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Rat brain (Na+-K+)atpase: Modulation of its ouabain-sensitive K+ -pnppase activity by thimerosal
0020-71 lX:83~010005-03$03,00/O Copyright 0 1983 Pergamon Press Ltd
I,lt. J. Biociwtu. Vol. 15, pp. 5 to 7, 1983 Printed in Great Britain. All rights reserved
RAT BRAIN (Na+-K+)ATPase: MODULATION OF ITS OUABAIN-SENSITIVE K+-PNPPase ACTIVITY BY THIMEROSAL R. N. A. H. LEWIS* and K. BOWLER Department of Zoology, University of Durham. Durham DHl 3LE, U.K. (Rrceiced
3 Nouemhrr
1981)
Abstract-l. The (Na’ + K+) ATPase activity of a rat brain synaptic membrane preparation was inhibited by 1O-5 M thimerosal. 2. The ouabain inhibitable K*-PNPPase activity of thimerosal treated membranes was compared with that of untreated membranes with respect to sensitivity to temperature, ouabain, K+ and ATP. 3. All those kinetic characteristics were substantially altered by treatment with thimetosai.
INTRODUCTION
was determined by procedures adapted from McGrath (1972). Membranes were treated with thimerosal as reported by Henderson & Askari (1977). Membranes were rapidly added to a solution containing IO-’ M thimerosal and 50mM Tris (pH 7.4 at 37”C), to give a membrane protein concentration of 300-600 pg cmm3. At 10 min incubation at 37”C, the mixture was diluted lo-fold with an ice cold low ionic strength buffer (10mM imidazole, 1 mM EDTA pH 7.2) and the membranes were sedimented by centrifugation at 1~~~~ far 30min. The sedimented membranes were then washed by three cycles of homogenisation in low ionic strength buffer and centrifugation in ice cold low ionic strength buffer. The post treatment of the thimerosal treated membranes with dithiothreitol was the same as that described by Henderson & Askari (1976).
The use of specific protein targeted modifiers as probes in mechanistic studies of enzyme systems is widespread, and many such reagents have been used to study the (Na+-K+)ATPase (ATP phosphohydrolase, EC 3.6.1.3), Schwartz et al. (1975). Henderson & Askari (1976, 1977) have suggested that thimerosal (ethylmercurithiosalicylat~) may be particularly useful in such work since they reported, under controlled conditions, a mild treatment with thimerosal can produce a sample devoid of (Na+-K+) ATPase activity but with the “partial” reactions of that enzyme apparently unaffected. This present paper reports some studies on one of the “partial” reactions of the (Na+-K+)ATPase in a thimerosal treated preparation of rat brain synaptic membranes. The kinetic properties studied were those relevant to the overall reaction mechanism of the (Na’-K’)ATPase with the intention of evaluating the effect of thimerosal on the enzyme and its potential usefulness in further mechanistic studies.
REWLTS The (Nat-K+)ATPase and K+-activated p-nitrophenyl phosphatase of rat brain synaptic membrane preparations were both affected by thimerosaf treatment, Table 1. The (Na+-K’)ATPase was compietely inhibited by 1O-5 M thimerosal whilst 2S’/, of the K+-PNPPase remained. Both activities were fully re-
MATERIALS AND METHODS Table 1. The effect of thimerosal
treatment on the (Na+K+)ATPase and K+-PNPPase activity of a’synaptic membrane preparation obtained from rat brain
Synaptic membranes were prepared from rat brain according to the methods of Lowe & Smart (1977). (Na+-Kf) ATPase activity was assayed as the ouabain sensitive release of inorganic phosphate under reaction conditions described in Jegensen (1974). Ouabain sensitive K+-PNPPase (K+-activated p-nitrophenyl phosphatase) was assayed by the ouabain inhibitable release of p-nitrophenol from a medium containing 20mM KCi, 5 mM MgCi2. 5 mM p-nitrophenyl phosphate and 50 mM Tris (pH 7.5 at assay temperature). In assays at low ouabain concentrations (< 10e3 M) a I2 min preincubation period of the membranes with ouabain in an appropriate K’-free assay medium was used to avoid the problems of slow ouabain binding and the effect of K’ on the rate of equilibration with ouabain (Akerd, 1971; Wallick & Schwartz, 1974). In such assays. the reaction was started by the addition of a solution containing KC1 and the appropriate ouabain concentration. Inorganic phosphate was determined as described by Atkinson et cd. (1973) and protein
Enzyme (Na+-K+)ATPase
activity K+-PNPPase
Untreated control membranes
87.5 & 9.3
IS.7 t 2.3
Thimerosal treated membranes
0.4 + 0.3
3.8 + 0.9
Treated membranes were preincubated with 1iY5 M thimerosal in the presence of 50 mM Tris (pH 7.4) for 10 min at 37-C. This suspension was diluted IO-fold with ice-cold 10mM imidazole buffer pH 7.2 containing I mM EDTA. After sedimentation and washing, the membranes were used to assay for enzyme activity. This was determined as the ouabain inhibitable fraction of the totai ATPase or K*-PNPPase activity. Enzyme activity in units of pmol of product mg. protein-’ hr-’ and are shown as the mean of four preparations + 1 SD.
* Present address: Department of Biochemistry, University of Alberta. Edmonton, Alberta, Canada T69 2H7. 5
6
R. N. A. H. LEWIS and K. BOWLER
I 0
I
I
3
6
I
I
12
c 15
[K’] rnc Fig. 1. The effect of pmolar concentration of ATP on K+ activation of K+-PNPPase from rat brain synaptic membranes with or without pretreated 10m5 M thimerosal. -+-Thimerosal treated membranes + 3 FM ATP, -Wthimerosal treated membranes, ---A-untreated control membranes + 3 PM ATP, --Vuntreated control membranes. Means of 4 control and treated preparation +l SD.
stored by post-treatment of the preparations with dithiothreitol. The K+-PNPPase activity of treated and
untreated membranes were characterised with respect to their sensitivities to temperature, ouabain and K+. The K+ sensitivity of the two preparations both described complex curves between 0.115 and 15 mM K+. Both curves approached saturation near 15 mM KC (see Fig. 1). The thimerosal treated preparation was significantly more sensitive to K+ as it tended towards saturation at lower K+ concentrations. The untreated preparation gave an apparently sigmoid shaped curve, however it could not be fitted to a Hilltype plot. Figure 2 shows the response of the two preparations to ouabain differed both quantitatively and
qualitatively. The native, untreated, preparation described a biphasic response with K+-PNPPase activity decaying through SO:/: k’,,,, near lo- M ouabain. In contrast the activity of the thimerosal treated membranes was considerably less ouabain sensitive and described a monophasic response which decayed through 50% V,,, at about 10m4 M ouabain. Figure 3 shows the temperature dependence of the K’-PNPPase of thimerosal treated and untreated membrane preparations in the form of Arrhenius plots. The untreated preparations gave a non-linear Arrhenius plot which fitted a smooth curve. The apparent activation energies at the upper (approx. 35’C) and lower (approx. 5’C) temperatures were 55 kJmol_ 1 and 200 kJmol_ ’ respectively. In marked contrast the behaviour of the treated sample could be best described by a linear function with an apparent activation energy of 80 kJmol- ‘. As has been suggested by Henderson & Askari (1977) the effect of thimerosal on the (Na+-K+)ATPase may be the result of a blockading of an essential sulphydryl group at a low affinity. non-hydrolytic, ATP binding site, the above experiments were carried out in the presence of 3 pm ATP. Previous work by Pitts (1974) has shown this concentration of ATP is sufficient to saturate the low affinity site without significant binding of the nucleotide to the hydrolytic site of the enzyme. These experiments show the inclusion of 3 /LM ATP in the assay media had no effect on the maximal activities nor temperature sensitivities of the enzyme of thimerosal treated or untreated preparations. As can be seen from Figs 1 and 2 in the thimerosal treated preparations 3 /tM ATP had no effect on ouabain nor potassium sensitivities of the enzyme. However in the untreated membrane preparations 3 PM ATP increased the sensitivity to K’. The effects however were only qualitative for the shapes of the curves obtained in the presence and absence of ATP were similar.
I
0 -10
I -8
I -6
I\* ,1, -4
3.2
-2
Log,, Duabain] Fig. 2. The effect of pmolar concentrations of ATP on ouabain inhibition of K+-PNPPase from rat brain synaptic membranes with or without pretreated IO-” M thimerosal. -t Thimerosal treated membranes +3 /tM ATP, -mthimerosal treated membranes. -Auntreated control membranes + 3 PM ATP. -V-untreated control membranes. Means of 4 control and treated preparations *SD.
1 3.29
I 3 38
1 3.47
I 3 56
I. 3.65
I/Temp.K-‘x103 Fig. 3. The effect of pmolar concentrations of ATP on the temperature kinetics of K’-PNPPase from rat brain synaptic membranes with or without pretreated IO ’ M thimerosal. a_Thimerosal treated membranes + 3 ,IM ATP, thimerosal treated membranes. -W-Untreated control membranes + 3 /tM ATP. untreated control membranes. Mean of 4 control and treated prepnraparations the enzyme activity was normalised to IOO”,, at 37 C and activities at other iemperatures are expressed :ts :I percentage of activit! at 37 c‘.
Effect of thimerosal
on Na’-K’
DISCUSSION
of the role
of the low affinity
and K’-PNPPase
ATP
bindine
7
activity
site in the overall reaction K+ )ATPase.
The observations reported here emphasize the high sensitivity of the (Na+-K+)ATPase to thimerosal. The rat brain synaptic membrane preparation used was considerably more sensitive than that reported for a guinea-pig microsomal enzyme by Henderson & Askari (1976). The inhibition of ATP hydrolysis was observed at a lower concentration. The lowest thimerosal concentration (lo-’ M) required to cause complete (Na+-K+)ATPase inhibition also reduced the activity of the K+-PNPPase “partial” reaction. That this residual K+-PNPPase activity is substantially altered with respect to its kinetic response to ouabain, K+ and temperature is of significance. In particular the change in shape of the Arrhenius plot from a smooth curve to a linear plot is of interest. The nonlinear Arrhenius plots usually obtained for (Na+K+)ATPase are usually attributed to a phase change in the lipid matrix of the membrane, Grisham & Barnett (1973), however the data shown in Fig. 3 suggest that protein mediated events cannot be excluded as having a role in the temperature kinetics of this enzyme. It is significant that the observed effects of pretreatment with thimerosal can be reversed completely with post incubation with dithiothreitol. This implicates the involvement of a sulphydryl group at a site remote from the phosphorylation sites on the enzyme. The failure to modify the kinetic properties of the K+-PNPPase of the treated preparation using micromolar concentrations of ATP is consistent with the suggestion that a sulphydryl group is directly involved at the lower affinity regulatory ATP binding site of the enzyme, Henderson & Askari (1977). Thus thimerosal could prove to be a useful tool in the evaluation
ATPase
mechanism
of the (NaF-
REFERENCES
AKERA T. (1971) Quantitative aspects of the interaction between ouabain and the Na+K+ ATPase. Biochim. biophp. Acta 245, 55-62. ATKINSON A., GATENBY A. D. & LOVE A. G. (1973) The determination of inorganic phosphate in biological SYSterns. Biochim. biophys. Acta 320, 195-204. GRISHAM C. M. & BARNETT R. E. (1973) Role of lipid-phase transitions in the regulation of the (sodium-potassium) adenosine triphosphatase. Biochemistry 12, 2635-2637. HENDERSON G. R. & ASKARI A. (1976) Transport ATPase: Thimerosal inhibits the Na+K+ dependent ATPase activitv without diminishing the Na’-dependent ATPase actiiity. Biochrru. hiophy.5: Rrs. Co~~~~m~.69, 499-505. HENIIERSON G. R. & ASRARI A. (1977) Transport ATPase: Further studies on the properties of the thimerosal treated enzyme. Archs B&he& Eioph~s. 182, 221-226. JORGENSEN P. L. (1974) Isolation of the (Na+-K+)ATPase. In Mvrhods it1 Enz~nolo(l~ XXXII. (Edited by COLOWIC~~ S. P. & KAPLAN N. 0.) .oo. . 277-290. Academic Press. New York. LOWE A. G. & SMART J. W. (1977) The presteadv state hydrolysis of ATP by porcine brain (Ng+-K+) hependent ATPase. Biochim. hiophys. Acta 481, 695-705. MCGRATH R. (1972) Protein measurement by ninhydrin determination of amino acids released by alkaline hydrolysis. Analyt. Biochem. 49, 95-102. PITTS B. J. R. (1974) The relationship of the K’-activated phosphatase to the Na+. K’ ATPase. Ann. N.Y. Acud. Sci. 242, 293-304. SCHWARTZ A., LINDENMAYER G. E. & ALLEN J. C. (1975) The sodium potassium adenosine triphosphatase: Pharmacological, Physiological and Biochemical Aspects. Phurmuc. Rec. 27, 3-134. WALLICK E. T. & SCHWARTZ A. (1974) Thermodynamics of the rate of binding of ouabain to the sodium, potassium adenosine triphosphate. J. hid. Chern. 249, 5141-5147.
I,lt. J. Biociwtu. Vol. 15, pp. 5 to 7, 1983 Printed in Great Britain. All rights reserved
RAT BRAIN (Na+-K+)ATPase: MODULATION OF ITS OUABAIN-SENSITIVE K+-PNPPase ACTIVITY BY THIMEROSAL R. N. A. H. LEWIS* and K. BOWLER Department of Zoology, University of Durham. Durham DHl 3LE, U.K. (Rrceiced
3 Nouemhrr
1981)
Abstract-l. The (Na’ + K+) ATPase activity of a rat brain synaptic membrane preparation was inhibited by 1O-5 M thimerosal. 2. The ouabain inhibitable K*-PNPPase activity of thimerosal treated membranes was compared with that of untreated membranes with respect to sensitivity to temperature, ouabain, K+ and ATP. 3. All those kinetic characteristics were substantially altered by treatment with thimetosai.
INTRODUCTION
was determined by procedures adapted from McGrath (1972). Membranes were treated with thimerosal as reported by Henderson & Askari (1977). Membranes were rapidly added to a solution containing IO-’ M thimerosal and 50mM Tris (pH 7.4 at 37”C), to give a membrane protein concentration of 300-600 pg cmm3. At 10 min incubation at 37”C, the mixture was diluted lo-fold with an ice cold low ionic strength buffer (10mM imidazole, 1 mM EDTA pH 7.2) and the membranes were sedimented by centrifugation at 1~~~~ far 30min. The sedimented membranes were then washed by three cycles of homogenisation in low ionic strength buffer and centrifugation in ice cold low ionic strength buffer. The post treatment of the thimerosal treated membranes with dithiothreitol was the same as that described by Henderson & Askari (1976).
The use of specific protein targeted modifiers as probes in mechanistic studies of enzyme systems is widespread, and many such reagents have been used to study the (Na+-K+)ATPase (ATP phosphohydrolase, EC 3.6.1.3), Schwartz et al. (1975). Henderson & Askari (1976, 1977) have suggested that thimerosal (ethylmercurithiosalicylat~) may be particularly useful in such work since they reported, under controlled conditions, a mild treatment with thimerosal can produce a sample devoid of (Na+-K+) ATPase activity but with the “partial” reactions of that enzyme apparently unaffected. This present paper reports some studies on one of the “partial” reactions of the (Na+-K+)ATPase in a thimerosal treated preparation of rat brain synaptic membranes. The kinetic properties studied were those relevant to the overall reaction mechanism of the (Na’-K’)ATPase with the intention of evaluating the effect of thimerosal on the enzyme and its potential usefulness in further mechanistic studies.
REWLTS The (Nat-K+)ATPase and K+-activated p-nitrophenyl phosphatase of rat brain synaptic membrane preparations were both affected by thimerosaf treatment, Table 1. The (Na+-K’)ATPase was compietely inhibited by 1O-5 M thimerosal whilst 2S’/, of the K+-PNPPase remained. Both activities were fully re-
MATERIALS AND METHODS Table 1. The effect of thimerosal
treatment on the (Na+K+)ATPase and K+-PNPPase activity of a’synaptic membrane preparation obtained from rat brain
Synaptic membranes were prepared from rat brain according to the methods of Lowe & Smart (1977). (Na+-Kf) ATPase activity was assayed as the ouabain sensitive release of inorganic phosphate under reaction conditions described in Jegensen (1974). Ouabain sensitive K+-PNPPase (K+-activated p-nitrophenyl phosphatase) was assayed by the ouabain inhibitable release of p-nitrophenol from a medium containing 20mM KCi, 5 mM MgCi2. 5 mM p-nitrophenyl phosphate and 50 mM Tris (pH 7.5 at assay temperature). In assays at low ouabain concentrations (< 10e3 M) a I2 min preincubation period of the membranes with ouabain in an appropriate K’-free assay medium was used to avoid the problems of slow ouabain binding and the effect of K’ on the rate of equilibration with ouabain (Akerd, 1971; Wallick & Schwartz, 1974). In such assays. the reaction was started by the addition of a solution containing KC1 and the appropriate ouabain concentration. Inorganic phosphate was determined as described by Atkinson et cd. (1973) and protein
Enzyme (Na+-K+)ATPase
activity K+-PNPPase
Untreated control membranes
87.5 & 9.3
IS.7 t 2.3
Thimerosal treated membranes
0.4 + 0.3
3.8 + 0.9
Treated membranes were preincubated with 1iY5 M thimerosal in the presence of 50 mM Tris (pH 7.4) for 10 min at 37-C. This suspension was diluted IO-fold with ice-cold 10mM imidazole buffer pH 7.2 containing I mM EDTA. After sedimentation and washing, the membranes were used to assay for enzyme activity. This was determined as the ouabain inhibitable fraction of the totai ATPase or K*-PNPPase activity. Enzyme activity in units of pmol of product mg. protein-’ hr-’ and are shown as the mean of four preparations + 1 SD.
* Present address: Department of Biochemistry, University of Alberta. Edmonton, Alberta, Canada T69 2H7. 5
6
R. N. A. H. LEWIS and K. BOWLER
I 0
I
I
3
6
I
I
12
c 15
[K’] rnc Fig. 1. The effect of pmolar concentration of ATP on K+ activation of K+-PNPPase from rat brain synaptic membranes with or without pretreated 10m5 M thimerosal. -+-Thimerosal treated membranes + 3 FM ATP, -Wthimerosal treated membranes, ---A-untreated control membranes + 3 PM ATP, --Vuntreated control membranes. Means of 4 control and treated preparation +l SD.
stored by post-treatment of the preparations with dithiothreitol. The K+-PNPPase activity of treated and
untreated membranes were characterised with respect to their sensitivities to temperature, ouabain and K+. The K+ sensitivity of the two preparations both described complex curves between 0.115 and 15 mM K+. Both curves approached saturation near 15 mM KC (see Fig. 1). The thimerosal treated preparation was significantly more sensitive to K+ as it tended towards saturation at lower K+ concentrations. The untreated preparation gave an apparently sigmoid shaped curve, however it could not be fitted to a Hilltype plot. Figure 2 shows the response of the two preparations to ouabain differed both quantitatively and
qualitatively. The native, untreated, preparation described a biphasic response with K+-PNPPase activity decaying through SO:/: k’,,,, near lo- M ouabain. In contrast the activity of the thimerosal treated membranes was considerably less ouabain sensitive and described a monophasic response which decayed through 50% V,,, at about 10m4 M ouabain. Figure 3 shows the temperature dependence of the K’-PNPPase of thimerosal treated and untreated membrane preparations in the form of Arrhenius plots. The untreated preparations gave a non-linear Arrhenius plot which fitted a smooth curve. The apparent activation energies at the upper (approx. 35’C) and lower (approx. 5’C) temperatures were 55 kJmol_ 1 and 200 kJmol_ ’ respectively. In marked contrast the behaviour of the treated sample could be best described by a linear function with an apparent activation energy of 80 kJmol- ‘. As has been suggested by Henderson & Askari (1977) the effect of thimerosal on the (Na+-K+)ATPase may be the result of a blockading of an essential sulphydryl group at a low affinity. non-hydrolytic, ATP binding site, the above experiments were carried out in the presence of 3 pm ATP. Previous work by Pitts (1974) has shown this concentration of ATP is sufficient to saturate the low affinity site without significant binding of the nucleotide to the hydrolytic site of the enzyme. These experiments show the inclusion of 3 /LM ATP in the assay media had no effect on the maximal activities nor temperature sensitivities of the enzyme of thimerosal treated or untreated preparations. As can be seen from Figs 1 and 2 in the thimerosal treated preparations 3 /tM ATP had no effect on ouabain nor potassium sensitivities of the enzyme. However in the untreated membrane preparations 3 PM ATP increased the sensitivity to K’. The effects however were only qualitative for the shapes of the curves obtained in the presence and absence of ATP were similar.
I
0 -10
I -8
I -6
I\* ,1, -4
3.2
-2
Log,, Duabain] Fig. 2. The effect of pmolar concentrations of ATP on ouabain inhibition of K+-PNPPase from rat brain synaptic membranes with or without pretreated IO-” M thimerosal. -t Thimerosal treated membranes +3 /tM ATP, -mthimerosal treated membranes. -Auntreated control membranes + 3 PM ATP. -V-untreated control membranes. Means of 4 control and treated preparations *SD.
1 3.29
I 3 38
1 3.47
I 3 56
I. 3.65
I/Temp.K-‘x103 Fig. 3. The effect of pmolar concentrations of ATP on the temperature kinetics of K’-PNPPase from rat brain synaptic membranes with or without pretreated IO ’ M thimerosal. a_Thimerosal treated membranes + 3 ,IM ATP, thimerosal treated membranes. -W-Untreated control membranes + 3 /tM ATP. untreated control membranes. Mean of 4 control and treated prepnraparations the enzyme activity was normalised to IOO”,, at 37 C and activities at other iemperatures are expressed :ts :I percentage of activit! at 37 c‘.
Effect of thimerosal
on Na’-K’
DISCUSSION
of the role
of the low affinity
and K’-PNPPase
ATP
bindine
7
activity
site in the overall reaction K+ )ATPase.
The observations reported here emphasize the high sensitivity of the (Na+-K+)ATPase to thimerosal. The rat brain synaptic membrane preparation used was considerably more sensitive than that reported for a guinea-pig microsomal enzyme by Henderson & Askari (1976). The inhibition of ATP hydrolysis was observed at a lower concentration. The lowest thimerosal concentration (lo-’ M) required to cause complete (Na+-K+)ATPase inhibition also reduced the activity of the K+-PNPPase “partial” reaction. That this residual K+-PNPPase activity is substantially altered with respect to its kinetic response to ouabain, K+ and temperature is of significance. In particular the change in shape of the Arrhenius plot from a smooth curve to a linear plot is of interest. The nonlinear Arrhenius plots usually obtained for (Na+K+)ATPase are usually attributed to a phase change in the lipid matrix of the membrane, Grisham & Barnett (1973), however the data shown in Fig. 3 suggest that protein mediated events cannot be excluded as having a role in the temperature kinetics of this enzyme. It is significant that the observed effects of pretreatment with thimerosal can be reversed completely with post incubation with dithiothreitol. This implicates the involvement of a sulphydryl group at a site remote from the phosphorylation sites on the enzyme. The failure to modify the kinetic properties of the K+-PNPPase of the treated preparation using micromolar concentrations of ATP is consistent with the suggestion that a sulphydryl group is directly involved at the lower affinity regulatory ATP binding site of the enzyme, Henderson & Askari (1977). Thus thimerosal could prove to be a useful tool in the evaluation
ATPase
mechanism
of the (NaF-
REFERENCES
AKERA T. (1971) Quantitative aspects of the interaction between ouabain and the Na+K+ ATPase. Biochim. biophp. Acta 245, 55-62. ATKINSON A., GATENBY A. D. & LOVE A. G. (1973) The determination of inorganic phosphate in biological SYSterns. Biochim. biophys. Acta 320, 195-204. GRISHAM C. M. & BARNETT R. E. (1973) Role of lipid-phase transitions in the regulation of the (sodium-potassium) adenosine triphosphatase. Biochemistry 12, 2635-2637. HENDERSON G. R. & ASKARI A. (1976) Transport ATPase: Thimerosal inhibits the Na+K+ dependent ATPase activitv without diminishing the Na’-dependent ATPase actiiity. Biochrru. hiophy.5: Rrs. Co~~~~m~.69, 499-505. HENIIERSON G. R. & ASRARI A. (1977) Transport ATPase: Further studies on the properties of the thimerosal treated enzyme. Archs B&he& Eioph~s. 182, 221-226. JORGENSEN P. L. (1974) Isolation of the (Na+-K+)ATPase. In Mvrhods it1 Enz~nolo(l~ XXXII. (Edited by COLOWIC~~ S. P. & KAPLAN N. 0.) .oo. . 277-290. Academic Press. New York. LOWE A. G. & SMART J. W. (1977) The presteadv state hydrolysis of ATP by porcine brain (Ng+-K+) hependent ATPase. Biochim. hiophys. Acta 481, 695-705. MCGRATH R. (1972) Protein measurement by ninhydrin determination of amino acids released by alkaline hydrolysis. Analyt. Biochem. 49, 95-102. PITTS B. J. R. (1974) The relationship of the K’-activated phosphatase to the Na+. K’ ATPase. Ann. N.Y. Acud. Sci. 242, 293-304. SCHWARTZ A., LINDENMAYER G. E. & ALLEN J. C. (1975) The sodium potassium adenosine triphosphatase: Pharmacological, Physiological and Biochemical Aspects. Phurmuc. Rec. 27, 3-134. WALLICK E. T. & SCHWARTZ A. (1974) Thermodynamics of the rate of binding of ouabain to the sodium, potassium adenosine triphosphate. J. hid. Chern. 249, 5141-5147.