The steady state level of phosphorylated intermediate in relation to the two sodium-dependent adenosine triphosphatases of calf brain microsomes.

Abstract During ATP hydrolysis, microsomal preparations form an acid-stable protein-phosphate complex. Since there is evidence that two different independent enzymatic sites catalyze the hydrolysis of ATP in calf brain microsomes, it was reasoned that either one or both of these might contribute to the formation of this complex. To determine the actual relationships, the phosphorylation reaction was compared with hydrolysis at each of the ATPase sites—at different levels of Na+, K+, and ATP and at different temperatures. The kinetic analysis indicates that it is hydrolysis at Site I, the Na+, -K+ ATPase, which correlates with the steady state level of protein-phosphate complex. The results add support to the hypothesis that this complex is an intermediate in the hydrolysis of ATP by the Na+-K+ ATPase—the site that takes part in Na+ and K+ transport across biological membranes. The second ATPase, Site II, is a Na+-ATPase inhibited by K+; the kinetic analysis indicates that this site does not contribute substantially to the steady state level of acid-stable phosphorylated intermediate. It is suggested that Site II may be related to a sodium pump such as the one in red blood cells described by J. F. Hoffman and F. M. Kregnow (Ann. N. Y. Acad. Sci., 137, 566 (1966)).

In an earlier paper, we presented evidence that two different ouabain-sensitive enzymatic sites independently catalyze the hydrolysis of ATP in calf brain microsomes (2) ; similarly, Czerwinski, Gitelman, and Welt have presented evidence for these two different sites in rat erythrocytes (3). One of these (Site I) is the Na+-K+ ATPase considered to be part of the mechanism for Na+ and Kf transport across biological membranes (4, 5 To answer this, we compared hydrolysis at each of the sites with the phosphorylat,ion reaction, as functions of Naf, K+, ATP, and temperature.
The results support the conclusion that it is Site I, the Na+-K+ ATPase, which forms the protein-phosphate intermediate; the kinetic analysis shows how the formation and breakdown of this intermediate relate to steps in the mechanism of hydrolysis at this site.

Preparation of Calf Brain Microsomes
Calf brain microsomes were prepared by a modification of the method of Schoner et al. (11) as previously described (3).

Preparation of A T32P
ATs2P was prepared by a modification of the method of Glynn and Chappell (12) as previously described (3).

Measurement of Acid-stable EnzymeJzP Complex
The procedure for showing 32P labeling of protein from AT3*P was a modification of the method described by Post,Sen,and Rosenthal (13). The amount of phosphate complex was determined by measuring the specific activity of acid-precipitated protein after incubation in the appropriate reaction media. In a typical experiment 3.0 ml of enzyme suspension (3.5 mg of protein per ml) were added to 100 ml of a cold solution having the following composition: 100 mM NaCl, 5 mM MgC12, 100 mM imidazole, pH 7.3. For a single experiment, 5.0 ml of this solution were used with 0.05 ml of water or 0.05 ml of 1.0 M KCl.
The mixture was brought to 37" by incubation for 15 min All centrifugations were at 10,000 x g for 20 min at 4".
To release the incorporated 32P, the final precipitate was heated for 15 min at 100" in 5.0 ml of a basic solution: 0.1 M NaOH, 0.2 M Na2C02, 0.1 mM K2HP04. When the solution cooled, 3.0 ml were assayed for 32Pi as described for measurement of hydrolysis of AT32P (3).

Measurement of AT32P Hydrolysis
The method for measuring the rate of release of 32Pi from AT32P has been described previously (3). The reaction conditions were the same as those used for measuring phosphorylation except that the protein concentration was lower and the total volume was 10 ml. in experiments in which the ATP concentration was lower, the level of K+ was high enough to inhibit Site II and again make its contribution negligible. Site II, Naf ATPase-In general, to determine this activity, the rate of ATP hydrolysis with and without Na+ was measured in the absence of K+.

Conditions for Separately
The activity at Site II is the difference between these rates with and without Na+.
The specific conditions for particular experiments are given in the legends of the figures.
In the absence of K+, Site I does not hydrolyze ATP.

Mg++
ATPase-This is the ouabain-insensitive activity measured in the absence of monovalent cations.
Determination of V,,, and K, at different temperatures K, and V,,, were determined from reciprocal plots of velocity against ATP concentration. At each temperature, the rates of hydrolysis were measured under optimal ion conditions for that temperature.
Preliminary estimates made at each temperature gave the approximate K, value. For the experimental determinations, substrate concentrations were then chosen to cover a range from 0.25 to 4 times the K,.
For each determination of vnmx and K, six substrate concentrations were used and the reciprocal plots were linear.

RESULTS
AND  (2) where E -Psat is the concentration of complex when both Na+ and K+ are at optimal levels. This relationship is shown in Fig. 1 where khTa$ is the proportionality constant when the Na+ level is optimal, and where E.ATP,,, is the concentration of unphosphorylated sites when both Naf and K+ are at optimal levels. The relationship is shown in Fig. 2

63
Here k& was the same as in Equation 2, and, at any concentration of ATP v E-P -= V max E -Pat (7) as shown in Fig. 3 Fig. 1. The calculation of kNa+ is described in the text.
in the text. tration.
The function is complex (as is the sodium dependence of over-all hydrolysis, Fig. l), suggesting the involvement of more than one Na+ in the formation of E N P. Temperature Dependence of Hydrolysis by Different Sites-h past work (14,15), there has been some difficulty in correlating hydrolysis and phosphorylation at low temperatures. Moreover, the effects of K+ at low temperature have been difficult to interpret (16,17). It seemed to us that these problems might come from the failure to recognize the relatively high contribution that Site II, the Na+ ATPase, makes to over-all hydrolysis at low t,emperature.
This led us to study each of the ATPases in calf brain microsomes separately as a function of temperature.  (background  activity  insensitive to Na+, K+, and ouabain) changed with temperature.
A notable feature in these Arrhenius plots is the nonlinearity in the curve for Site I, the Na+-K+ ATPase. This curvature has been observed before in rabbit and rat brain microsomes even when total hydrolysis was measured without any attempt to distinguish Sites I and II (15,18). The sharp falling off of the curve is not a reflection of cation or substrate inhibition of Site I at low temperature; at each temperature the measurements were made under optimal conditions for that temperature, and the reciprocal plots showed no evidence for inhibition at any temperature. Instead, there is probably a reversible, temperature-induced conformational change.
This phenomenon has been reported for a number of other enzyme systems, e.g. Reference 9. For the Na+-K+ ATPase, between 25 and 35", the energy of activation was 12 kcal per mole; between 1 and 11" it was 51 kcal per mole.
For the Na+ ATPase over the entire temperature range the energy of activation was 19 kcal per mole; for the ?rlg++ ATPase it was 14 kcal per mole. ing phosphorylation and hydrolysis at 9", the same relationships were found to hold as those described for 37"; that is, the data at 9" fit the steady state relationships given in Equation  10. We conclude that hydrolysis at Site I occurs through the same mechanism at 9" as at 37". Fig. 7 shows the temperature dependence of K, for each of the enzymes.
The curve for Site I shows a break similar to that seen in the V,,, curve (Fig. 6). Here again this probably reflects the temperature-induced conformational change affecting Site I. Temperature Dependence of Phosphorylation Reaction-Compar-As shown in Fig. 8 there was no significant change in E or (E N P)S&t with temperature, and the ratio (E -P),,JE was constant.
Since (E -P),,,/E is a function of &a~&& and k& as given in Equation  12, the constancy of this ratio suggests that both these rate constants-for formation and breakdown Conditions were the same as for Fig. 2 except for the indicated temperature. 0, with 10 mM K+; this gives a measure of E -Psst as described in the text. l , without K+; gives a measure of E as described in the text. of E N P-change proportionately.
Moreover, they must both decrease when temperature is lowered because Vmax, which equals kKzat (E -P)sst, falls with temperature.
Interpretation of K-t EJects at Low Temperature- Table   I shows some typical effects of K+ at low temperature.
Observations of this kind have been reported for rabbit brain (15), pig brain (17), and human red blood cells (16). To interpret the apparently complex effects of K+ on the ATPase at low temperature it is helpful to emphasize three points: (a) at lower temperatures under optimal conditions, Site II accounts for a greater proportion of the total ATP hydrolysis (Fig. 6) ; (a) Site II at all temperatures has a much lower K, (Fig. 7) ; and (c) K+ inhibits Site II and activates Site I (3).
With these features in mind, consider for example the effect of K+ at 0" and 10 PM ATP (Table I).
With Na+ but no K+, all At the lowest level of ATP in Table I (1 PM), Site I is far more unsaturated (Km = 2.5 x lOA M at 0") than Site II (K, = 2.5 X 10-S M at 0"). Under these conditions, the K+ activation of Site I will not completely offset the inhibition of Site II. The over-all effect is therefore inhibition by K+. Thus, as shown by the examples in Table I, with these two enzymes operating independently, K+ can activate, inhibit, or have no effect at all on the over-all rate.
General Conclusions-The evidence for two independent ouabain-sensitive ATPase sites in calf brain microsomes was presented by us in an earlier paper (3). Before this Czerwinski et al. (2) had inferred the presence of a second ATPase from their kinetic studies on rat erythrocytes, and Kanazawa, Saito, and Tonomura (15) had obtained similar data from their work on rabbit brain microsomes, although they interpreted it somewhat differently.
More recently, a number of reports have appeared, on electric organ (20) and kidney (21), which we believe support the argument for two Na+-dependent enzymatic sites. From all of this, it seems to us that Site II, the Na+-ATPase, as well as Site I, may have wide general distribution in biological membranes.
In any event, when Site II exists in a preparation, the recognition of its presence helps to analyze the kinetics of ATP hydrolysis.
For example, in this paper, we have shown that the acid-stable enzyme-phosphate complex of calf brain microsomes is an intermediate in the mechanism of hydrolysis by Site I, not only at 37", but at low temperature as well. Without recognizing Site II, the correlation of over-all hydrolysis with E -P formation is not nearly so good at 37" and cannot be made at all when the temperature is low (14,15). Also, as we have shown here and in the earlier paper (3), a consideration of two independent sites explains certain complexities in the kinetics of over-all hydrolysis and in the cation effects, particularly at low temperature.
The correlation betweeu hydrolysis at Site I and the phosphorylation reaction adds further support to the proposal of Post et al. (13)  at this site, then, remains unknown. In considering the biological function of Site II, there are many Na+-dependent membrane transport systems in which it could take part.
One possibility that seems particularly promising is a Na+ pump in the red blood cell described by Hoffman and Kregnow (22). It has the following properties: (a) it is activated by external Na+, optimal levels being about 150 mM; (b) it transports Na+ from inside to outside the cell; (c) it is ATPdependent; (d) it needs no K+; and (e) it is inhibited by ouabain, and this effect is antagonized by K+. For comparison, Site II is activated by the same high levels of Na+ that activate this pump, much higher levels than the Na+-K+ ATPase requires. It seems from this that Site II might be activated in z&o by external Na+.
Also, similar to this Naf pump, Site II needs no K+, is inhibited by ouabain, and-despite the fact that its activity requires no K+-the effect of ouabain is antagonized by K+.l Whether or not Site II is actually a part of this Na+ pump described by Hoffman and Kregnow remains to be seen.