Studies of high potassium and low potassium sheep erythrocyte membrane sodium-adenosine triphosphatase. Interactions with oligomycin, adenosine triphosphate, sodium, and potassium.

Abstract The effects of oligomycin on Na+-ATPase of high K (HK) and low K (LK) sheep erythrocyte membranes have been investigated. Activation of LK Na+-ATPase is observed with ATP ≤ 0.2 µm; activation of HK is observed with ATP ≤ 0.02 µm. Inhibition occurs with higher ATP. At 0.2 µm ATP, oligomycin stimulation of LK Na+-ATPase is associated with a 3- to 4-fold increase in the 32P-"intermediate;" inhibition of HK is associated with only a slight increase (1.3-fold) in 32P-"intermediate." The effects of oligomycin are similar for HK and LK in that activation occurs at low catalytic rates (≤ 20 pmoles mg-1 min-1); inhibition occurs at higher rates, irrespective of the means of altering the rate (varying ATP or Na+ concentration, or both; stimulation of LK with specific isoimmune antiserum). Oligomycin counteracts K+-inhibition (LK) and K+ counteracts oligomycin inhibition (HK). The results are consistent with a reaction sequence involving oligomycin-sensitive conformational changes of phosphorylated and probably unphosphorylated intermediate, i.e. [see PDF for equation] and [see PDF for equation] the resulting shift in equilibrium can, at low catalytic rates, be evidenced in stimulation of Na+-ATPase. Interaction of HK and LK Na+-ATPase with Na+, K+, and ATP are interdependent and markedly different for the two; at constant ATP (0.2 µm), HK is more sensitive to activation by Na+ and less sensitive to inhibition by K+ than LK ATPase. Although effects of both K+ and oligomycin are dependent on ATP concentration, a difference in affinity for ATP in addition to, or as a result of, a different relative affinity for Na+ and K+ may be the basis for the distinctions between HK and LK membranes.


SUMMARY
The effects of oligomycin on Na+-ATPase of high K (HK) and low K (LK) sheep erythrocyte membranes have been investigated.
Activation of LK Na+-ATPase is observed with ATP 5 0.2 pM; activation of HK is observed with ATP 5 0.02 PM. Inhibition occurs with higher ATP. At 0.2 pM ATP, oligomycin stimulation of LK Naf-ATPase is associated with a 3-to 4-fold increase in the 3ZP-"intermediate;" inhibition of HK is associated with only a slight increase (1.3-fold) in 32P-"intermediate." The effects of oligomycin are similar for HK and LK in that activation occurs at low catalytic rates (5 20 pmoles mg-l min-I); inhibition occurs at higher rates, irrespective of the means of altering the rate (varying ATP or Na+ concentration, or both; stimulation of LK with specific isoimmune antiserum). Oligomycin counteracts K+-inhibition (LK) and K+ counteracts oligomycin inhibition (HK). The results are consistent with a reaction sequence involving oligomycin-sensitive conformational changes of phosphorylated and probably unphosphorylated intermediate, i.e. EiP + EOP and Ei + E,,; the resulting shift in equilibrium can, at low catalytic rates, be evidenced in stimulation of Na+-ATPase. Interaction of HK and LK Na+-ATPase with Naf, K+, and ATP are interdependent and markedly different for the two; at constant ATP (0.2 KM), HK is more sensitive to activation by Na+ and less sensitive to inhibition by K+ than LK ATPase.
Although effects of both Kf and oligomycin are dependent on ATP concentration, a difference in affinity for ATP in addition to, or as a result of, a different relative affinity for Naf and K+ may be the basis for the distinctions between HK and LK membranes. A preliminary report has been presented (1). occurrcncc of a genrtic modification of the ion transport system in red cells of certain species, e.g. high potassium (HK) and low potassium (LK) sheep crythrocytcs (a), (b) the evidence in red cells for a close relationship between ion transport and menbrane-bound N&activated ATl'ase activity (3-G), and (cj the relative purity of mammalian erythrocyte mcmbrancs conlpared t,o many other membrane preparations. Previous stud& showed that Na+-plus I~+-ATPase activity of HK' and LK sheep red cells correlated quantitatively with the Na+, I(+-pump activity of the two types of ~11s (7,8), i.e. both activities were several-fold higher in IIK cells as compared to LK ~11s.
A similar difference was all apparent in the partial reactions associated with "transport ATPax" (9). The steady state level of Na+-dependent membrane phosphorylation (phosphorylatcd intermediate) was approsimatelg 7-fold higher in HK than LK membranes; a similar ratio was found for the number of ouabaiu-binding sites in the tn-o types of cells (10). In addition to the quantitative difference between IIK and LK membrane Na+-ATPase, kinetic differcnccs were also found when the system was studied at low ATI? concentration (9). In particular, the two types of membranes differed in their response to I(+; the HK type was activated b>-low levels of I<+ and inhibited only wit,h relatively high concentrations, whereas the LK type was markedly inhibited by I(+, even at low concentrations.
In the prcscnt study we hare continued the investigation of these quantitative and kinetic diffcrcnces to elucidate further the nature of this genetic modification which may be of fundamental importance in understanding the Nn+, K+--1TPasc enzyme system.

METHOIIS
Mcmbr~anes were prepared from fresh, sheep erythrocytes as described previously (9). The volume of the suspensions, equivalent to one-half of t,he original packed ccl1 volume, contained 2 to 4 mg of protein per ml; fulther dilutions were done as indicated, with 2 in;11 Tris-HCl (pH 7.4). l'roccdures for labeling membranes with SZP using [8-%, y-"'?P]ATP and for exchange rates with oligomycin present w-cre more similar in magnitude than the Na+-dependent ;\TI' hydrolysis rates; i.e. the HK:LK activity ratio for exchange was 2.7, whereas that for hydrolysis was approximately 10. XIorcover, these studies (9) and those of others (12)(13)(14)(15)(16)(17) suggested that oligomycin ir1hibit.s the multistage Na+-ATPase at a step othrl than the sodium-stimulated phosphorylation of an "intcrmcdiate." In the prcseut study, we examined further the cffccts of oligomycin on the two types of membranes to determine \\-hethcl their difference is reflected in a difference iu rate of a step prior to, or subsequent to, the step affected by oligomycin.
The effects of oligomycin on the Na+-stimulated phosphor~lation C%intermediate) are shown in Fig. 1. At 0.2 ~CLJI -iTP, oligomycin markedly increased the Y' bound in LK (3. to 4fold), but only slightly increased that bound in HK.
-it lower ATP (0.02 PM), oligompcin increased phosphorylation in HK as well as in LK, but to a lesser extent (N1.7.fold).
To tletermincl whether these efYccts represented increased or decreased catalytic center activity, the Na+~ATPnse rate was also mra-;ured under identical conditions.
The Na+--\TI-'ase divided by the Sa+stimulated increment iu phosphorylation, i.e. the turnover 01 catalytic center act'ivity is shown in Fig. 2 for experiments carried out at 0.2 pM ATP.
In the absence of oligomycin, the catalytic center activity of HK was about a-fold that of LK. In the prcscncc of oligomycin, the turnover of HK XV dccreased and that of LK was not changed, with the result that the turnover of the two types in the presence of oligomycin tended to bc similar.
Oligomycin increased the level of phosphorylated intermediatje in LK membranes ( Fig. 1) and did not alter its turnover (Fig. 2) due to a stimulatiou of Na+-ATPase activity. This is shorrn in Fig. 3  2. Effects of oligomycin on catalytic center activity of HK and LK Na+-ATPase.
Catalytic center activity was determined from (a) the amount of "2P-intermediate and (b) Na+--1TPase activity measured in the same reaction vessel, as described in Fig. 1 Figs. 1 and 2. In Fig. 3 the Na+-STPase activity without oligomycin is the control and is represented as 100%.
Values of Na+-ATI'ase with oligomycin are represented as a percentage of the control; i.e. values above the 100% lint represent stimulation, and vnlucs below rrpresent inhibition.
However, the curves for both systems \V(YCI similar with respect to the apparent stimulation observed at the lowest *iTI' concentrations followed by a tendency toward inhibition at higher ATP concentrations. Similar results were obtained with higher lllgz+ concentrations (0.1 mM). Activating effec?n could not be observed by varying the concentrations of oligoniyc~in.
When the Nat concentration was varied, it was also found that the oligomycin response could be altered. This may rcflcct ai1 influence of the inhibitor on the kinetics of PJa+ activation, as described by Robinson (17) and shown in Fig. 4. In t,hc> absence of oligomycin, the Nn+-activation curve for IIK mc~ml~n~cs approximates Michaelis-Mcnten kinetics and is markedly diffcreiit from that obtained with LK, e.g. 20 m&r T\'s+ markedly stimulated HK but failed to increase the activity of IX above that observed wit.hout added Na+. Furthermore, the activity of LK is lowered by the addition of small amounts of 511' (5 5 m&r) to values below that observed without nddcd Na+. If the activities are compared to the activity without added Xa+ but with 50 rnM K+ added to counteract possible act,ivation by trace amounts of Na+, the Na+-response curve appears to consist of two components; one is activated by low levels of Naf, whereas the other is activated by >20 mM Na+.
With oligomycin present, the sensitivity to activation by Na+ was markedly increased in bot,h HK and LK membranes.
The data in I?&. 4 also show that without added Na+, oligomycin stimiilatrs ATl'asc activity, particularly in HK membranes. This stimulatory effect was abolished when 50 mM KC1 was inclutkxl in the medium to obliterate effects of small amounts of P\'a+.
It has been shown previously that oligomycin inhibits I(+stimulated hydrolysis of the 32P-intermediate (11,12). The interac: tion of oligomycin and K+ with the membrane Na+-ATJ'ase assayed at 0.2 pM ATP and 50 rnM NaCl arc shown in 4. l<Xfect of Na+ concent,ration and oligomycin on ATPase activity of HK and LK membranes.
act oligomycin inhibition, i.e. the percentage inhibition was less with I~+ prcxclnt; with LK mcmbrancs, K-inhibition was countcr:&xl by oligomycin. These results arc compatible with inhibition by oligomycin at a step in which thcrc is a transformation of an intcrmediatc or site to a I~+-sensitive state, whether the I(+. cffcct is iiiliibit,ory or stiniulatory.
Txteraction wiih A TI', ;L'a+, and Ii+-The f'orcgoing r(lsu1t.s liavc shown that the oligoni,vc~in response is dqx~ridcnt on the -iTI' and, to an cxtcnt, the Na+ c,ollc~c~ntratiolls and is countcr- is tlecreaxt~l by K+ (18, 19), an effect coulit(~ra('t(xl by N:r-b. These observatioils have raised the possibility lrhat the basis for the diffcrcnccs betn-ccn HK and LK transport systems is their relative aflinitics for ATl' which in turn affect the K, for Na+ and K-k or vic:e versa, or both. Rlichnclis const,ants (K *rP) for Na-l-l\Tl'asc wcrc dcterminrd with a range of ATl' concentrations from 0.01 to 2 +M. $lthough l,he values obtained wcrc sinrilar to the reported dissociation constant for ATP binding (18, 19), variation in values determined with lnqxnxtions from different animals prcicluded a meaningful comparison of IIK and LK; J<ATP for LK was 0.13 f 0.04 PM (three animals), whereas for IIK it was 0.06 f 0.02 pnr (four anitnnls) . How-ever, the rcsporlse of Xa+-ATl'ase to Kf was found to be dcpendcnt on .ITl' concentration, particularly with l-II< membranes, and to be different for the two types of mcmbrancs as shown in Fig. 5. As the ,\TP concentration dccrcased, xtimulatory effects of K+ dccrcasctl and inhibitory effects incrcasctl.
Wl~i the Na+ concciitration was varied, the r~~~lx~i~~ to KS n-as altc~rc~tl as sliown in Fig. 6. I>ecrca&g the Sa+ increased inhibition by Km in both ILK and LK membranes. The interplay of both :u*tivnting and inhibitory cffrcts of K+, and the action of ?;:I+ 011 these effec2t.q are al-;o alqxlnnt with IIK menibranes. X:1' iiot only decreased K+ inhibition as described above, l)iit it also tlccrcascd K+ activation as intlicnted by the decrcxsc in I<+ ac~tivntion (1 nut KCl) as X:1+ was increased from 5 to 20 mu. With I mx Na+, the rcsporrsc pattern for IIK tc~ndcd to rcscmble that of LK measured with 20 m&f Na+. Wh(n similar data are plotted as activity versus Xa+ concclntration (Fig. 7), an apparent decrease in activity at, low K-a+ levels is cvitk~nt, iii LK in the absence of K' and is more marked Tf'ase-11Xen LK mcmbrancs arc treated with specific isoimmunc (alIti-L) serum, the Na+, K(+ pump and iVa+-ATPax actiyit\ arc increased sereral-fold (20)(21)(22). IIowc~r, t,he kinetirs of the Na+-ATPase new sites arc not identical wit,h those of IlK but rcscmhle those of LK membranes, with respect to the I(+response pattern (22). WC hare compared the effect of oligom!-ciu on the activity of anti-L-stimulated LK Na+-ATPase (Table II).
In the cont>rol or nouimmune scrunl-treated membranes, Na+-~Yl'l'asc activit,y (0.2 FM .Vrl') was incrcnsed by oligomyciil as in Fig. 3   with the .<y+ttm and vice versa, and increases the alqn-cnt affinity for Sn-, as observed in these studies. Decreased inhibition of Na+, K+-XTPasc by oligomyc?n as the -1TP concentration is decreased has been observed by Robinson (li).
IIe suggested two possible routes of hydrolysis: hydrolysib of EiP (lo\\-V,,,, Pathway A4 of Fig. 8) and hydrolysis of Rol' (high r,1,,,, Pathway R of Fig. 8). His model does not necessarily include Ei + E0. &it low ATP concentration, hydrolysis of EJ' could be relatively faster than that of E:,J', but the over-all rate limited by rapid conversion of EiP to Eel'. With oligomycin, the equilibrium would be shifted towards Bi forms; Pathway 13 would be dccrcased, and Pathway ;1 would still function and appear as a stimulation, particularly if interaction of ATl with E< is no longer decreased by the equilibrium E; with EC,. Other possibilities cannot be ruled out; in particular, if oligomycin does not inhibit completely KJ' + EOP (and Ei + Bo), a concomitant increase in initial phosphorylatlon (Ei + JCJ') would be manifested in tither activation or inhibition depending upon whether the transition step (Eel' + ITOP) is relatively faster or slower than the phosphorylation step, respectively.
That the effects of oligomycin are dependent on the absolute activity, peg se, are indicated when the effects are examined as shown in Fig. 9. Here all values for Na+-;ITPasc activity obtained at various -1TP concentrations and at constant Na+ concentration (50 mnr) are plotted. The Na+-ATPase in the presence of oligomycin is shown as a percentage of the control. The open symbols represent LK sheep (each symbol represents a different animal), and the closed symbols represent HK sheep. As shown, there is considerable overlap of the values of IIK and LK.
The data are depicted in this manner in order to cmphasize that T&en the absolute Naf-ATPase activity is greater than about 20 pmoles mg-l min-I, the effect of oligomycin is inhibitory.
The dependence of the oligomycin effect on t.he absolute activity is apparent not only when t,he activity is altered by changes in substrate and activator (Naf) concentrations, but also when the activity is altered (increased) by the specific reaction of LK-t.ype membranes with specific isoimmune serum as shown in Table II. ..Iv-=HK ooav~=LK r CO/JfrO/: No -ATPase ( pmoles mg-I mid) F~ti. 9. Effects of oligomycin on Na+-ATPase of HK and LK membranes.
Each sy&ol represents the activity, determined in a seuarnte exneriment. of an HK (closed sz/mboZs~ or an LK Cohen sl/~lboZ.s) sheep; each &flerent symbol represents a different animsl. Values are taken from experiments carried out at various ATP concentrations as described in Fig. 3.
AITP concentration affected the rcspor~se of Naf-XTPasc not only to oligomycin but also to I(+; howcvcr, the Kf-response l)attcrn for LK remained different from that for HK membranes, ~cn when the A1TP concentration was varied by two orders of magnitude.
It is plausible that a difference in affinity for ATP, in addition to or as a result of different affinities for Xa+ and K+ is a basic kinetic distinction between the two types of membrane Sa'-.\l'Pasc and Na+, K+-pump systems. When a constant ;Yl'l' concentration (0.2 PM), the K+ response of HK rcscmbled that of LK when the Na:K concentration ratio for HK was 1 '20th that of LK (Fig. 6). The pattern of I(+ response with vari0u.s Sa+ concentrations indicated an effect of Na+ not only on I<+ activation, but also on K+ inhibition, both types of resl)onsc bcitlg clearly evident in HK mcmbrancs assayed with 101~ -Yrl' f onccntration.
Xthough low XT1 concentrations arc helpful for csarnining certain distinct effects of X-a+ and K+, the necessity of using fragmc~nted cell membranes to study the ATPase system does not allow the separate analysis of the effects of Na+ and of K+ on each side of the membrane.
It is not possible to determine whetlrcr HK and LK membranes arc different with respect to the interaction of one or both of the ionic species with one or both sides of the membranes.
In studies of the pump with intact cells, Hoffman and Tosteson obscrvcd differences in both the affinity of the pump for extracellular K+ and for intracellular Sa+ or I<+, or both (25). On the basis of their studies, they concluded that their data were compatible with a model consisting of rapidly equilibrating puml)-ion complexes.
They proposed that the rate of an actual transport step would be governed by the "fraction of pump sites which are loaded with appropriate ions on bot'h the cis and tram surfaces." In the present scheme, differences in affinities for Naf or K+, or both, between IIK and LK membranes may be associated with a difference in the steady state equilibrium between components of the Na+-ATPase system, i.e. EiP + E:,I' and Ei + Ro. The former step would probably be associated with K+ activation due to K+-activated hydrolysis of EoP; the latter step, with K+ inhibition, would be due to inberact,ion of K+ with EO. Evidence to support these effects of K+ have been obtained recently by Sicgcl and Goodwin (26). il difference in the equilibrium of components would be a conscqucnce of a difference in rate of a single step and would be reflected also in a quantitative difference in over-all Xa+--1TPase activity, as described previously (9).