initially since some heavy metals accelerate hydrolysis of acetyl phos- phate (16). With respect to stability of acyl phosphopeptides during storage at -45”, the chelator did not provide protection at neutral pH and it was necessary to bring the pH of samples close to pH 2 or 3 before freezing them.

Comparison 01” pH Hydrolysis Profile of Active Site Phos- photripeptide, Pr4, with That of Pro-Asp(P)-Lys-At each pH the two [32P]phospllotripeptides were incubated in a single re- action vessel. After incubation they were separated from each other and from Pi by paper electrophoresis. The hydrolysis profiles were almost identical (Fig. 2). Only from pH 7 to 9 was Pro-Asp(P)-Lys significantly more labile than Pr4 and even so the maximum effect was only an increase in the rate constant of 1.4.fold. This difference may be due to the difference in the protonation of the terminal amino groups of the 2 molecules. In this pH range, Pr4 loses a proton and Pro-Asp(P)-Lys re- tains one as judged from electrophoretic mobility. This point is discussed later.

Comparison of pH Hydrolysis Pro$le of Actizle Site Phospho- tripeptide, Pr4, with That of Pro-Glu(P)-Lys-At each pH the two [32P]pllosl)hotripeptides were incubated in a single reaction vessel. Below pH 3 and above pH 11 the rates of hydrolysis were the same. Elsewhere the profiles were obviously dif- ferent (Pig. 3). From pH 4 to 7 Pro-Glu(I’)-Lys TX-as 2- to 3- fold more stable. About pH 8 there was a cross-over. From pH 9 to 10.3 the model synthetic peptide was 3-fold more labile

PH

0.003 ’ 0 2 4 6 8 IO 12

PH

as in Fig. 1. The half-solid circles are common to the values for both substances where there was no significant difference between them. The vertical bars show 12 S.E. from the mean of two ex- periments. Bars are not shown where they would overlap the symbols. The probability that the rate constants at pH 7,8, and 9 are the same for the two substances is less than 0.001. Values estimated in carbonate-bicarbonate buffer are identified sepa- rately (V, q ).

FIG. 3 (right). Comparison of the pH hydrolysis profile of the limit phosphotripeptide of (Na+ + K+)-ATPase, Pr4 (A, v), with that, of Pro-Glu(P)-Lys (0, q ) estimated simultaneously as in Fig. 2. The points marked with half-solid circles are common to both substances. The vertical bars show 52 S.E. from the mean of three experiments except where they overlap the symbols. Values estimated in carbonate-bicarbonate buffer are identified separately (v, 0).

except in the presence of carbonate-bicarbonate buffer, in which all three phosphopeptides had the same relatively low rate of hydrolysis (compare Fig. 2).

Comparison of Isoelectric pH of Three Phosphotripeptides- The isoelectric pH of these peptides is useful for characterization since it lies half-way between the pK values of the secondary ionization of the phosphate group and the ionization of the terminal carboxyl group (5). It does not depend on electro- phoretic mobility. Since aspartic acid with carboxyl pK values of 1.88 and 3.65 is a stronger acid than glutamic acid with pK values of 2.19 and 4.25, one would expect the isoelectric pH of Pro-Asp(P)-Lys to be lower than that of Pro-Glu(P)-Lys. It was (Table I). The isoelectric pH of the aspartyl phospho- peptide was 3.8 and that of the glutamyl phosphopeptide was 4.0. The isoelectric pH of Pr4 was 3.8. These data show another point of correspondence between Pr4 and Pro-Asp(P)- Lys and of difference between Pr4 and Pro-Glu(P)-Lys.

In these experiments the absolute value of the isoelectric point may not be entirely reliable because of the difference between the temperature of measurement of the buffer (23’) and the temperature of the electrophoresis (2’) and because of uneven motion of the spots in accordance with differences in the wetness of the paper and endosmosis, but between pH 2.8 and 4.5 there was, in every experiment but one, a distinct difference between the motion of Pro-Glu(P)-Lys and that of the other two phosphotripeptides (for examples see later Fig. 5 and also Fig. 2 in Ref 6). The relative difference in the iso- electric points was consistently demonstrable. Near pH 8 the differences in mobility reflect differences in the ionization of the NHz-terminal group. As a secondary amine proline has a higher pK than other amino acids.

Slow Attack by Carboxypeptidase B on Natural Phosphotri- peptide, Pr&--Previously it was shown that Pr4 and Pro-Asp(P)- Lys were much less sensitive to carboxypeptidase B than was Pro-Glu(P)-Lys (6). This insensitivity was taken as evidence

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TABLE I Electrophoretic mobility of [32P]phosphotripeptides

Mobility was estimated by paper electrophoresis at 2” for 2 hours at 80 volts per cm followed by radioautography. Mobility of the center of the spot toward the cathode is in the positive direction with reference to caffeine as a neutral marker of endos- mosis. The isoelectric pH was estimated from the best straight line by least squares through the points at pH values 2.8, 4.0, and 4.5. The buffers are given under “Methods.”

PH -

_- Pro-Glu (P)-Lys’” Pr4b Pro-Asp(P)-LysC

2.0 +23.5 2.8 +15.2 3.5 t-4.0 4.0 -1.1 4.5 -4.9 5.0 -11.0 6.0 -13 8.0 -12 8.5 -17

Peptide mobility

‘llz

122.6 +12.8

+2.3 -3.2 -7.7

-13.2 -16 -25 -33

+23.0 +12.9

-3.3 -8.5

-14 -14

0 Isoelectric pH 4.0. b Isoelectric pH 3.8. c Isoelectric pH 3.8.

for an aspartyl phosphate residue in Pr4. It might be argued on the contrary that this insensitivity could be evidence against a COOH-terminal position for the lysine in Pr4, discounting the effects of trypsin for the moment. With a highly radioactive preparation of Pr4 and a very high concentration of carboxy- peptidase B it was possible to show slow release of a new [a2P]- phosphopeptide with an electrophoretic mobility at pH 4 which indicates loss of the basic amino acid (Fig. 4). This experiment confirms the COOH-terminal position of the lysine and em- phasizes the resistance of its peptide bond to carboxypeptidase B in the presence of a phosphate group on an adjacent aspartyl residue. Carboxypeptidase B (0.5 pg in 10 ~1 at pH 8.0) re- leased lysine from both Pro-Glu-Lys and Pro-Asp-Lys during incubation for 16 hours at 23”.

The NH%-terminal ends of all three phosphotripeptides were resistant to the action of leucine aminopeptidase in several un- successful attempts to show an effect.

Comparison of Pr4 from Kidney with That from Brain-The experiments of Kahlenberg et al. (3) were done with the (Na+ + K+)-ATPase of guinea pig brain as the source of the denatured phosphoprotein. In order to determine whether the difference between their results and ours was due to a difference between brain and kidney, we compared Pr4 from guinea pig brain with Pr4 from guinea pig kidney with respect to (a) isoelectric point, (b) sensitivity to carboxypeptidase B, and (c) comparison of the pH-hydrolysis profile with that of Pro-Glu(P)-Lys. The results were the same in all three respects regardless of the tissue from which the enzyme was prepared. Fig. 5 shows the insensitivity of Pr4 from either tissue of the guinea pig to the action of carboxypeptidase B. The corresponding insensitivity of Pro- Asp(P)-Lys is shown elsewhere (6).

Comparison of Sensitivity of Phosphate Bond to Hydroxylamine before and after Denaturation-Up to this point all experiments were done on the phosphoenzyme obtained by denaturation with acid. But denaturation itself might induce a transphos- phorylation from the true binding site in the native phospho- enzyme. Denaturation at neutral pH with dodecyl sulfate ap-

Pr4 ORIGIN

ii) (-)

FIG. 4 (upper). Slow attack of carboxypeptidase B on the limit phosphotripeptide of (Na+ + K+)-ATPase, Pr4. [3V’] Pr4 was incubated at 23” with about 0.5 mg of porcine carboxypeptidase B (CBP-B) (Worthington) in 30~1 of 0.1 M K1HPOd. The reaction mixture was cautiously adjusted close to pH 7.2 with 1 M KOH as judged by pH paper. Sample A is an undigested control. Sam- ples B, C, and D were digested for 10,20, and 40 min, respectively. Sample E was incubated for 60 min and then treated with 10 ~1 of 2 M hydroxylamine. Sample F was incubated for 15 min with an equal amount of carboxypeptidase A in place of carboxypeptidase B. Aliquots were applied to paper for electrophoresis at pH 4 and subsequent radioautography as described under “Meth- ods.” The arrow points to the phosphopeptide released by car- boxypeptidase B. The distance between it and Pr4 is 19.5 cm. Pi moved to the left beyond the edge of the film.

FIG. 5 (lower). Comparison of digestion with carboxypeptidase B (CBP-B) of t,he limit phosphotripeptide of (Na+ + K+)-ATPase, Pr4, made from guinea pig brain with that made from guinea pig

I kidney and with Pro-Glu(P)-Lys,-GLU-. Eachsample, initially in 15 pl of 10 mM HCl, was adjusted with 3 ~1 of 1.26 M NH&ICOI to approximately pH 7.5 as judged with pH paper. To each test sample was added 0.01 mg of porcine carboxypeptidase B (Worth- ington) in 10 ~1 of 0.1 M NaCl and to each control were added 10 ~1 of 0.1 M NaCl. All samples were incubated for 40 min at 23”. Aliquots were applied to paper for electrophoresis at pH 2.9 and the paper was radioautographed.

pears to produce the same type of phosphate bond as does denaturation at acid pH (6, 17, 18). Within this limited range the manner of denaturation does not affect the result. Con- firmatory evidence has also been sought in the literature with respect to the reactivity of the native phosphoenzyme with hydroxylamine, which forms hydroxamate derivatives from acyl phosphates with release of Pi. It was anticipated that hydroxylamine would react with the active site phosphate to form a stable hydroxamate and an inhibited form of the enzyme. The actual results were more complex (1, 2). Hydroxylamine may stimulate or inhibit (Na+ + K+)-ATPase activity. As a stimulator it may substitute for K+ or itself decompose and release NHJ+, which can substitute for K+ (19). The use of N-methyl hydroxylamine excluded the latter possibility and exposed a partial inhibition of phosphorylation (20). Complete inhibition has been obtained with addition of a metal such as Ca2+ to the reaction mixture (21).

In order to exclude the possibility in new experiments that

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0 5 IO 15 SECONDS AFTER UNLABELED ATP

FIG. 6. Acceleration by hydroxylamine of dephosphorylation of the phosphoenzyme of (Na+ + K+)-ATPase previously inhibited with N-ethylmaleimide. The enzyme was treated with N-ethyl- maleimide by a modification of the method of Fahn et al. (22). About 150 pmoles of inhibited (Na+ + K+)-ATPase was phos- phorylated by 40 nmoles of [yJ2P]ATP in the presence of 16 pmoles of NaCl, 1 pmole of MgCl?, and 10 Fmoles of imidazole glycylgly- tine (pH 7.5) in a volume of 0.8 ml at 0” and 5 s before zero time. At zero time further formation of [32P]phosphoenzyme was inter- rupted by a chase of 1 rmole of unlabeled MgATP in 0.1 ml which also contained 0.1 /*mole of KC1 to dephosphorylate immediately any native phosphoenzyme which might be present (0). After 5 s were added either 100 pmoles of Tris-Cl (pH 7.5) as a control (m), 100 pmoles of NHsOH.HCl neutralized with Tris (X), or 2 pmoles of MgADP (A). The last addition was made in order to confirm the effectiveness of the previous treatment with N-ethyl- maleimide, which replaces sensitivity to K+ in the native enzyme with sensitivity to ADP in the poisoned enzyme. The volume of these additions was 0.1 ml. The reaction was stopped with acid and radioactivity in the washed precipitate was estimated. Back- ground phosphorylation, with KC1 replacing NaCl, was sub- tracted. The rate constant for spontaneous dephosphorylation in the absence of NHgOH is k = 0.072 (s)-1 and in its presence k = 0.122 (s)-I, giving a difference of k = 0.05 (s)-’ or 3.0 (min)-I.

hydroxylamine might act like K+, the enzyme was treated with N-ethylmaleimide (22). After such treatment a [Y?]phospho- enzyme is obtained (from [32P]ATP in the presence of Na+ and 11g2+) which is insensitive to K+ (but is sensitive to splitting by ADP). In this form the active site is the same as in the native enzyme (23, 24). This treated enzyme was phosphorylated with [3?P]hTP and the [32P]phosphoenzyme was chased with a solution of unlabeled ATP containing 0.1 rnM Kf to dephos- phorylate immediately any residual native phosphoenzyme in the reaction mixture. During the subsequent spontaneous dephosphorylation of the treated phosphoenzyme, addition of 0.1 M hydroxylamine accelerated dephosphorylation at pH 7.5 and 0”. The increment in the rate constant of dephosphoryla- tion n-as 3.0 (min)+ (Fig. 6). Under the same conditions the increment in the rate constant for dephosphorylation of the limit phosphotripeptide, Pr4, was 0.066 (min)+ (Table II). The phosphate bond was 45.fold more sensitive to hydroxylamine before denaturation than it was after isolation of the active site phosphopeptide. This result showed that before denaturation the phosphoenzyme was highly sensitive to hydroxylamine by a mechanism not involving substitution of hydroxylamine for K+.

In order to look for an inhibited form of the enzyme as a

TA~~L-E II

Splitling of limit phosphotripeptide, Pr4, by 0.1 M hydroxylamine at pH Y.5 and 0”

To Pr4 in 15 ~1 of 0.01 ZVI HCl were added 15 ~1 of 0.2 M NHZOH. HCl freshly neutralized with Tris to pH 7.56. The reaction was stopped with 2 ,~l of 2 M HzSO~ at the times indicated and 15 pl were applied to paper for electrophorcsis at pH 2. After radio- autography Pr4 and Pi were cut out and counted. There was no significant hydrolysis in the absence of hydroxylamine or in the presence of acidified hydroxylamine. At zero time 57% of the radioactivity was in Pr4 and the results given have been nor- malized with respect to this value. The points fit an exponential curve with a correlation coefficient of 0.991 and a rate constant of 0.066 (min)-I.

Time PI4 Time Pr‘l

min % min %

0 100 6 62 1 84 8 54 2 82 10 49 3 73 16 34 4

I 70

I /

product of this reaction, the turnover of the phosphate group was estimated in the presence of hydroxylamine. In two experi- ments under the conditions of Fig. 6, except that hydroxylamine was added before ATP, the treated enzyme was phosphorylated with unlabeled ATP and 60 s later carrier-free [3ZP]ATP was added. Formation of the [3ZP]phosphoenzyme was about 1.4- fold faster in the presence of hydroxylamine than in its absence There was no reduction in the steady state level of the [““PI- phosphoenzyme in the presence of hydroxylamine such as is observed in the untreated enzyme (20-22). Phosphorylation was immediate when [32P]ATP was added to the dephospho- enzyme in the presence or absence of hydroxylamine. Since hydroxylamine increased the rate of turnover of the phospho- enzyme without inhibition of phosphorylation, there was no evidence in support of the formation of a stable derivative of the phosphoenzyme. Paper electrophoresis at pH 6 of the supernatant from two similar experiments (without acid de- naturation) showed a faint radioactive spot moving faster than a2Pi. Its formation required treatment of the enzyme with N- ethylmaleimide, hydroxylamine, and Na+ in the presence of Mg2f and [32P]ATP. It is possible that hydroxylamine attacks preferentially the P-O bond before denaturation, as water does in the native enzyme (25), and attacks the C-O bond after denaturation. Methanol and ethanol in acid solution attack The P-O bond after denaturation (26).

DISCUSSION

The active site limit phosphotripeptide of (Na+ + K+)-ATP is identifiable at present only by 32P which is held by a labile acyl phosphate bond. The quantities used in these experiments were less than 1 nmole and there was massive contamination by nonradioactive peptides. Previous experiments on the electro- phoretic mobility of the phosphopeptide, on its chemical re- activity and on the manner of its release by proteolytic enzymes

Thr

( > (6) have narrowed the choice of structures to Ser -X(P)-Lys where X is either aspartic or glutamic acid. To resolve this uncertainty, analogous model phosphotripeptides were syn- thesized and compared with the unknown active site phospho- tripeptide, Pr4, with respect to pH phosphohydrolysis profile,

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isoelectric point, and sensitivity of the lysinc bontl to cleavage by csrbosypeptidase B. The synthetic peptides, Pro-Glu(P)- Lys ant1 Pro-Asp(P)-Lys clearly differed from each other in these tests. Pro-Glu(P)-Lys was about 2.5-fold more stable at pH values 4 to 7 and about 2.5-fold more labile at p1-I values 9 to 11 than was Pro-Asp(P)-Lys (compare the circles in Fig. 2 with the circles in Fig. 3). The isoelectric point of l’ro-Glu(P)- Lys was 0.2 unit higher than that of Pro-Asp(P-Lys (Table I). Pro-Glu(P)-Lys was sensitive to carboxypeptidase B (Fig. 5 and Ref. 6) and Pro-bsp(P-Lys was resistant (6). These differences are functions of the phosphate group. In particular, the lysine bond of Pro-Asp-Lys was sensitive to carbosypeptidase B (6). These differences in function depend on a simple difference in structure, the presence (glutamic acid) or abscncc (nspartic acid) of a single methyl~~ne group in the aliphatic chain between the acyl phosphate bond and the backbone of the pcptidc.

The unknown l)hosphotripeptide, Pr4, differs from both of the model pcptidc; in that the proline rcsiduc is replaced by a serine or threonine residue. With respect to one of the model peptidcs this is the only difference; kvith respect to the other there is also the difference of the presence or absence of a meth- ylene group in the nliphatic chain of the central amino acid. The problem is therefore to clecide whether Pr4 has this distinc- tive methylcnc group or not,. With respect to the three tests of reactivity, l’r4 differs from Pro-Glu(P)-Lys in the same ways that Prl~-;\s1)(l’)-J,!-s does (Figs. 3 and 5, Table 1). On one

Thr

( ) hand, if l’r-l is Scr -Glu(l’)-Lys, then all three differences in Thr

reactivity must be assigned to replacement of prolinc by ( > Ser and this replacement. must have the same effect on the three functions oT the phosphate group as does deletion of a methylene group adjsccnt to the ncylphosphate group. This possibility is

Thr unlikely. On the other hand, if Pr4 is ( ) Ser --lsp(P)-Lys, t,hen these tliffcrences in the functions of the phosphate group can be assigned to the absence of the methylcne group by analogy with the effect of this methylene group on the functions of the phosphate group in the two model phosphopcptides. This choice for the structure of Pr4 implies that the replacement of proline by serine or threonine has relatively little effect on the functions of the phosphate group. In fact the only ap- parent diffcrcnce between Pro-lsp(l’)-J,ys and 1’1.4 was a less than 1.4.fold greater stability of Pr4 to hydrolysis between pH values 7 and 9 (Fig. 2). An interpretation for this dif- ference in reactivity is offered in the nest paragraph. In con- clusion, three functions of the phosphate group in the active site phosphotripcptide, I’+ from (r\‘a+ + I<+)-.Vl%se can be related much more directly to those of two analogous model phosphotripeptides if it is assumed that the central residue in Pr4 is @-asparty phosphate than if it is assumed that the central residue is y-glutamyl phosphate.

Interpretation of the specific shapes of the $1 hydrolysis profiles is outside the scope of this paper. Hovxvcr, in the case of the differcncc bet\xen the profile for 1% and that for I’ro- Asp(P)-Lys, it may be helpful to offer an interpretation con- sistent with the data. Between pH 6 and 10 the rate constant for Pro~Asp(I’-Lys changed very little, whereas that for Pr4 showed a decline of up to 30 T0 between pI1 7 and 9 (Fig. 2). Similarly betn-een $1 6 and 8 the electrophorctic mobility of Pro-Asp(l’)-Lys changed x-cry little, whereas that for Pr4 in- creased 1.5.fold (Table I) ; this increase indicates loss of positive charge from t11r termillnl amino group, which has a pK about 8

(5). The pK of the imino group of proline is I.5 units higher than that of the amino group of threonine or serine; consequently, Pro-Asp(l’)-Lys should retain its SH?-terminal proton in this range of ~1-1 where I’r4 loses its proton. The presence of such a proton xould tend to withdraw electrons from the acyl phos- phate bond. Di Sabato and Jencks (11) have shown that electron-withdrawing groups accelerate hydrolysis of acyl phosphates (their Table III). Absence of the proton could therefore leave Pr4 more stable than Pro-Asp(P)-Lys. Five atoms intervcnc between the terminal amino group and the carboxyl group linked to the phosphate; consequently the effect should not be large.

With respect to the sensitivity to hydrosylamine of an acyl phosphate on the enzyme before denaturation, it has been as- sumed that “hydrosylamine is inaccessible to the susceptible . phosphate residue” (1) .’ smcc formation of a stable enzyme- hydroxamate was not demonstrated (20). The present experi- ments showed that precautions to esclutlc a K+-like action (27) of hydroxylamine permitted demonstration of a sensitivity of the functional phosphoenzyme to hytlrosylamine 45.fold greater than that of the isolated phosphopeptide (Fig. 6 and Table II). Furthermore, preliminary experiments suggested that the products of the reaction include the insoluble dephosphoenzyme and a small soluble phosphorylated molecule (“Results,” test falla\\-ing Fig. 6). Therefore, it is likely that the acyl phosphate bond is stable to acid denaturation.

To our knowledge an identification of an aspartyl phosphate at the active site of an enzyme has not been previously reported in the literature. The y-carbosyl group of a glutamic acid residue has been reported as the phosphate binding site of ATP citrate lyase of rat liver (28). Acetate kinase (29) and 3.P-glycerate kinase (30) aiso each have a phosphocnzyme in which the phosphate is probably linked to a carbosyl group. Each phosphoenzyme has a U-shaped pH hydrolysis profile but no one of these profiles can be entirely superimposed upon another according to the data available. Enzymes are also known which bind acyl phosphates as transient intermediates during the reaction sequence. For instance, glutamine syn- thetase binds bound y-glutamyl phosphate (31), ATP citrate lyase binds citryl phosphate (32), and succinyl-CoA synthetase binds succinyl phosphate (33).

(Sa+ + I(+)-ATPase can be phosphorylated at the same active site either by ATl’ or by Pi. It is phosphorylated b> ATP in the presence of Ka+ and by Pi in the presence of K+ (34). Thus, it appears to l)ind the phosphate alternatively in high and low energy levels according to the binding of n‘a+ or K+, respectively. Such control of the energy level of the active site can be fitted into a tentative reaction sequence for the active transport of Na+ outward and I(+ inward across a cell membrane by this enzyme (34).

Ackno&dgments-We are indebted especially to Frances Gonsoulin and also to ,Jean Sorrells for attentive technical ns- sistance. We are indebted to James I’. Brineaus for preparing the (KS+ + K+)-ATPase. 11-e are indebted to Betty Orcutt for performing one of the experiments. We are indebted to Sidney Colowick, Jane II. Park, Giovanni Di Edwaid J. Hill for help in revising the manuscript.

Sabato, and

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Robert L. Post and Shoji KumePotassium Ion Transport Adenosine Triphosphatase

Evidence for an Aspartyl Phosphate Residue at the Active Site of Sodium and

1973, 248:6993-7000.J. Biol. Chem. 

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