ARCHIVES
OF
BIOCHEMISTRY
Transport
AND
BIOPHYSICS
175, 185-189 (1976)
ATPase of Erythrocyte Membrane: Sensitivities of Na+,K+ATPase and K+-Phosphatase Activities to Ouabain’ W. HUANG
Department
of Pharmacology
and
AND A. ASKARI
Therapeutics,
Medical
College
of Ohio,
Toledo,
Ohio
43614
Received January 19, 1976 Cardiac glycosides are inhibitors of Na+,K+-ATPase, and K+-phosphatase activities of the transport enzyme. Previous studies have shown that when the sensitivities of these two activities to ouabain are compared by the addition of varying concentrations of the drug to the assay media, the K+-phosphatase is significantly less sensitive than Na+,K+ATPase. This work was done to seek an explanation for this phenomenon. 3-O-Methylfluorescein phosphate was used as substrate for the continuous fluorimetric assay of K+phosphatase obtained from human red cells. When ouabain was added to the assay medium, a time-dependent inhibition of K+-phosphatase was observed. The rate of inhibition was also influenced by the order of additions of K+ and ouabain. In view of these results, several enzyme samples exposed to ouabain for varying lengths of time were prepared, and their Na+,K+-ATPase and K+-phosphatase activities were then determined. A good correlation between the extent of inhibition of the two activities was obtained. These results prove that the previously observed discrepancies between the sensitivities of Na+,K+-ATPase and K+-phosphatase to ouabain are due to the different kinetics of drug interaction with the enzyme under the different conditions of the two assays and that once a certain level of ouabain binding to the enzyme is achieved, both activities are equally inhibited. Preparations of (Na+ adenosinetriphosphatase
+
K+)-activated
(Na+,K+-ATPase, ATP phosphohydrolase, EC 3.6.1.3) from various tissues also catalyze the K+activated hydrolysis of p-nitrophenylphosphate (l), acetylphosphate (2), carbamylphosphate (21, umbelliferone phosphate (3), and 3-O-methylfluorescein phosphate (4). Several laboratories have reported that this K+-phosphatase activity remains associated with highly purified enzyme preparations (5-7). Thus, in spite of some early doubts (8, 9), it is apparent now that the K+-phosphatase activity is a partial reaction catalyzed by the purified enzyme which contains two major polypeptides and phospholipids (10-12). Recent work in our laboratory (4) demonstrated that when the fluorigenic sub-
’ This work was supported by NIH Research Grant HL-19129 awarded by the National Heart and Lung Institute, PHS-DHEW.
strate 3-O-methylfluorescein phosphate is used for the assay of K+-phosphatase, and when the Na+,K+-ATPase and K+-phosphatase activities of several partially purified enzyme preparations and crude tissue fractions are compared, a good correlation between the two activities of the different preparations is obtained. These findings suggested that in situations where the assay of Na+,K+-ATPase activity is difficult or impossible to perform (either because of the low specific activity of the enzyme or due to limitations of sample size), the highly sensitive fluorimetric assay of K+MFPase2 may be used to determine the level of this enzyme in various samples. A potential value of the K+-MFPase assay may be envisioned in those experiments where a tissue is exposed to a cardiac glycoside, and the possible relationship be* Abbreviation used: K+-MFPase, 3-O-methylfluorescein phosphatase. 185
Copyright 8 All rights of
1976 by reproduction
Academic Press, Inc. in any
form
reserved.
K+-dependent
186
HUANG
AND ASKARI
tween the induced pharmacologic effect and the inhibition of the membrane-bound enzyme is sought (e.g., 13-15). The question arises, however, whether the Na+,K+ATPase and K+-phosphatase activities of the enzyme are similarly affected by cardiac glycosides. Judah et al. (1) were the first to demonstrate that the K+-phosphatase (assayed with p-nitrophenylphosphate as the substrate), like the Na+,KTATPase, is inhibited by cardiac glycosides. However, in several subsequent studies when the effects of varying concentrations of a cardiac glycoside on K+-phosphatase and Na+,K+-ATPase activities were compared, it was found that the K+-phosphatase activity is significantly less sensitive than the Na+,K+-ATPase to the inhibitory effect of the drug (8,16-21). Since the addition of Na+ and ATP to the assay mixture for K+-phosphatase activity increases the sensitivity of this activity to ouabain (8, 18, 21, 25), and since Na+ and ATP increase the rate of ouabain binding to the enzyme (27), it has been suggested (25,27) that the above observations on the different drug sensitivities of K+-phosphatase and Na+,K+-ATPase may be due to the different rates of ouabain binding to the enzyme under the conditions of the two assays. This hypothesis, however, has not been rigorously proved; the possibility that after ouabain binding to the enzyme the two activities may be inhibited to different extents has not been ruled out. The experiments presented here were done to clarify these questions. MATERIALS
AND METHODS 3-O-methylfluorescein 3-@Methylfluorescein, phosphate, ATP, and ouabain were obtained from Sigma Chemical Co. (St. Louis, MO.). Fluorimetric determintion of 3-0-methylfluorescein phosphatase activity was done by the continuous recording procedure as described before (3, 4). K+-MFPase activity of each enzyme sample was determined at 37°C under the following standard conditions. The enzyme suspension (lo-50 ~1) was added to 2.5 ml of a solution containing 0.8 PM substrate, 4 mM MgCl,, 1 mM EDTA, 80 rnrd Tris-HCl (pH 7.6). Progress of the reaction was recorded for 5 min. A concentrated KC1 solution (10 ~1) was then added to obtain a final concentration of 10 mM KC1 in the mixture, and the recording was continued for another 5 min. The KC-
dependent activity was calculated from the difference between the slopes of the two resulting straight lines. For certain experiments deviations from the above standard conditions are noted in the text. The Na+,K+-ATPase activity of the enzyme was assayed by the measurement of released inorganic phosphate (22) after incubation at 37°C in a medium containing 2 rnru ATP, 3 mM MgCl,, 1 mM EDTA, 100 mM NaCl, 25 mM KCl, and 80 mM Tris-HCl (pH 7.61, in a total volume of 2.5 ml. To determine the Na+,K+-independent portion of the activity, KC1 and NaCl were omitted from the reaction mixture. For these assays, reaction time was 20 min or less, and the amount of substrate used was no more than 20% of the initial amount. It should be noted that under the standard conditions of both assays, Na+,K+-ATPase and K+-MFPase, the amount of product formed was a linear function of time for all control and partially inhibited enzyme samples. Thus, enzyme preparations containing bound ouabain behaved as irreversibly inhibited enzymes in the course of both assays. Hemoglobin-free membranes from human red cells were prepared as described before (23). Membrane preparations with partially inhibited enzyme activities were obtained either by the exposure of hemoglobin-free membranes to ouabain or by the exposure of intact red cells to the drug prior to the preparation of membranes. Isolated membranes were added to 10 vol of a solution containing 10e6 M ouabain, 1 mM EDTA, 2 mM MgCl,, 2 mM TrisH,PO,, and 50 mM Tris-HCl (pH 7.4). After incubation at 37°C for 5-20 min, membranes were sedimented by centrifugation in cold at 15,OOOg for 15 min. They were then washed three times in 50 vol of a cold solution of 1 mM Tris-EDTA (pH 7.4). Exposure of intact cells to ouabain was achieved by suspending whole blood in 10 vol of isotonic saline containing lo-’ M ouabain and incubating the mixture at 37°C for 5-30 min. After centrifugation the cells were washed twice in 15 vol of cold saline. Membranes were then prepared from these cells in the cold. RESULTS
AND DISCUSSION
The different sensitivities of K+-phosphatase and Na+,K+-ATPase activities to cardiac glycosides that are added to the assay media have been observed with enzyme preparations from a variety of tissues (8,16-19) including that of the human red cells (20, 21). We chose this particular enzyme for the present studies because of the extreme ease with which it can be prepared and handled. The notoriously low specific activity of this preparation was also of particular interest, because it
OUABAIN
SENSITIVITY
is, in fact, with enzyme samples of low activity obtained from drug-exposed tissues that one may wish to use the highly sensitive assay of K+-MFPase activity in preference to the assay of Na+,K+-ATPase activity. Effect of Ouabain on K+-MFPase Activity of the Red Cell Membranes
Since 3-0-methylfluorescein phosphate is a newly found substrate for K+-phosphatase, it was of interest to determine whether ouabain affects the hydrolysis of this substrate in the same manner that it affects the hydrolysis of the previously used substrates of the enzyme. In Fig. 1 the continuous recording assay of the enzyme is shown. Two assays were carried out in separate cuvettes. The results are superimposed for easier comparison. Since the red cell enzyme is a crude preparation, there is considerable phosphatase activity in the presence of Mg2+ alone (A). Addition of K+ produces an increase in the activity (B). When ouabain is added after the addition of K+, no inhibition of activity within the period of observation is obtained (C). However, the addition of ouabain 10 min prior to the addition of KC (D) produces almost complete inhibition of the K+-dependent activity (E). One may wonder why an activating effect of ouabain on phosphatase activity in the absence of K+ is not observed in experi-
OF TRANSPORT
ATPASE
187
ments of Fig. 1. We have previously reported (4, 24, 25) that such effects of ouabain on 3-0-methylfluorescein phosphatase and umbelliferone phosphatase are obtained when enzyme preparations from sources other than red cells are used. It may be that for unknown reasons the red cell enzyme behaves differently. However, a more likely explanation is that in this crude preparation the small activating effect of ouabain is masked by a large background of contaminating Mgz+-dependent phosphatase activities. In the experiments of Fig. 2 three identical reaction mixtures were set up, and the Mg2+-dependent rate was recorded for each (A). Ouabain was added to all three simultaneously. K+ was added to one immediately atier the addition of ouabain (B), and to the others (C and D) 5 and 10 min after the addition of ouabain. As the results indicate, the degree of inhibition by ouabain is dependent on the duration of incubation of the enzyme with ouabain prior to the addition of K+. The combined data of Figs. 1 and 2 clearly show that if a one-point assay of K+-MFPase activity is done, depending on the order of addition of the reagents, and the choice of reaction time, one could draw drastically different conclusions about the sensitivity of this reaction to ouabain. It should be noted that the general characteristics of the above data are not at all
0.6
0.6
7 as B = .E $ & $ A e2
0.5. 0.4.
0.3 0.2
h 0.1
Minutes
1. Effects of K+ and ouabain on the continuous recording assay of 3XLmethylfluorescein phosphatase activity of the human red cell membranes. Experimental conditions are described in the text. FIG.
FIG. 2. Dependence of the inhibitory effect of 0.4 rniu ouabain on the duration of exposure of enzyme to ouabain. K+ concentration was 10 mru. Other conditions as described in the text.
188
HUANG
AND ASKARI
..
inconsistent with what is known about the factors which affect the interaction of cardiac glycosides with this enzyme system. The time-dependency of cardiac glycoside binding to the enzyme, and the inhibitory effects of K+ on the rate of binding are well-established (27). Comparison of Kf-MFPase and Na+,KATPase Activities of Enzyme Preparations Containing Bound Ouabain
Previous studies (31, 32) have established that when ouabain is added to the enzyme under the conditions of Na+,KfATPase assay, the drug-induced inhibition of Na+,K+-ATPase activity is time-dependent and that the time-course of inhibition is affected by the concentrations of the various ligands in the assay medium. These observations, the data we have presented above, and a variety of other reports (8, 18, 21, 2.5) all provide support for the suggestion that the previously reported discrepancies between the sensitivities of K+-phosphatase and Nat ,K+-ATPase activities to cardiac glycosides that are added to the assay media are due to the different kinetics of interaction of the drug with the enzyme under the different conditions of the two assays. Nevertheless, the possibility had to be considered that at any steady-state level of ouabain-enzyme complex the K+-phosphatase activity of the enzyme may be less inhibited than the Na+,K+-ATPase activity. To test this alternative, the following experiments were done. Red cell membranes were incubated with ouabain for different lengths of time to obtain different levels of ouabain binding to the enzyme. The preparations were then washed in cold and assayed for K+MFPase and Na+,K+-ATPase under standard conditions. In addition, intact red cells were incubated with ouabain for various lengths of time, and thoroughly washed in cold. Membranes were then prepared from the cells and assayed .as above. In each experiment the activities of the ouabaintreated enzyme were compared with those of a control preparation which had been similarly incubated and washed, but in the absence of ouabain. Figure 3 shows a good correlation between the extent of in-
OY
Per cent inhibition
of K+-MFPare
3. Relationship between inhibition of Na+,K+-ATPase and inhibition of K+-MFPase in enzyme samples containing different amounts of bound ouabain. The samples were obtained either by the exposure of isolated membranes to ouabain (01 or by the exposure of intact cells to ouabain and subsequent preparation of membranes (Al. Details of experimental conditions are given in the text. FIG.
hibitions of the two activities in the various preparations. The above data clearly establish that once a certain degree of binding of ouabain to an enzyme preparation has been achieved, Nat ,K+-ATPase and K+-MFPase activities are equally inhibited. Therefore, it is concluded that the determination of the extent of inhibition of K+-MFPase in a tissue that is exposed to a cardiac glycoside can be taken as the extent of inhibition of the transport enzyme and any cellular function that may be correlated with the activity of this enzyme. Of course, this conclusion is strictly applicable to the case of the human red cell enzyme. Similar studies with appropriate preparations should be done if the K+-MFPase assay is to be used for the study of the interaction of cardiac glycosides with the transport enzymes of other tissues. However, it is reasonable to expect the same type of results with the enzymes of all tissues which, like the human red cell enzyme, are highly sensitive to cardiac glycosides (27). It should also be emphasized that the correlation between the extent of inhibitions of Na+,K+-ATPase and K+-phosphatase activities of a ouabain-treated enzyme may only be observed when the K+-phos-
OUABAIN
SENSITIVITY
phatase is assayed under the conditions described here. No data are available to indicate that a similar correlation is obtainable if other substrates, or different conditions, are used for the assay of K+phosphatase. REFERENCES 1. JUDAH, J. D., AHMED, K., AND MCLEAN, A. E. M. (1962) Biochim. Biophys. Acta 66,472-480. 2. IZUMI, F., NAGAI, K., AND YOSHIDA, H. (1966)J.
B&hem. Z’okya 60,533-537. 3. Pwrs, B. J. R., AND ASKARI, A. (1971) B&him. Biophys. Acta 227, 453-459. 4. HUANG, W., AND ASKARI, A. (1975) Anal. Biothem. 66, 265-271. 5. UESUGI, S., DULAK, N. C., DIXON, J. F., HEXUM, T. D., DAHL, J. 0. L., PERDUE, J. F., AND HOKIN, L. E. (1971) J. Biol. Chem. 246, 531543. 6. JORGENSEN, P. L., SKOU, J. C., AND SOLOMONSON, L. P. (1971) B&him. Biophys. Acta 233, 381-394. 7. Pms, B. J. R., LAN-E, L. K., AND SCHWARTZ, A. (1973) Biochim. Biophys. Res. Commun. 53, 1060-1066. 8. ISRAEL, Y., AND TITUS, E. (1967) B&him. Biophys. Acta 139, 450-459. 9. ALBERS, R. W., AND KOVAL, G. J. (1966)J. Biol. Chem. 241, 1896-1898. 10. KYTE, J. (1971) J. Biol. Chem. 246,4157-4165. 11. HOKIN, L. E., DAHL, J. L., DEUPREE, J. D., DIXON, J. F., HACKNEY, J. F., AND PERDUE, J. F. (1973) J. Biol. Chem. 248, 2593-2605. 12. LANE, L. K., COPENHAVER, J. H., JR., LINDENMAYER, G. E., AND SCHWARTZ, A. (1973) J. BioZ. Chem. 248, 7197-7200. 13. BESCH, H. R., JR., ALLEN, J. C., GLICK, G., AND SCHWARTZ, A. (1970) J. Pharmacol. Exp. Z’her. 171, 1-12.
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TRANSPORT
ATPASE
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14. Akera, T., LARSEN, F. S., AND BRODY, T. M. (1970) J. Pharmacol. Rxp. Ther. 173, 145-151. 15. NECHAY, B. R. (1974)Ann. N. Y. Acad. Sci. 242, 501-518. 16. FUJITA, M., NA~AO, T., TASHIMA, Y., MIZUNO, N., NAGANO, K., AND NAKAO, M. (1966) Biochim. Biophys. Acta 117, 42-53. 17. BADER, H., AND SEN, A. K. (1966) B&him. Biophys. Acta 118, 116-123. 18. YOSHIDA, H., NAGAI, K., OHASHI, T., AND NAKAGAWA, Y. (1969) B&him. Biophys. Acta 171, 178-185. 19. FREY, M., Prprs, B. J. R., AND ASKARI, A. (1973) B&hem. Pharmacol. 22, 9-15. 20. GARRAHAN, P. J., POUCHAN, M. I., AND REGA, A. F. (1969) J. Physiol. 202, 305-327. 21. GARRAHAN, P. J., POUCHAN, M. I., AND REGA, A. F. (1970) J. Mem. BioZ. 3, 26-42. 22. FISKE, C. H., AND SUBBAROW, Y. (1925) J. Biol.
Chem. 66, 375-400. 23. HUANG, W., AND ASKARI, A. (1975) Life Sci. 16, 1253-1262. 24. PITTS, B. J. R., AND ASKARI, A. (1971) Biochim. Biophys. Acta 225, 388-391. 25. PITTS, B. J. R., AND ASKARI, A. (1973) Arch. B&hem. Biophys. 154, 476-482. 26. PITTS, B. J. R. (1974)Ann. N. Y. Acad. Sci. 242,
293-304. 27. SCHWARZ, A., LINDENMAYER, G. E., AND ALLEN, J. C. (1975) Pharmacol. Rev. 27, 3-134. 28. MATSUI, H., AND SCHWARTZ, A. (1968) Biochim. Biophys. Acta 151, 655-663. 29. AKERA, T., BRODY, T. M., So, R. H., TOBIN, T., AND BASKIN, S. I. (1974)Ann. N. Y. Acad. Sci. 242, 617-634. 30. SIEGEL, G. J., AND JOSEPHSON, L. (1972) Eur. J.
Biochem. 25, 323-335. 31. ALLEN, J. C., AND SCHWARTZ, A. (1970) J. Mol. Cell. Cardiol. 1, 39-45. 32. ALBERS, R. W., KOVAL, G. J., AND SIEGEL, G. J. (1968) Mol. Pharmacol. 4, 324-336.
OF
BIOCHEMISTRY
Transport
AND
BIOPHYSICS
175, 185-189 (1976)
ATPase of Erythrocyte Membrane: Sensitivities of Na+,K+ATPase and K+-Phosphatase Activities to Ouabain’ W. HUANG
Department
of Pharmacology
and
AND A. ASKARI
Therapeutics,
Medical
College
of Ohio,
Toledo,
Ohio
43614
Received January 19, 1976 Cardiac glycosides are inhibitors of Na+,K+-ATPase, and K+-phosphatase activities of the transport enzyme. Previous studies have shown that when the sensitivities of these two activities to ouabain are compared by the addition of varying concentrations of the drug to the assay media, the K+-phosphatase is significantly less sensitive than Na+,K+ATPase. This work was done to seek an explanation for this phenomenon. 3-O-Methylfluorescein phosphate was used as substrate for the continuous fluorimetric assay of K+phosphatase obtained from human red cells. When ouabain was added to the assay medium, a time-dependent inhibition of K+-phosphatase was observed. The rate of inhibition was also influenced by the order of additions of K+ and ouabain. In view of these results, several enzyme samples exposed to ouabain for varying lengths of time were prepared, and their Na+,K+-ATPase and K+-phosphatase activities were then determined. A good correlation between the extent of inhibition of the two activities was obtained. These results prove that the previously observed discrepancies between the sensitivities of Na+,K+-ATPase and K+-phosphatase to ouabain are due to the different kinetics of drug interaction with the enzyme under the different conditions of the two assays and that once a certain level of ouabain binding to the enzyme is achieved, both activities are equally inhibited. Preparations of (Na+ adenosinetriphosphatase
+
K+)-activated
(Na+,K+-ATPase, ATP phosphohydrolase, EC 3.6.1.3) from various tissues also catalyze the K+activated hydrolysis of p-nitrophenylphosphate (l), acetylphosphate (2), carbamylphosphate (21, umbelliferone phosphate (3), and 3-O-methylfluorescein phosphate (4). Several laboratories have reported that this K+-phosphatase activity remains associated with highly purified enzyme preparations (5-7). Thus, in spite of some early doubts (8, 9), it is apparent now that the K+-phosphatase activity is a partial reaction catalyzed by the purified enzyme which contains two major polypeptides and phospholipids (10-12). Recent work in our laboratory (4) demonstrated that when the fluorigenic sub-
’ This work was supported by NIH Research Grant HL-19129 awarded by the National Heart and Lung Institute, PHS-DHEW.
strate 3-O-methylfluorescein phosphate is used for the assay of K+-phosphatase, and when the Na+,K+-ATPase and K+-phosphatase activities of several partially purified enzyme preparations and crude tissue fractions are compared, a good correlation between the two activities of the different preparations is obtained. These findings suggested that in situations where the assay of Na+,K+-ATPase activity is difficult or impossible to perform (either because of the low specific activity of the enzyme or due to limitations of sample size), the highly sensitive fluorimetric assay of K+MFPase2 may be used to determine the level of this enzyme in various samples. A potential value of the K+-MFPase assay may be envisioned in those experiments where a tissue is exposed to a cardiac glycoside, and the possible relationship be* Abbreviation used: K+-MFPase, 3-O-methylfluorescein phosphatase. 185
Copyright 8 All rights of
1976 by reproduction
Academic Press, Inc. in any
form
reserved.
K+-dependent
186
HUANG
AND ASKARI
tween the induced pharmacologic effect and the inhibition of the membrane-bound enzyme is sought (e.g., 13-15). The question arises, however, whether the Na+,K+ATPase and K+-phosphatase activities of the enzyme are similarly affected by cardiac glycosides. Judah et al. (1) were the first to demonstrate that the K+-phosphatase (assayed with p-nitrophenylphosphate as the substrate), like the Na+,KTATPase, is inhibited by cardiac glycosides. However, in several subsequent studies when the effects of varying concentrations of a cardiac glycoside on K+-phosphatase and Na+,K+-ATPase activities were compared, it was found that the K+-phosphatase activity is significantly less sensitive than the Na+,K+-ATPase to the inhibitory effect of the drug (8,16-21). Since the addition of Na+ and ATP to the assay mixture for K+-phosphatase activity increases the sensitivity of this activity to ouabain (8, 18, 21, 25), and since Na+ and ATP increase the rate of ouabain binding to the enzyme (27), it has been suggested (25,27) that the above observations on the different drug sensitivities of K+-phosphatase and Na+,K+-ATPase may be due to the different rates of ouabain binding to the enzyme under the conditions of the two assays. This hypothesis, however, has not been rigorously proved; the possibility that after ouabain binding to the enzyme the two activities may be inhibited to different extents has not been ruled out. The experiments presented here were done to clarify these questions. MATERIALS
AND METHODS 3-O-methylfluorescein 3-@Methylfluorescein, phosphate, ATP, and ouabain were obtained from Sigma Chemical Co. (St. Louis, MO.). Fluorimetric determintion of 3-0-methylfluorescein phosphatase activity was done by the continuous recording procedure as described before (3, 4). K+-MFPase activity of each enzyme sample was determined at 37°C under the following standard conditions. The enzyme suspension (lo-50 ~1) was added to 2.5 ml of a solution containing 0.8 PM substrate, 4 mM MgCl,, 1 mM EDTA, 80 rnrd Tris-HCl (pH 7.6). Progress of the reaction was recorded for 5 min. A concentrated KC1 solution (10 ~1) was then added to obtain a final concentration of 10 mM KC1 in the mixture, and the recording was continued for another 5 min. The KC-
dependent activity was calculated from the difference between the slopes of the two resulting straight lines. For certain experiments deviations from the above standard conditions are noted in the text. The Na+,K+-ATPase activity of the enzyme was assayed by the measurement of released inorganic phosphate (22) after incubation at 37°C in a medium containing 2 rnru ATP, 3 mM MgCl,, 1 mM EDTA, 100 mM NaCl, 25 mM KCl, and 80 mM Tris-HCl (pH 7.61, in a total volume of 2.5 ml. To determine the Na+,K+-independent portion of the activity, KC1 and NaCl were omitted from the reaction mixture. For these assays, reaction time was 20 min or less, and the amount of substrate used was no more than 20% of the initial amount. It should be noted that under the standard conditions of both assays, Na+,K+-ATPase and K+-MFPase, the amount of product formed was a linear function of time for all control and partially inhibited enzyme samples. Thus, enzyme preparations containing bound ouabain behaved as irreversibly inhibited enzymes in the course of both assays. Hemoglobin-free membranes from human red cells were prepared as described before (23). Membrane preparations with partially inhibited enzyme activities were obtained either by the exposure of hemoglobin-free membranes to ouabain or by the exposure of intact red cells to the drug prior to the preparation of membranes. Isolated membranes were added to 10 vol of a solution containing 10e6 M ouabain, 1 mM EDTA, 2 mM MgCl,, 2 mM TrisH,PO,, and 50 mM Tris-HCl (pH 7.4). After incubation at 37°C for 5-20 min, membranes were sedimented by centrifugation in cold at 15,OOOg for 15 min. They were then washed three times in 50 vol of a cold solution of 1 mM Tris-EDTA (pH 7.4). Exposure of intact cells to ouabain was achieved by suspending whole blood in 10 vol of isotonic saline containing lo-’ M ouabain and incubating the mixture at 37°C for 5-30 min. After centrifugation the cells were washed twice in 15 vol of cold saline. Membranes were then prepared from these cells in the cold. RESULTS
AND DISCUSSION
The different sensitivities of K+-phosphatase and Na+,K+-ATPase activities to cardiac glycosides that are added to the assay media have been observed with enzyme preparations from a variety of tissues (8,16-19) including that of the human red cells (20, 21). We chose this particular enzyme for the present studies because of the extreme ease with which it can be prepared and handled. The notoriously low specific activity of this preparation was also of particular interest, because it
OUABAIN
SENSITIVITY
is, in fact, with enzyme samples of low activity obtained from drug-exposed tissues that one may wish to use the highly sensitive assay of K+-MFPase activity in preference to the assay of Na+,K+-ATPase activity. Effect of Ouabain on K+-MFPase Activity of the Red Cell Membranes
Since 3-0-methylfluorescein phosphate is a newly found substrate for K+-phosphatase, it was of interest to determine whether ouabain affects the hydrolysis of this substrate in the same manner that it affects the hydrolysis of the previously used substrates of the enzyme. In Fig. 1 the continuous recording assay of the enzyme is shown. Two assays were carried out in separate cuvettes. The results are superimposed for easier comparison. Since the red cell enzyme is a crude preparation, there is considerable phosphatase activity in the presence of Mg2+ alone (A). Addition of K+ produces an increase in the activity (B). When ouabain is added after the addition of K+, no inhibition of activity within the period of observation is obtained (C). However, the addition of ouabain 10 min prior to the addition of KC (D) produces almost complete inhibition of the K+-dependent activity (E). One may wonder why an activating effect of ouabain on phosphatase activity in the absence of K+ is not observed in experi-
OF TRANSPORT
ATPASE
187
ments of Fig. 1. We have previously reported (4, 24, 25) that such effects of ouabain on 3-0-methylfluorescein phosphatase and umbelliferone phosphatase are obtained when enzyme preparations from sources other than red cells are used. It may be that for unknown reasons the red cell enzyme behaves differently. However, a more likely explanation is that in this crude preparation the small activating effect of ouabain is masked by a large background of contaminating Mgz+-dependent phosphatase activities. In the experiments of Fig. 2 three identical reaction mixtures were set up, and the Mg2+-dependent rate was recorded for each (A). Ouabain was added to all three simultaneously. K+ was added to one immediately atier the addition of ouabain (B), and to the others (C and D) 5 and 10 min after the addition of ouabain. As the results indicate, the degree of inhibition by ouabain is dependent on the duration of incubation of the enzyme with ouabain prior to the addition of K+. The combined data of Figs. 1 and 2 clearly show that if a one-point assay of K+-MFPase activity is done, depending on the order of addition of the reagents, and the choice of reaction time, one could draw drastically different conclusions about the sensitivity of this reaction to ouabain. It should be noted that the general characteristics of the above data are not at all
0.6
0.6
7 as B = .E $ & $ A e2
0.5. 0.4.
0.3 0.2
h 0.1
Minutes
1. Effects of K+ and ouabain on the continuous recording assay of 3XLmethylfluorescein phosphatase activity of the human red cell membranes. Experimental conditions are described in the text. FIG.
FIG. 2. Dependence of the inhibitory effect of 0.4 rniu ouabain on the duration of exposure of enzyme to ouabain. K+ concentration was 10 mru. Other conditions as described in the text.
188
HUANG
AND ASKARI
..
inconsistent with what is known about the factors which affect the interaction of cardiac glycosides with this enzyme system. The time-dependency of cardiac glycoside binding to the enzyme, and the inhibitory effects of K+ on the rate of binding are well-established (27). Comparison of Kf-MFPase and Na+,KATPase Activities of Enzyme Preparations Containing Bound Ouabain
Previous studies (31, 32) have established that when ouabain is added to the enzyme under the conditions of Na+,KfATPase assay, the drug-induced inhibition of Na+,K+-ATPase activity is time-dependent and that the time-course of inhibition is affected by the concentrations of the various ligands in the assay medium. These observations, the data we have presented above, and a variety of other reports (8, 18, 21, 2.5) all provide support for the suggestion that the previously reported discrepancies between the sensitivities of K+-phosphatase and Nat ,K+-ATPase activities to cardiac glycosides that are added to the assay media are due to the different kinetics of interaction of the drug with the enzyme under the different conditions of the two assays. Nevertheless, the possibility had to be considered that at any steady-state level of ouabain-enzyme complex the K+-phosphatase activity of the enzyme may be less inhibited than the Na+,K+-ATPase activity. To test this alternative, the following experiments were done. Red cell membranes were incubated with ouabain for different lengths of time to obtain different levels of ouabain binding to the enzyme. The preparations were then washed in cold and assayed for K+MFPase and Na+,K+-ATPase under standard conditions. In addition, intact red cells were incubated with ouabain for various lengths of time, and thoroughly washed in cold. Membranes were then prepared from the cells and assayed .as above. In each experiment the activities of the ouabaintreated enzyme were compared with those of a control preparation which had been similarly incubated and washed, but in the absence of ouabain. Figure 3 shows a good correlation between the extent of in-
OY
Per cent inhibition
of K+-MFPare
3. Relationship between inhibition of Na+,K+-ATPase and inhibition of K+-MFPase in enzyme samples containing different amounts of bound ouabain. The samples were obtained either by the exposure of isolated membranes to ouabain (01 or by the exposure of intact cells to ouabain and subsequent preparation of membranes (Al. Details of experimental conditions are given in the text. FIG.
hibitions of the two activities in the various preparations. The above data clearly establish that once a certain degree of binding of ouabain to an enzyme preparation has been achieved, Nat ,K+-ATPase and K+-MFPase activities are equally inhibited. Therefore, it is concluded that the determination of the extent of inhibition of K+-MFPase in a tissue that is exposed to a cardiac glycoside can be taken as the extent of inhibition of the transport enzyme and any cellular function that may be correlated with the activity of this enzyme. Of course, this conclusion is strictly applicable to the case of the human red cell enzyme. Similar studies with appropriate preparations should be done if the K+-MFPase assay is to be used for the study of the interaction of cardiac glycosides with the transport enzymes of other tissues. However, it is reasonable to expect the same type of results with the enzymes of all tissues which, like the human red cell enzyme, are highly sensitive to cardiac glycosides (27). It should also be emphasized that the correlation between the extent of inhibitions of Na+,K+-ATPase and K+-phosphatase activities of a ouabain-treated enzyme may only be observed when the K+-phos-
OUABAIN
SENSITIVITY
phatase is assayed under the conditions described here. No data are available to indicate that a similar correlation is obtainable if other substrates, or different conditions, are used for the assay of K+phosphatase. REFERENCES 1. JUDAH, J. D., AHMED, K., AND MCLEAN, A. E. M. (1962) Biochim. Biophys. Acta 66,472-480. 2. IZUMI, F., NAGAI, K., AND YOSHIDA, H. (1966)J.
B&hem. Z’okya 60,533-537. 3. Pwrs, B. J. R., AND ASKARI, A. (1971) B&him. Biophys. Acta 227, 453-459. 4. HUANG, W., AND ASKARI, A. (1975) Anal. Biothem. 66, 265-271. 5. UESUGI, S., DULAK, N. C., DIXON, J. F., HEXUM, T. D., DAHL, J. 0. L., PERDUE, J. F., AND HOKIN, L. E. (1971) J. Biol. Chem. 246, 531543. 6. JORGENSEN, P. L., SKOU, J. C., AND SOLOMONSON, L. P. (1971) B&him. Biophys. Acta 233, 381-394. 7. Pms, B. J. R., LAN-E, L. K., AND SCHWARTZ, A. (1973) Biochim. Biophys. Res. Commun. 53, 1060-1066. 8. ISRAEL, Y., AND TITUS, E. (1967) B&him. Biophys. Acta 139, 450-459. 9. ALBERS, R. W., AND KOVAL, G. J. (1966)J. Biol. Chem. 241, 1896-1898. 10. KYTE, J. (1971) J. Biol. Chem. 246,4157-4165. 11. HOKIN, L. E., DAHL, J. L., DEUPREE, J. D., DIXON, J. F., HACKNEY, J. F., AND PERDUE, J. F. (1973) J. Biol. Chem. 248, 2593-2605. 12. LANE, L. K., COPENHAVER, J. H., JR., LINDENMAYER, G. E., AND SCHWARTZ, A. (1973) J. BioZ. Chem. 248, 7197-7200. 13. BESCH, H. R., JR., ALLEN, J. C., GLICK, G., AND SCHWARTZ, A. (1970) J. Pharmacol. Exp. Z’her. 171, 1-12.
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