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Cardiac (microsomal) Na + K adenosinetriphosphatase and its possible relationship to the active Na + K transport system
ARCHIVES
OF
BIOCHEMISTRY
Cardiac Possible
AND
(Microsomal) Relationship
BIOPHYSICS
Na
372-382 (1962)
99,
+ K Adenosinetriphosphatase
to the Active
JOSEPH V. AUDITORE From the Department
of Pharmacology, Received
Na
AND
Meharry May
+ K Transport
LEROY Medical
and
Its
System’
MURRAY2 College, Nashville,
Tennessee
17, 1962
An ATPase has been found in the high-speed sediment (52,000 X g) isolated from a rabbit ventricle sucrose homogenate containing 0.1% deoxycholate, whose activity depends on the relative concentrations of four naturally occurring cations, namely, Na, K, Mg, and Ca. In the presence of Mg, the addition of Na and K separately stimulated enzyme activity slightly. When Na and K are present together with Mg, an intense stimulation of enzyme activity occurs with the optimum Na: K ratio being approximately 10: 1. In the presence of isotonic Na + K solutions, the K,,, values for Na and K are 1 and 56 mM respectively. High K in the presence of Na inhibited ATPase activity, and the inhibition was analyzed as the competitive type. Demonstrated is the complex relationship that exists when Na and K are both present. Ammonium can substitute for K but not for Na. Calcium inhibited the Na-K ATPase. The influence of G-strophanthin in the presence of Mg, Mg + Na, Mg + K, and Mg + Na + K is reported. Half-maximal inhibition occurs at 6.3 X IO-7 M G-strophanthin. The similarities between the Na-K activated ATPase and the active, linked, Na + K transport, system are discussed. INTRODUCTION
Skou (1, 2) reported the presence of an adenosinetriphosphatase (ATPase) in submicroscopic particles of the peripheral crab nerve which is highly dependent on the relative concentrations of Na, K, Mg, and Ca for its activity. Mg appears to be an absolute requirement for enzyme activity while Ca is purely inhibitory. In the presence of Mg, Na but not K alone stimulates enzyme activity to some extent. Intense stimulation occurs, however, when Mg, Na, and K are present together. Of interest is that the Na + K active component can be inhibited by low concentrations of G-strophanthin, a 1 This investigation was supported by Grants RG-8508 and B-3520 from the National Institutes of Health, Division of Research Grants, U. S. Public Health Service and the Tennessee Heart Association. 2 With the technical assistance of Littleton Wade.
supposedly specific inhibitor of the active Na + K transport system (3). These properties have led Skou to suggest a pmsible connection between this ATPase and the active, linked, Na + K transport system in nerve tissue. The question arises whether a similar ATPase exists in muscle cells. If the enzyme is involved in transport, then its presence in cardiac muscle where Na + K active transport occurs may be assumed. This investigation reports the presence of an ATPase in cardiac muscle which possesses two active components, one active in the presence of Mg and the other in the presence of Mg + Na + K. The complex relationship that exists between Na, K, Mg, and other cations has been investigated, and the similarities between the enzyme and the active Na + K transport system are compared. A preliminary study of this work has been reported (4, 5).
372
NA MATERIALS
AND
PREPARATION
METHODS
OF ENZYME
Each batch of enzyme was prepared from a 10% homogenate which was made by homogenizing several fresh ventricles in a medium containing 0.25 M sucrose, 0.1% DOC, 0.005 1M EDTA, and 0.030 mM L-histidine, pH 6.0. The homogenization was carried out in an all-glass hand homogenizer submerged in an ice bath. The pH of the final tissue homogenate was 6.2. After removing the nuclear debris and mitochondrial fractions from the homogenate, the supernatant fluid was centrifuged at 52,000 X g for 45 min. The obtained high-speed pellet in this investigation will be referred to as “microsomal.” This fraction was washed one time with a 0.25 A4 sucrose, 0.05% DOC, 0.001 M EDTA, 0.030 mM L-histidine solution. The microsomal fraction was finally resuspended in a small amount of cold isotonic sucrose. All experiments were carried out with freshly prepared enzyme. ENZYME
will be seen that when deoxycholate is omitted from the homogenizing medium, the enzyme cannot be detected in either the mitochondrial or microsomal fraction. The enzyme appears, however, only in the microsomal fraction upon the addition of 0.1% DOC to the sucrose homogenizing medium. Deoxycholate causes fragmentation of the already susceptible heart mitochondria, and its presence presumably produces further contamination of the microsomal fraction by mitochondrial fragments. From such facts it may be argued that the microsomal Na-K-stimulated ATPase actually may have had its origin in the mitochondrion. That mitochondrial contamination of the microTABLE
RESULTS OF THE
KA-K
ATPASE
Several experiments were performed to determine whether the Na-K activated ATPase originates from the mitochondrion or microsomal particle. From Table I it
LOCALIZATION
I
@ a 3:: "0 E, E 6.M
ASSAY
The total volume of the reaction mixture was kept constant at 2.5 ml. and buffered with imidazole (80 mM) to which was added a small amount of HCl to obtain pH 6.7. The time of incubation was 60 min. at 37°C. The reaction mixture was charged with 15 wmoles Mg and 7.5 pmoles ATP. Various concentrations of Na, K, NH4 , Mg, and Ca were tested in this system. All cations were added in the chloride form. The reaction was stopped by the addition of 1.5 ml. of 8% HClO, . The filtered solution was analyzed for inorganic phosphate using Fiske and SubbaRow’s method (6) employing amidol as the reducing agent. ATP was rendered Na and K free by passing it through a Dowex 50 cation-exchange resin column in the hydrogen form. The free acid was brought to pH 7.0 with Tris. Protein determinations were carried out on fresh enzyme preparations employing Lowry’s et al. method (7). Specific activity is expressed as micromoles of inorganic phosphorus liberated from ATP/mg. protein/hr.
LOCALIZATION
373
+ K ADENOSINETRIPHOSPHATASE
5
I
OF NA-K-ACTIVATED -
ATPASE
Specific activity Preparation
MgATP
"N","T'Kf
-
--
SD. 3
4
24”
Mitochondria Microsomes
AGb Mitochon-
dria Microsomes 3
(Tc
Mitochondria Mitochondria debris Mitochondria supernatant Microsomes Microsome supernatant
I3.87 f
S.D.
1.03
7.49 f
l! 5.23 f
1.1214.48
I( 1.95 f
1.66
1.20
f
1.57
9.69 f
1.37
(1.43 f
0.9711.95
7.06 f
0.45
9.38 f
1.2412.04
Y& 0.94
6.24 f
0.52
f
2.28
3.64 zk 1.38 3.92 f
0.81
7.42 f 1’0.32 f
-
0.82 9.74 f 2.0718.23 f
0.23 2.04
a Fractions isolated from sucrose-EDTA-histidine. a Fractions isolated from sucrose-EDTAhistidine plus 0.1% deoxvcholate. c Mitochondrial and microsomal fractions washed one time with 0.25 M sucrose, resuspended in 0.25 M sucrose + 0.1% deoxycholate for 2 hr. at 4”C., and fractions recovered by centrifugation. ,”
Y
374
AUDITORE
AND MURRAY
somal fraction does exist is supported from studies using succinic dehydrogenase activity as a measurement of mitochondrial contamination. Fractionation studies revealed that approximately 22% of the total recovered cellular succinic dehydrogenase activity was localized in the high-speed sediment. This degree of contamination was determined, however, from experiments in which DOC was omitted since DOC inhibited the succinoxidase system. Mitochondrial contamination of heart microsomes has been reported (8, 9). To circumvent this problem, it was decided at this point to add DOC to oncewashed mitochondrial and microsomal suspensions isolated from a 0.25 M sucroseEDTA-histidine homogenizing medium. The results of these experiments (C) are also reported in Table I. The addition of 0.1% DOC to such mitochondria produced a negligible quantity of mitochondrial fragments as evidenced by the sediment (mitochondria debris) which formed at 52,000 X g from the mitochondrial supernatant. This small amount of sediment contained ATPase activity which was stimulated only slightly by Na + K. The final mitochondrial supernatant also contained ATPase activity, but it could not be stimulated further by Na + K. When fresh sucrose-isolated microsomes are treated with 0.1% DOC, the Na-Kactivated ATPase makes its appearance. The bulk of this ATFase was found in the final microsomal supernatant. Although more experimental data are needed to make final conclusions regarding the exact origin of the Na-K ATPase, the data do suggest, however, that the enzyme is probably a part of the sarcoplasmic reticulum. The authors also wish to re-emphasize that the enzyme preparation used to carry out the characterization studies which follow was the high-speed sediment that formed at 52,000 X g from a nuclear-debris plus mitochondrial-free supernatant. The reported data suggest that this high-speed sediment probably contains a mixture of a known reported mitochondrial ATPase (10, 11) and an unknown ATPase probably located in the microsomal particles.
LIBERATION
OF INORGANIC PHOSPHATE
Figure 1 shows the enzymic rate of hydrolysis of ATP as a function of time with Mg alone in the reaction mixture and with Mg + Na + K. It will be noted that as time increases so does the Mg + Na + K:Mg activation ratio. The dotted line corresponds to the terminal inorganic phosphate of ATP. INFLUEMX
OF PH ON ATPASE ACTIVITY
The influence of pH on ATPase activity with Mg and Mg + Na + K in the medium is shown in Fig. 2A. The ATPase activity represented by the solid curves was determined at the pH indicated. To determine the stability of the enzyme at the indicated pH values, the enzyme was exposed for 15 min. at 37°C. and subsequently adjusted with Tris and/or HCl to a standard pH of 6.8 and the reaction was started. Extending the exposure time to 90 min. failed to change the appearance of the curves (data not shown). When activity is measured in the presence of Mg and at the pH values indicated, a single optimum pH is obtained. Upon exposing the enzyme to the various pH values and subsequent adjustment of pH the optimum shifts to pH 5..5 and is accompanied by a general increase in activity on the acid side and a decrease on the alkaline side when compared to the “nonexposed” curve. Addition of Na + K expands the optimum pH of both the “nonexposed” and “exposed enzyme systems.” In the “non-exposed” determination the Mg + Na + K:Mg activation ratio varied from 1.82 at pH 4.6 to 2.27 at pH 6.5-6.9 and then decreased to 1.70 at 8.7 and 1.09 at 9.4. The activation ratio increases from 1.16 at pH 4.6 to 1.96 at pH 6.9 and then decreases to 1.36 at 9.4 from the “exposed” determination. The Mg + Na + K:Mg control was 1.82. From these experiments it can be said that the enzyme possesses a maximum pH around 9.0. However, the Na-K active component of the enzyme (difference between the Mg and Mg + Na + K curves) shows a maximum pH about 7.5. This is in agreement with the results of Skou (I), Post et al. (la), and Dunham and Flynn (13). The pH curves
NA
f
375
K ADENOSINETRIPHOSPHATASE
x’ h& n-M/I) Ni,
140 mM/I, It714 mM/I
X
/
.* 0
1
120 TIME
FIG. 1. Liberation of inorganic reaction was run in imidazole-HCl
I
I
60
I80
(min)
phosphate from ATP, buffer, pH 6.7.
3 mM, as a function
of time.
The
8 -
Determined
--l
Adjusted
at pH to pH 6.8
Mg6mM,ATP3mM Na 140mt.4,
IO
K 14mM
5
WITH
CN-
0
FIG. 2. Relationship between enzyme activity and pH. Reaction mixture contained ATP, 3 mM; Mg, 6 mM, determined at pH;-adjusted to pH 6.8; 8, without CN: B, with CN.
376
AUDITORE
AND MURRAY
also suggest the presence of more than one enzyme. Figure 2B shows the influence of NaCN (10 mM) on pH-activity curves. Cyanide has been shown to inhibit almost completely kidney alkaline phosphatase (14). The experiments revealed that CN failed to inhibit the Mg base line of either the alkaline or acid ATPase activities but did suppress partially that increase in enzyme activity produced by Na + K. DEPENDENCE
ON MAGNESIUM
The influence of Mg on enzyme activity is shown in Fig. 3. Mg activates the enzyme, IO
and maximal activation is obtained when the Mg:ATP ratio is equal to one. Halfmaximal activation occurred at 0.1 mM, and there was little inhibition at high Mg concentrations. INFLUENCE OF SODIUM AND ALONE ON MG-DEPENDENT
POTASSIUM ENZYME
The experiments reported in Fig. 4 show that Na and K ions alone in the presence of Mg stimulate enzyme activity, but it should be emphasized that the stimulation is very slight indeed. Maximum stimulation is obtained when the concentration of Na and K ions reaches 80 mM, and inhibition does not occur at high concentrations. ISOTONIC EXPERIMENTS
8
0
1
I 4
I 8
I 16
I 12
MAGNESIUM
I 20
In order to simulate an isotonic condition similar to that found in the intact cell, Na and K were interchanged maintaining a constant concentration of 154 mM. Figure 5 reveals that as the K is increased in the face of decreasing amounts of Na, the ATPase activity increases rapidly at first, reaches a maximum, and then falls off. Illustrated also is the intense stimulation of enzyme activity when both Na and K are present. Maximum activation is obtained at approximately 14 mM of K in the presence of 140 mM of Na, a ratio of 1: 10. Under these experimental conditions half-maximal activation occurs when the K is 1 mM and Na is 56 mM.
I 24
mM
FIG. 3. Enzyme activity in relation to the concentration of magnesium. Reaction mixture contained imidazole-HCI buffer, pH 6.7; and ATP, 3 m&f.
I- POTASSIUM
31
I
40
I
80
I
120
1
I
200
I60 IONS
I
I
I
I
240
280
320
360
mM
FIG. 4. Enzyme activity in relation to the concentration of sodium and potassium. Reaction mixture contained imidazole-HCl buffer, pH 6.7, ATP, 3 mM; and Mg, 6 mlM.
377
NA $ K ADENOSINETRIPHOSPHATASE
-
3O
k2 MAXIMAL
I 20 I 140
AT I mM OF K+
I 40 I 120
POTASSIUM I I 60 80 I
mM
I
100 80 SODIUM
I 100
I 140
I 120
I
16C
I
I
I
I
60
40
20
0
mM
FIG. 5. The influence of isotonic solutions of Na and K on ATPase activity. Na and K were exchanged for each other to achieve an isotonic condition similar to that which prevails with the intact cell. Reaction mixture also contained imidazole-HCl buffer, pH 6.7; ATP, mM; and Mg, 6 mM.
Skou (I) and Dunham and Glynn (13) reported a similar K, value for K, and under similar experimental conditions Post et al. (12) reported a K, of 3 mM for K and 24 mM for Na. It will be seen later (Figs. 6 and 7) that the K, is actually dependent upon the concentration of both Na and K. The K, of Na changes considerably compared to that of potassium (Fig. 7). ENZYME ACTIVITY IN RELATION AND POTASSIUM
TO SODIUM
These experiments were designed to study the relationship between Na and K. More specifically, they were designed to determine the optimum Na:K ratio, to determine whether Na and K are antagonistic to each other, and to determine the influence of several Na: K ratios on enzyme activity similar to those which might occur intracellularly as a result of a disturbance in the Na : K concentration gradient. The experiment is reported in Fig. 6. It will be seen that as K is increased in the presence of various constant concentrations of Na, enzyme activity rises sharply at low K levels, reaches a maximum, and then begins
to decline as the K is elevated. When the concentration of Na is 4 mM there is little activation. Maximum activation occurs at low K levels when the Na concentration is held at lo-80 mlM. In concentration range lo-160 mM Na, the optimal Na:K ratio is IO: 1. At 320 mM Na the ratio is slightly greater. It appears therefore that to obtain maximum activation the Na: K ratio should approach 10: 1. A high Na (320 mM) in the presence of small amounts of K did not stimulate the enzyme to its fullest extent. Low sodium (lo-20 mM) , on the other hand, in the presence of low K stimulated enzyme activity greatly. High levels of K at low Na inhibited enzyme activity almost to the Mg base line. It is obvious from these experiments that inhibition by high K decreases when the Na is elevated. The influence of K depends accordingly not only on the presence of Mg and Na but also on the concentration of sodium. A reciprocal plot of the rate of enzyme activity versus the K concentration of the lower K concentrations (lo-20 mmoles) revealed that the inhibition produced by the K ion is due to competitive type kinetics.
378
AUDITORE
AND
MURRAY
7
67-
.
IO mMNa 4 mMNa
5
I 20
41
I 40
I 60
I 100
I 60
POTASSIUM
I 120
I 140
I 160
mM
6. Enzyme activity in relat,ion to varying several constant concentrations of Na. Reaction 6.7; ATP, 3 mM; and Mg, 6 mM. FIG.
concentrations of K in the presence of mixture contained imidaaole-HCI, pH
I- OmMK’ e- 4 mM K+ 3- 12 mM K+ 4- 20mM K+ S- 60mM K+ 6-160mM Kt .-•
IO
I 20
I 40
I 60
I 100
I 60
SODIUM
.
I
I 120
I 140
.-. I I60
200
mM
FIG. 7. Enzyme activity in relation to varying concentrations several constant K concentrations. Reaction mixture contained 6.7; ATP, 3 mM; and Mg, 6 m2M.
It is of interest, however, that an analysis of the curve obtained with higher concentrations of K did not reveal simple competitive kinetics. It, appears that the Ki (8 mM) of the system is changing and that we are dealing with another type of inhibition. It will be seen from Fig. 7 that the K ion
I 160
of Na in the presence of imidazole-HCl buffer pH
also has an effect on the relation between enzyme activity and Na concentration. In these experiments the Na ion has been varied up to 200 mM in the presence of several constant concentrations of K. There is little activation of ATPase activity when no K is present. However, when as little as
NA + K ADENOSINETRIPHOSPHATASE
4 mM K is introduced into the system, the activity increases greatly attaining full activation when an Na of 40 mM is reached. When 12 mM K is added, the activity increases further but the increase is only slightly higher than with 4 mM K. Maximum activation is now at 120 mM Na suggesting again that an Na:K ratio of 10: 1 appears necessary to attain full activation. At 20 mM K the total activation is not as high as with the previous K concentrations, and inhibition is more obvious when 80 and 160 mM K are present in the medium. Inhibition is quite noticeable at low Na. Higher Na concentrations, however, cause the usual activating effect, but activation is only pa,rtial.
INFLUENCEOF AMMONIUM Figure 8 shows the influence of various concentrations of NH., on enzyme activity. NH4 alone stimulates enzyme activity very little. When the NH, ion is added in the presence of Na, there is a rapid rise in enzyme activity at low NH4, and it reaches its maximum at higher concentrations. The K, for NH4 is 9.5 mM. Experiments were also carried out to determine whether NH, could be substituted for Na. No increase in activity was obtained as K was increased (data not shown).
INFLUENCEOF STORAGE This study was undertaken to determine what effect storage would have on the enzyme activity in the presence of Mg alone,
FIG.
8. Enzyme activity
in relation to ammo-
nium alone and ammonium in presence of Na, 140 mM. Reaction mixture contained imidazoleHCI buffer, pH 6.7; ATP, 3 mM; and Mg, 6 mM.
0
I
I
I
I
I
I
40
80
120
160
200
240
TIME
IN HOURS
9. Enzyme activity as a function of time stored at 0°C. The following combinations of cations were put into the reaction mixt,ure in the presence of ATP, 3 mM: A Mg, 6 mM; Na, 140 m&f; K, 14 m&f. X Mg, 6 mM; Na, 80 mM. 0 Mg, 6 mM; K, 80 mM. 0 Mg, 6 mM. FIG.
Mg + Na, Mg + K, and Mg + Na + K. The results of such a study are shown in Fig. 9. The enzyme preparation was stored at O”C., and sampled at zero time and various time intervals thereafter. As can be seen from Fig. 9, there is a gradual fall in enzyme. activity with time at first greatly and then leveling off. The Mg + Na + K activity does not fall at a greater rate than that activity observed with Mg alone suggesting that it, is only the Mg-activated ATPase which is disappearing. However, after 120 hr. it begins to fall at a greater rate indicating that it is losing activity more rapidly than with Mg alone. The phenomenon is interesting, and a more detailed study is being carried out to learn more about this disappearance. It should be noted that the curve follows an equation of the following type y = 1~oC-“~where, yo is the initial concentration of enzyme, y, the concentration at time t, and k some time constant. Little difference was noted in the rate of fall of
380
AUDITORE
AND MURRAY
activity among the following systems: Mg alone, Mg + Na, and Mg + K. Of interest also is the peculiar hump that ,exists in the curve between 20 and 50 hr. suggesting the possible disappearance of :some stabilizing factor. It should also be pointed out that the greatest Na + K activation is obtained at roughly 50 hr. The Mg + Na + K:Mg ratio at this time was slightly greater than two.
ANTAGONISMBETWEEN MAGNESIUM
*-WITHOUT No+ OR K+ a-80mM Na+ A-80mM K+ a- 14mM K’, 140mM No+
AND
CALCIUM
From Fig. 10 it will be seen that concentrations of Ca greater than 0.2 mM inhibit the stimulation of enzyme activity by Mg. When the Mg is varied in the presence of Ca the activity increases, but it should be pointed out that the activity never attains that Mg level of activity which is observed in the complete absence of Ca. The inhibition appears to follow noncompetitive kinetics. These data also suggest that MgATP is the true substrate for the Na-K-activated enzyme. Experiments have also been carried out using Ca instead of Mg, and it appears that Ca may substitute for Mg, but with Ca in the medium the enzyme cannot be further stimulated by Na + K. INFLUENCE
OF G-STROPHANTHIN
Figure 11 shows the influence of G-strophanthin on the Na-K ATPase. In one phase of the experiment the influence of the cardiac glycoside on ATPase activity was
FIG. 11. Influence of G-strophanthin on Na-K ATPase. Reaction mixture contained imidazoleHCl buffer pH 6.7; ATP, 3 mM; and Mg, 6 mM. l ,withoutNaorK;0,80mMNa;A,SOmMK; A, 14 mM K and 140 mM Na.
tested in the presence of Mg alone. Under these experimental conditions no inhibition of ATPase activity was evident. Furthermore, G-strophanthin did not affect that small increase in enzyme activity when the enzyme was in the presence of Mg + Na or Mg + K. When Na and K are present in a 10: 1 ratio, the observed intense stimulation was inhibited by G-strophanthin. Almost complete inhibition of activity was observed at 1O-5 M with half-maximal concentration occurring at 6.3 X lo-’ M. DISCUSSION
FIG. 10. Relationship between enzyme activity and various concentrations of Mg and Ca in the presence of standard concentrations of Mg, Ca, Na, and K.
An ATPase has been demonstrated in the high-speed sediment the activity of which is highly dependent on four naturally occurring cations, namely, Na, K, Mg, and Ca. In the presence of Mg, Na and K added separately stimulate enzyme activity slightly. A preliminary study also reveals the presence of a similar enzyme in the rabbit auricle and human ventricle. The presence of the enzyme in other tissues has been reported (4,5, 12, 13, 15-19). The crab nerve enzyme reported by Skou (1, 2) is stimulated by Na alone but not by K. Using broken human erythrocyte membranes, Post et al. (12) found no stimulation by either Na or K,
NA + K ADEHOSINETRIPHOSPHATASE
381
while Dunham and Glynn (13) reported concentrations should lead to a correspondslight stimulation by K but not by Xa. The addition of Na + K together, however, has been found to enhance enzyme activity markedly of all enzyme preparations regardless of origin. To obtain maximum enzyme activity a Na:K ratio of 10 must prevail with the cardiac ATPase ; this differs considerably from the maximum Na : K ratio of 1 reported for the crab nerve enzyme. Competitive displacement of Na from its active site on the enzyme by K has been noted. High concentrations of K ions in the presence of Na inhibit the enzyme. The derived K; and K, values for K suggest that the K-stimulating site is distinct from the K-inhibitory site. Skou (1) originally proposed that the Na-K ATPase enzyme might be involved in the active transport of Na + K. Several years later Dunham and Glynn (13) and Post et al. (12) added further support to this proposal by showing that the erythrocyte enzyme and the active Na + K transport system possessed an unusual constellation of common properties. Although more correlatable data are needed to arrive at a definite conclusion regarding the identity of the reported Na-K ATPase with the active transport system in cardiac muscle, the discussion which follows, however, points out that the two systems have an unusual number of characteristics in common. The active Na + K transport system requires energy, and this energy is derived from energy-rich phosphate esters (20). Both the enzyme and the transport system use ATP, an energy-rich phosphate ester, as substrate. The active transport is a linked system, that is, requiring both Na and K; the enzyme also requires both Na and K for full activation. The cardiac Na-K-activated ATPase is present in that subcellular fraction which presumably in other tissues can be assumed to contain the active transport system (21). In the cardiac cell the intracellular concentrations of K and Na are approximately 60-80 and 20-30 mmoles/kg. tissue, respectively. Any change in these steady-state
ing change in enzyme activity. If we assume that the enzyme is double-faced, i.e., one face pointing intracellularly with a high affinity for Na and one extracellularly with a high affinity for K, then, as pointed out by Skou (2), the saturation of the intracellular face of the enzyme by Na depends on the concentration of both nTa and K. The polarity of the enzyme has recently been described by Glynn (22) and Whittam (23). From Fig. 7 one can see that at physiological cation concentrations the Na saturation is roughly .50% and that a decrease or increase in sodium concentration brings about a concomitant change in enzyme activity. The half-maximal concentration of Xa (30 mM) at physiological K concentration (80 mM) is roughly identical to the intracellular Na concentration of the cardiac cell. Furthermore, the competitive nature of Na and K reveals that as K levels are increased or decreased there results a corresponding change in enzyme activity. These above changes in enzyme activity are what one would expect if the enzyme operates as the Xa + K active transport system in living cell membranes. Moreover, too much K inhibits the enzyme noncompetitively and this may correspond to K toxicity. At the extracellular face of the enzyme the K affinity is presumably high. In vitro when the K concentration is approximately equal to the physiological extracellular concentration, the K saturation of the enzyme is roughly 90%. The K, value obtained for K under these conditions was slightly less than 1 indicating a high affinity of K for the enzyme. Furthermore, Post et al. (12) found that the ammonium ion can substitute for K but not for Na in both systems. We have not determined whether ammonium can substitute for K and not Na in the transport system, but we have demonstrated that it can only substitute for the K in the enzyme studies. Lastly, G-strophanthin, a specific inhibitor of the active Na + K transport system, also inhibits the Na + K-activated component of the reported ATPase enzyme. The concentration which produces half-maximal
382
AUDITORE
AND
inhibition of the Na + K ATPase approaches that concentration of G-strophanthin which promotes K loss in cardiac tissue (24). Of interest also is Klein’s (25) observation of a concomitant rise in Mg-dependent ATPase activity and a fall in ventricular Na during the development of the embryonic chick heart, but unfortunately he failed to correlate the latter event with the G-strophanthin-sensitive specific Na-K, ATPase . REFERENCES et Biophys. Acta 23, 1. SKOU, J. C., Biochim. 394 (1957). 2. SKOU, J. C., Biochim. et Biophys. Acta 42, 6 (1960). 3. SCHATZE~IANN, H. J., Helv. Physiol. et Pharmacol. Acta II, 346 (1953). 4. CAZORT, R. J., AND AUDITORE, J. V., Pharmacologist 2, 72 (1961). 5. AUDITORE, J. V., Proc. Sot. Exptl. Biol. Med. 110, 595 (1962). 6. FISKE, C. H., AND SUBBAROW, Y. S., J. Biol. Chem. 66, 375 (1925). LOWRY, 0. H., ROSEBROUGH, N. J., FARR, A. L., AND RANDALL, R. J., J. Biol. Chem. 193, 265 (1951). H~LSMANS, H. A., Biochim. et Biophys. Acta 54, 1 (1961). SIEKEVITZ, P., in “Methods in Enzymology”
10. II. 12.
13. 14.
15. 16.
17. 18.
19. 20. 21. 22. 23. 24. 25.
MURRAY (S. 0. Colowick and N. 0. Kaplan, eds.), Vol. V, p. 61. Academic Press, New York, 1962. LARDY, H. A., AND WELLMAN, H., J. Biol. Chem. 201, 357 (19531. PRESSMAN, B. C., AND L~RDY, H. A., J. Biol. Chem. 197, 547 (1952). POST, R. L., MERRITT, C. R., KINSOLVING, C. R., AND ALBRIGHT, C. D., J. Biol. Chem. 235, 1796 (1960). DUNHAM, E. T., AND GLYNN, I. M., J. Physiol. (London) 156, 274 (1961). BONTING, S. L., TSOODLE, A. D., DEBRUIN, H., AND MAYRON, B. R., Arch. Biochem. Biophys. 91, 130 (1960). SKOU, J. C., Biochim. et Biophys. Acta 58, 314 (1962). BONTING, S. L., SIMON, K. A., AND HAWKINS, N. M., Arch. Biochem. Biophys. 95, 416 (1961). JARNEFELT, J., Biochim. et Biophys. Acta 48, 104 (1961). TOSTESON, D. C., MOULTON, R. H., AND BLAUSTEIN, M., Federation Proc. 19, 128 (1960). ALDRIDGE, W. N., Biochem. J. 83, 527 (1962). HODGKIN, A. L., .&ND KEYNES, R. D., J. Physiol. (London) 128, 28 (1955). HANZON, V., AND TOCHI, G., Exptl. Cell Research 16, 256 (1959). GLYNN, I. M., J. Physiol. (London) 160, 18 (1962). WHITTAM, R., Biochem. J. 83, p29 (1962). TUTTLE, R. S., WITT, P. N., AND FARAH, A., J. Pharmacol. Exptl. Therap. 133, 281 (1961). KLEIN, R. L., Am. J. Physiol. 201, 858 (1961).
OF
BIOCHEMISTRY
Cardiac Possible
AND
(Microsomal) Relationship
BIOPHYSICS
Na
372-382 (1962)
99,
+ K Adenosinetriphosphatase
to the Active
JOSEPH V. AUDITORE From the Department
of Pharmacology, Received
Na
AND
Meharry May
+ K Transport
LEROY Medical
and
Its
System’
MURRAY2 College, Nashville,
Tennessee
17, 1962
An ATPase has been found in the high-speed sediment (52,000 X g) isolated from a rabbit ventricle sucrose homogenate containing 0.1% deoxycholate, whose activity depends on the relative concentrations of four naturally occurring cations, namely, Na, K, Mg, and Ca. In the presence of Mg, the addition of Na and K separately stimulated enzyme activity slightly. When Na and K are present together with Mg, an intense stimulation of enzyme activity occurs with the optimum Na: K ratio being approximately 10: 1. In the presence of isotonic Na + K solutions, the K,,, values for Na and K are 1 and 56 mM respectively. High K in the presence of Na inhibited ATPase activity, and the inhibition was analyzed as the competitive type. Demonstrated is the complex relationship that exists when Na and K are both present. Ammonium can substitute for K but not for Na. Calcium inhibited the Na-K ATPase. The influence of G-strophanthin in the presence of Mg, Mg + Na, Mg + K, and Mg + Na + K is reported. Half-maximal inhibition occurs at 6.3 X IO-7 M G-strophanthin. The similarities between the Na-K activated ATPase and the active, linked, Na + K transport, system are discussed. INTRODUCTION
Skou (1, 2) reported the presence of an adenosinetriphosphatase (ATPase) in submicroscopic particles of the peripheral crab nerve which is highly dependent on the relative concentrations of Na, K, Mg, and Ca for its activity. Mg appears to be an absolute requirement for enzyme activity while Ca is purely inhibitory. In the presence of Mg, Na but not K alone stimulates enzyme activity to some extent. Intense stimulation occurs, however, when Mg, Na, and K are present together. Of interest is that the Na + K active component can be inhibited by low concentrations of G-strophanthin, a 1 This investigation was supported by Grants RG-8508 and B-3520 from the National Institutes of Health, Division of Research Grants, U. S. Public Health Service and the Tennessee Heart Association. 2 With the technical assistance of Littleton Wade.
supposedly specific inhibitor of the active Na + K transport system (3). These properties have led Skou to suggest a pmsible connection between this ATPase and the active, linked, Na + K transport system in nerve tissue. The question arises whether a similar ATPase exists in muscle cells. If the enzyme is involved in transport, then its presence in cardiac muscle where Na + K active transport occurs may be assumed. This investigation reports the presence of an ATPase in cardiac muscle which possesses two active components, one active in the presence of Mg and the other in the presence of Mg + Na + K. The complex relationship that exists between Na, K, Mg, and other cations has been investigated, and the similarities between the enzyme and the active Na + K transport system are compared. A preliminary study of this work has been reported (4, 5).
372
NA MATERIALS
AND
PREPARATION
METHODS
OF ENZYME
Each batch of enzyme was prepared from a 10% homogenate which was made by homogenizing several fresh ventricles in a medium containing 0.25 M sucrose, 0.1% DOC, 0.005 1M EDTA, and 0.030 mM L-histidine, pH 6.0. The homogenization was carried out in an all-glass hand homogenizer submerged in an ice bath. The pH of the final tissue homogenate was 6.2. After removing the nuclear debris and mitochondrial fractions from the homogenate, the supernatant fluid was centrifuged at 52,000 X g for 45 min. The obtained high-speed pellet in this investigation will be referred to as “microsomal.” This fraction was washed one time with a 0.25 A4 sucrose, 0.05% DOC, 0.001 M EDTA, 0.030 mM L-histidine solution. The microsomal fraction was finally resuspended in a small amount of cold isotonic sucrose. All experiments were carried out with freshly prepared enzyme. ENZYME
will be seen that when deoxycholate is omitted from the homogenizing medium, the enzyme cannot be detected in either the mitochondrial or microsomal fraction. The enzyme appears, however, only in the microsomal fraction upon the addition of 0.1% DOC to the sucrose homogenizing medium. Deoxycholate causes fragmentation of the already susceptible heart mitochondria, and its presence presumably produces further contamination of the microsomal fraction by mitochondrial fragments. From such facts it may be argued that the microsomal Na-K-stimulated ATPase actually may have had its origin in the mitochondrion. That mitochondrial contamination of the microTABLE
RESULTS OF THE
KA-K
ATPASE
Several experiments were performed to determine whether the Na-K activated ATPase originates from the mitochondrion or microsomal particle. From Table I it
LOCALIZATION
I
@ a 3:: "0 E, E 6.M
ASSAY
The total volume of the reaction mixture was kept constant at 2.5 ml. and buffered with imidazole (80 mM) to which was added a small amount of HCl to obtain pH 6.7. The time of incubation was 60 min. at 37°C. The reaction mixture was charged with 15 wmoles Mg and 7.5 pmoles ATP. Various concentrations of Na, K, NH4 , Mg, and Ca were tested in this system. All cations were added in the chloride form. The reaction was stopped by the addition of 1.5 ml. of 8% HClO, . The filtered solution was analyzed for inorganic phosphate using Fiske and SubbaRow’s method (6) employing amidol as the reducing agent. ATP was rendered Na and K free by passing it through a Dowex 50 cation-exchange resin column in the hydrogen form. The free acid was brought to pH 7.0 with Tris. Protein determinations were carried out on fresh enzyme preparations employing Lowry’s et al. method (7). Specific activity is expressed as micromoles of inorganic phosphorus liberated from ATP/mg. protein/hr.
LOCALIZATION
373
+ K ADENOSINETRIPHOSPHATASE
5
I
OF NA-K-ACTIVATED -
ATPASE
Specific activity Preparation
MgATP
"N","T'Kf
-
--
SD. 3
4
24”
Mitochondria Microsomes
AGb Mitochon-
dria Microsomes 3
(Tc
Mitochondria Mitochondria debris Mitochondria supernatant Microsomes Microsome supernatant
I3.87 f
S.D.
1.03
7.49 f
l! 5.23 f
1.1214.48
I( 1.95 f
1.66
1.20
f
1.57
9.69 f
1.37
(1.43 f
0.9711.95
7.06 f
0.45
9.38 f
1.2412.04
Y& 0.94
6.24 f
0.52
f
2.28
3.64 zk 1.38 3.92 f
0.81
7.42 f 1’0.32 f
-
0.82 9.74 f 2.0718.23 f
0.23 2.04
a Fractions isolated from sucrose-EDTA-histidine. a Fractions isolated from sucrose-EDTAhistidine plus 0.1% deoxvcholate. c Mitochondrial and microsomal fractions washed one time with 0.25 M sucrose, resuspended in 0.25 M sucrose + 0.1% deoxycholate for 2 hr. at 4”C., and fractions recovered by centrifugation. ,”
Y
374
AUDITORE
AND MURRAY
somal fraction does exist is supported from studies using succinic dehydrogenase activity as a measurement of mitochondrial contamination. Fractionation studies revealed that approximately 22% of the total recovered cellular succinic dehydrogenase activity was localized in the high-speed sediment. This degree of contamination was determined, however, from experiments in which DOC was omitted since DOC inhibited the succinoxidase system. Mitochondrial contamination of heart microsomes has been reported (8, 9). To circumvent this problem, it was decided at this point to add DOC to oncewashed mitochondrial and microsomal suspensions isolated from a 0.25 M sucroseEDTA-histidine homogenizing medium. The results of these experiments (C) are also reported in Table I. The addition of 0.1% DOC to such mitochondria produced a negligible quantity of mitochondrial fragments as evidenced by the sediment (mitochondria debris) which formed at 52,000 X g from the mitochondrial supernatant. This small amount of sediment contained ATPase activity which was stimulated only slightly by Na + K. The final mitochondrial supernatant also contained ATPase activity, but it could not be stimulated further by Na + K. When fresh sucrose-isolated microsomes are treated with 0.1% DOC, the Na-Kactivated ATPase makes its appearance. The bulk of this ATFase was found in the final microsomal supernatant. Although more experimental data are needed to make final conclusions regarding the exact origin of the Na-K ATPase, the data do suggest, however, that the enzyme is probably a part of the sarcoplasmic reticulum. The authors also wish to re-emphasize that the enzyme preparation used to carry out the characterization studies which follow was the high-speed sediment that formed at 52,000 X g from a nuclear-debris plus mitochondrial-free supernatant. The reported data suggest that this high-speed sediment probably contains a mixture of a known reported mitochondrial ATPase (10, 11) and an unknown ATPase probably located in the microsomal particles.
LIBERATION
OF INORGANIC PHOSPHATE
Figure 1 shows the enzymic rate of hydrolysis of ATP as a function of time with Mg alone in the reaction mixture and with Mg + Na + K. It will be noted that as time increases so does the Mg + Na + K:Mg activation ratio. The dotted line corresponds to the terminal inorganic phosphate of ATP. INFLUEMX
OF PH ON ATPASE ACTIVITY
The influence of pH on ATPase activity with Mg and Mg + Na + K in the medium is shown in Fig. 2A. The ATPase activity represented by the solid curves was determined at the pH indicated. To determine the stability of the enzyme at the indicated pH values, the enzyme was exposed for 15 min. at 37°C. and subsequently adjusted with Tris and/or HCl to a standard pH of 6.8 and the reaction was started. Extending the exposure time to 90 min. failed to change the appearance of the curves (data not shown). When activity is measured in the presence of Mg and at the pH values indicated, a single optimum pH is obtained. Upon exposing the enzyme to the various pH values and subsequent adjustment of pH the optimum shifts to pH 5..5 and is accompanied by a general increase in activity on the acid side and a decrease on the alkaline side when compared to the “nonexposed” curve. Addition of Na + K expands the optimum pH of both the “nonexposed” and “exposed enzyme systems.” In the “non-exposed” determination the Mg + Na + K:Mg activation ratio varied from 1.82 at pH 4.6 to 2.27 at pH 6.5-6.9 and then decreased to 1.70 at 8.7 and 1.09 at 9.4. The activation ratio increases from 1.16 at pH 4.6 to 1.96 at pH 6.9 and then decreases to 1.36 at 9.4 from the “exposed” determination. The Mg + Na + K:Mg control was 1.82. From these experiments it can be said that the enzyme possesses a maximum pH around 9.0. However, the Na-K active component of the enzyme (difference between the Mg and Mg + Na + K curves) shows a maximum pH about 7.5. This is in agreement with the results of Skou (I), Post et al. (la), and Dunham and Flynn (13). The pH curves
NA
f
375
K ADENOSINETRIPHOSPHATASE
x’ h& n-M/I) Ni,
140 mM/I, It714 mM/I
X
/
.* 0
1
120 TIME
FIG. 1. Liberation of inorganic reaction was run in imidazole-HCl
I
I
60
I80
(min)
phosphate from ATP, buffer, pH 6.7.
3 mM, as a function
of time.
The
8 -
Determined
--l
Adjusted
at pH to pH 6.8
Mg6mM,ATP3mM Na 140mt.4,
IO
K 14mM
5
WITH
CN-
0
FIG. 2. Relationship between enzyme activity and pH. Reaction mixture contained ATP, 3 mM; Mg, 6 mM, determined at pH;-adjusted to pH 6.8; 8, without CN: B, with CN.
376
AUDITORE
AND MURRAY
also suggest the presence of more than one enzyme. Figure 2B shows the influence of NaCN (10 mM) on pH-activity curves. Cyanide has been shown to inhibit almost completely kidney alkaline phosphatase (14). The experiments revealed that CN failed to inhibit the Mg base line of either the alkaline or acid ATPase activities but did suppress partially that increase in enzyme activity produced by Na + K. DEPENDENCE
ON MAGNESIUM
The influence of Mg on enzyme activity is shown in Fig. 3. Mg activates the enzyme, IO
and maximal activation is obtained when the Mg:ATP ratio is equal to one. Halfmaximal activation occurred at 0.1 mM, and there was little inhibition at high Mg concentrations. INFLUENCE OF SODIUM AND ALONE ON MG-DEPENDENT
POTASSIUM ENZYME
The experiments reported in Fig. 4 show that Na and K ions alone in the presence of Mg stimulate enzyme activity, but it should be emphasized that the stimulation is very slight indeed. Maximum stimulation is obtained when the concentration of Na and K ions reaches 80 mM, and inhibition does not occur at high concentrations. ISOTONIC EXPERIMENTS
8
0
1
I 4
I 8
I 16
I 12
MAGNESIUM
I 20
In order to simulate an isotonic condition similar to that found in the intact cell, Na and K were interchanged maintaining a constant concentration of 154 mM. Figure 5 reveals that as the K is increased in the face of decreasing amounts of Na, the ATPase activity increases rapidly at first, reaches a maximum, and then falls off. Illustrated also is the intense stimulation of enzyme activity when both Na and K are present. Maximum activation is obtained at approximately 14 mM of K in the presence of 140 mM of Na, a ratio of 1: 10. Under these experimental conditions half-maximal activation occurs when the K is 1 mM and Na is 56 mM.
I 24
mM
FIG. 3. Enzyme activity in relation to the concentration of magnesium. Reaction mixture contained imidazole-HCI buffer, pH 6.7; and ATP, 3 m&f.
I- POTASSIUM
31
I
40
I
80
I
120
1
I
200
I60 IONS
I
I
I
I
240
280
320
360
mM
FIG. 4. Enzyme activity in relation to the concentration of sodium and potassium. Reaction mixture contained imidazole-HCl buffer, pH 6.7, ATP, 3 mM; and Mg, 6 mlM.
377
NA $ K ADENOSINETRIPHOSPHATASE
-
3O
k2 MAXIMAL
I 20 I 140
AT I mM OF K+
I 40 I 120
POTASSIUM I I 60 80 I
mM
I
100 80 SODIUM
I 100
I 140
I 120
I
16C
I
I
I
I
60
40
20
0
mM
FIG. 5. The influence of isotonic solutions of Na and K on ATPase activity. Na and K were exchanged for each other to achieve an isotonic condition similar to that which prevails with the intact cell. Reaction mixture also contained imidazole-HCl buffer, pH 6.7; ATP, mM; and Mg, 6 mM.
Skou (I) and Dunham and Glynn (13) reported a similar K, value for K, and under similar experimental conditions Post et al. (12) reported a K, of 3 mM for K and 24 mM for Na. It will be seen later (Figs. 6 and 7) that the K, is actually dependent upon the concentration of both Na and K. The K, of Na changes considerably compared to that of potassium (Fig. 7). ENZYME ACTIVITY IN RELATION AND POTASSIUM
TO SODIUM
These experiments were designed to study the relationship between Na and K. More specifically, they were designed to determine the optimum Na:K ratio, to determine whether Na and K are antagonistic to each other, and to determine the influence of several Na: K ratios on enzyme activity similar to those which might occur intracellularly as a result of a disturbance in the Na : K concentration gradient. The experiment is reported in Fig. 6. It will be seen that as K is increased in the presence of various constant concentrations of Na, enzyme activity rises sharply at low K levels, reaches a maximum, and then begins
to decline as the K is elevated. When the concentration of Na is 4 mM there is little activation. Maximum activation occurs at low K levels when the Na concentration is held at lo-80 mlM. In concentration range lo-160 mM Na, the optimal Na:K ratio is IO: 1. At 320 mM Na the ratio is slightly greater. It appears therefore that to obtain maximum activation the Na: K ratio should approach 10: 1. A high Na (320 mM) in the presence of small amounts of K did not stimulate the enzyme to its fullest extent. Low sodium (lo-20 mM) , on the other hand, in the presence of low K stimulated enzyme activity greatly. High levels of K at low Na inhibited enzyme activity almost to the Mg base line. It is obvious from these experiments that inhibition by high K decreases when the Na is elevated. The influence of K depends accordingly not only on the presence of Mg and Na but also on the concentration of sodium. A reciprocal plot of the rate of enzyme activity versus the K concentration of the lower K concentrations (lo-20 mmoles) revealed that the inhibition produced by the K ion is due to competitive type kinetics.
378
AUDITORE
AND
MURRAY
7
67-
.
IO mMNa 4 mMNa
5
I 20
41
I 40
I 60
I 100
I 60
POTASSIUM
I 120
I 140
I 160
mM
6. Enzyme activity in relat,ion to varying several constant concentrations of Na. Reaction 6.7; ATP, 3 mM; and Mg, 6 mM. FIG.
concentrations of K in the presence of mixture contained imidaaole-HCI, pH
I- OmMK’ e- 4 mM K+ 3- 12 mM K+ 4- 20mM K+ S- 60mM K+ 6-160mM Kt .-•
IO
I 20
I 40
I 60
I 100
I 60
SODIUM
.
I
I 120
I 140
.-. I I60
200
mM
FIG. 7. Enzyme activity in relation to varying concentrations several constant K concentrations. Reaction mixture contained 6.7; ATP, 3 mM; and Mg, 6 m2M.
It is of interest, however, that an analysis of the curve obtained with higher concentrations of K did not reveal simple competitive kinetics. It, appears that the Ki (8 mM) of the system is changing and that we are dealing with another type of inhibition. It will be seen from Fig. 7 that the K ion
I 160
of Na in the presence of imidazole-HCl buffer pH
also has an effect on the relation between enzyme activity and Na concentration. In these experiments the Na ion has been varied up to 200 mM in the presence of several constant concentrations of K. There is little activation of ATPase activity when no K is present. However, when as little as
NA + K ADENOSINETRIPHOSPHATASE
4 mM K is introduced into the system, the activity increases greatly attaining full activation when an Na of 40 mM is reached. When 12 mM K is added, the activity increases further but the increase is only slightly higher than with 4 mM K. Maximum activation is now at 120 mM Na suggesting again that an Na:K ratio of 10: 1 appears necessary to attain full activation. At 20 mM K the total activation is not as high as with the previous K concentrations, and inhibition is more obvious when 80 and 160 mM K are present in the medium. Inhibition is quite noticeable at low Na. Higher Na concentrations, however, cause the usual activating effect, but activation is only pa,rtial.
INFLUENCEOF AMMONIUM Figure 8 shows the influence of various concentrations of NH., on enzyme activity. NH4 alone stimulates enzyme activity very little. When the NH, ion is added in the presence of Na, there is a rapid rise in enzyme activity at low NH4, and it reaches its maximum at higher concentrations. The K, for NH4 is 9.5 mM. Experiments were also carried out to determine whether NH, could be substituted for Na. No increase in activity was obtained as K was increased (data not shown).
INFLUENCEOF STORAGE This study was undertaken to determine what effect storage would have on the enzyme activity in the presence of Mg alone,
FIG.
8. Enzyme activity
in relation to ammo-
nium alone and ammonium in presence of Na, 140 mM. Reaction mixture contained imidazoleHCI buffer, pH 6.7; ATP, 3 mM; and Mg, 6 mM.
0
I
I
I
I
I
I
40
80
120
160
200
240
TIME
IN HOURS
9. Enzyme activity as a function of time stored at 0°C. The following combinations of cations were put into the reaction mixt,ure in the presence of ATP, 3 mM: A Mg, 6 mM; Na, 140 m&f; K, 14 m&f. X Mg, 6 mM; Na, 80 mM. 0 Mg, 6 mM; K, 80 mM. 0 Mg, 6 mM. FIG.
Mg + Na, Mg + K, and Mg + Na + K. The results of such a study are shown in Fig. 9. The enzyme preparation was stored at O”C., and sampled at zero time and various time intervals thereafter. As can be seen from Fig. 9, there is a gradual fall in enzyme. activity with time at first greatly and then leveling off. The Mg + Na + K activity does not fall at a greater rate than that activity observed with Mg alone suggesting that it, is only the Mg-activated ATPase which is disappearing. However, after 120 hr. it begins to fall at a greater rate indicating that it is losing activity more rapidly than with Mg alone. The phenomenon is interesting, and a more detailed study is being carried out to learn more about this disappearance. It should be noted that the curve follows an equation of the following type y = 1~oC-“~where, yo is the initial concentration of enzyme, y, the concentration at time t, and k some time constant. Little difference was noted in the rate of fall of
380
AUDITORE
AND MURRAY
activity among the following systems: Mg alone, Mg + Na, and Mg + K. Of interest also is the peculiar hump that ,exists in the curve between 20 and 50 hr. suggesting the possible disappearance of :some stabilizing factor. It should also be pointed out that the greatest Na + K activation is obtained at roughly 50 hr. The Mg + Na + K:Mg ratio at this time was slightly greater than two.
ANTAGONISMBETWEEN MAGNESIUM
*-WITHOUT No+ OR K+ a-80mM Na+ A-80mM K+ a- 14mM K’, 140mM No+
AND
CALCIUM
From Fig. 10 it will be seen that concentrations of Ca greater than 0.2 mM inhibit the stimulation of enzyme activity by Mg. When the Mg is varied in the presence of Ca the activity increases, but it should be pointed out that the activity never attains that Mg level of activity which is observed in the complete absence of Ca. The inhibition appears to follow noncompetitive kinetics. These data also suggest that MgATP is the true substrate for the Na-K-activated enzyme. Experiments have also been carried out using Ca instead of Mg, and it appears that Ca may substitute for Mg, but with Ca in the medium the enzyme cannot be further stimulated by Na + K. INFLUENCE
OF G-STROPHANTHIN
Figure 11 shows the influence of G-strophanthin on the Na-K ATPase. In one phase of the experiment the influence of the cardiac glycoside on ATPase activity was
FIG. 11. Influence of G-strophanthin on Na-K ATPase. Reaction mixture contained imidazoleHCl buffer pH 6.7; ATP, 3 mM; and Mg, 6 mM. l ,withoutNaorK;0,80mMNa;A,SOmMK; A, 14 mM K and 140 mM Na.
tested in the presence of Mg alone. Under these experimental conditions no inhibition of ATPase activity was evident. Furthermore, G-strophanthin did not affect that small increase in enzyme activity when the enzyme was in the presence of Mg + Na or Mg + K. When Na and K are present in a 10: 1 ratio, the observed intense stimulation was inhibited by G-strophanthin. Almost complete inhibition of activity was observed at 1O-5 M with half-maximal concentration occurring at 6.3 X lo-’ M. DISCUSSION
FIG. 10. Relationship between enzyme activity and various concentrations of Mg and Ca in the presence of standard concentrations of Mg, Ca, Na, and K.
An ATPase has been demonstrated in the high-speed sediment the activity of which is highly dependent on four naturally occurring cations, namely, Na, K, Mg, and Ca. In the presence of Mg, Na and K added separately stimulate enzyme activity slightly. A preliminary study also reveals the presence of a similar enzyme in the rabbit auricle and human ventricle. The presence of the enzyme in other tissues has been reported (4,5, 12, 13, 15-19). The crab nerve enzyme reported by Skou (1, 2) is stimulated by Na alone but not by K. Using broken human erythrocyte membranes, Post et al. (12) found no stimulation by either Na or K,
NA + K ADEHOSINETRIPHOSPHATASE
381
while Dunham and Glynn (13) reported concentrations should lead to a correspondslight stimulation by K but not by Xa. The addition of Na + K together, however, has been found to enhance enzyme activity markedly of all enzyme preparations regardless of origin. To obtain maximum enzyme activity a Na:K ratio of 10 must prevail with the cardiac ATPase ; this differs considerably from the maximum Na : K ratio of 1 reported for the crab nerve enzyme. Competitive displacement of Na from its active site on the enzyme by K has been noted. High concentrations of K ions in the presence of Na inhibit the enzyme. The derived K; and K, values for K suggest that the K-stimulating site is distinct from the K-inhibitory site. Skou (1) originally proposed that the Na-K ATPase enzyme might be involved in the active transport of Na + K. Several years later Dunham and Glynn (13) and Post et al. (12) added further support to this proposal by showing that the erythrocyte enzyme and the active Na + K transport system possessed an unusual constellation of common properties. Although more correlatable data are needed to arrive at a definite conclusion regarding the identity of the reported Na-K ATPase with the active transport system in cardiac muscle, the discussion which follows, however, points out that the two systems have an unusual number of characteristics in common. The active Na + K transport system requires energy, and this energy is derived from energy-rich phosphate esters (20). Both the enzyme and the transport system use ATP, an energy-rich phosphate ester, as substrate. The active transport is a linked system, that is, requiring both Na and K; the enzyme also requires both Na and K for full activation. The cardiac Na-K-activated ATPase is present in that subcellular fraction which presumably in other tissues can be assumed to contain the active transport system (21). In the cardiac cell the intracellular concentrations of K and Na are approximately 60-80 and 20-30 mmoles/kg. tissue, respectively. Any change in these steady-state
ing change in enzyme activity. If we assume that the enzyme is double-faced, i.e., one face pointing intracellularly with a high affinity for Na and one extracellularly with a high affinity for K, then, as pointed out by Skou (2), the saturation of the intracellular face of the enzyme by Na depends on the concentration of both nTa and K. The polarity of the enzyme has recently been described by Glynn (22) and Whittam (23). From Fig. 7 one can see that at physiological cation concentrations the Na saturation is roughly .50% and that a decrease or increase in sodium concentration brings about a concomitant change in enzyme activity. The half-maximal concentration of Xa (30 mM) at physiological K concentration (80 mM) is roughly identical to the intracellular Na concentration of the cardiac cell. Furthermore, the competitive nature of Na and K reveals that as K levels are increased or decreased there results a corresponding change in enzyme activity. These above changes in enzyme activity are what one would expect if the enzyme operates as the Xa + K active transport system in living cell membranes. Moreover, too much K inhibits the enzyme noncompetitively and this may correspond to K toxicity. At the extracellular face of the enzyme the K affinity is presumably high. In vitro when the K concentration is approximately equal to the physiological extracellular concentration, the K saturation of the enzyme is roughly 90%. The K, value obtained for K under these conditions was slightly less than 1 indicating a high affinity of K for the enzyme. Furthermore, Post et al. (12) found that the ammonium ion can substitute for K but not for Na in both systems. We have not determined whether ammonium can substitute for K and not Na in the transport system, but we have demonstrated that it can only substitute for the K in the enzyme studies. Lastly, G-strophanthin, a specific inhibitor of the active Na + K transport system, also inhibits the Na + K-activated component of the reported ATPase enzyme. The concentration which produces half-maximal
382
AUDITORE
AND
inhibition of the Na + K ATPase approaches that concentration of G-strophanthin which promotes K loss in cardiac tissue (24). Of interest also is Klein’s (25) observation of a concomitant rise in Mg-dependent ATPase activity and a fall in ventricular Na during the development of the embryonic chick heart, but unfortunately he failed to correlate the latter event with the G-strophanthin-sensitive specific Na-K, ATPase . REFERENCES et Biophys. Acta 23, 1. SKOU, J. C., Biochim. 394 (1957). 2. SKOU, J. C., Biochim. et Biophys. Acta 42, 6 (1960). 3. SCHATZE~IANN, H. J., Helv. Physiol. et Pharmacol. Acta II, 346 (1953). 4. CAZORT, R. J., AND AUDITORE, J. V., Pharmacologist 2, 72 (1961). 5. AUDITORE, J. V., Proc. Sot. Exptl. Biol. Med. 110, 595 (1962). 6. FISKE, C. H., AND SUBBAROW, Y. S., J. Biol. Chem. 66, 375 (1925). LOWRY, 0. H., ROSEBROUGH, N. J., FARR, A. L., AND RANDALL, R. J., J. Biol. Chem. 193, 265 (1951). H~LSMANS, H. A., Biochim. et Biophys. Acta 54, 1 (1961). SIEKEVITZ, P., in “Methods in Enzymology”
10. II. 12.
13. 14.
15. 16.
17. 18.
19. 20. 21. 22. 23. 24. 25.
MURRAY (S. 0. Colowick and N. 0. Kaplan, eds.), Vol. V, p. 61. Academic Press, New York, 1962. LARDY, H. A., AND WELLMAN, H., J. Biol. Chem. 201, 357 (19531. PRESSMAN, B. C., AND L~RDY, H. A., J. Biol. Chem. 197, 547 (1952). POST, R. L., MERRITT, C. R., KINSOLVING, C. R., AND ALBRIGHT, C. D., J. Biol. Chem. 235, 1796 (1960). DUNHAM, E. T., AND GLYNN, I. M., J. Physiol. (London) 156, 274 (1961). BONTING, S. L., TSOODLE, A. D., DEBRUIN, H., AND MAYRON, B. R., Arch. Biochem. Biophys. 91, 130 (1960). SKOU, J. C., Biochim. et Biophys. Acta 58, 314 (1962). BONTING, S. L., SIMON, K. A., AND HAWKINS, N. M., Arch. Biochem. Biophys. 95, 416 (1961). JARNEFELT, J., Biochim. et Biophys. Acta 48, 104 (1961). TOSTESON, D. C., MOULTON, R. H., AND BLAUSTEIN, M., Federation Proc. 19, 128 (1960). ALDRIDGE, W. N., Biochem. J. 83, 527 (1962). HODGKIN, A. L., .&ND KEYNES, R. D., J. Physiol. (London) 128, 28 (1955). HANZON, V., AND TOCHI, G., Exptl. Cell Research 16, 256 (1959). GLYNN, I. M., J. Physiol. (London) 160, 18 (1962). WHITTAM, R., Biochem. J. 83, p29 (1962). TUTTLE, R. S., WITT, P. N., AND FARAH, A., J. Pharmacol. Exptl. Therap. 133, 281 (1961). KLEIN, R. L., Am. J. Physiol. 201, 858 (1961).