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Purification of cardiac (Na+,K+)-activated adenosine triphosphatase from rat
ANALYTICAL
BIOCHEMISTRY
175,284-288
Purification
( 1988)
of Cardiac (Na+,K+)-Activated Triphosphatase from Rat’
Adenosine
TAKAHIDE WATANABE, YUKO TAWADA, AND MUNEKAZU
SHIGEKAWA
Department ofMolecular Physiology, National Cardiovascular Center Research Institute, Suita, Osaka 565. Japan Received May 26,1988 A procedure is described for preparation of highly active (Na+,K+)-ATPax from rat heart which has a specific activity of 200-600 amol PJmg/h. The procedure is simple and can be applied to small amounts of heart muscle. (- 1 g). The ATPase activity was more than 90% sensitive to ouahain (at concentrations up to 1 mM). The ouabain sensitivity is biphasic with about 20% of the ATPase activity being inhibited at -3 X lo-’ M ouabain. Q 1988 Academic Pits,
Inc.
WORDS: protein/enzyme purification; microsomes; membrane-bound brane solubilization; detergents (surfactants). KEY
proteins; mem-
Purification and characterization of (Na+, K+)-ATPase (ATP phosphohydrolase, EC 3.6.1.3) from cardiac tissue are important because of its probable role as the receptor for cardiac glycosides in cardiac muscle. This enzyme has been highly purified from mammalian kidney ( 1,2) and brain (3) and from nonmammalian tissues such as the rectal gland of dog fish shark (4), electric organ of the electric eel (5) and brine shrimp (6) and has been reported to have specific activities ranging from 400 to 2200 pmol PJmglh. Despite its pharmacological importance, (Na+,K+)-ATPase has been only partially purified from cardiac muscle. Matsui and Schwartz (7) reported a purification procedure using the deoxycholate and NaI treatments which yielded a preparation with a specific activity of around 30 pmol Pi/mg/h. Pitts and Schwartz (8) later improved the purification method and obtained an enzyme preparation with a specific activity of up to 400 pmol Pi/mg/h from bovine heart. These procedures, however, are rather complex and difficult to apply to the
hearts from small animals. We report here a simple purification method by which the enzyme with specific activities of 200-600 pmol Pi/mg/h could easily be obtained from a small amount of heart muscle.
‘This work was supported by a Research Grant from Cardiovascular Diseases (61-2) from the Ministry of Health and Welfare and a Grant-in-Aid from Science and Technology Agency.
*Abbreviations used: Chaps, 3-[3-(chlolamidopropyl)dimethylammoniol-I-propanesulfonate; Mops, 4-morpholinepropansulfonic acid: SDS, sodium dodecyl sulfate.
0003-2697188 $3.00 Copyright 0 1988 by Academic Press. Inc. All rights of reproduction in any form reserved.
MATERIALS
AND METHODS
Preparation of sarcolemmal vesicles. Ventricles were dissected from hearts of Wistar rats (250-300 g). Sarcolemmal vesicles were prepared according to the method of Pitts (9) with a slight modification. The microsomal fraction was suspended in 30% sucrose solution containing 0.3 M KCl, 50 mM sodium pyrophosphate, and 0.1 M T&-Cl (pH 8.3). A solution containing 160 mM KC1 and 20 mM Mops2 (pH 7) (KCl/Mops) was layered over the top of this suspension and centrifuged at 95,OOOg for 60 min. The white band at the interface between KCl/Mops and 30% sucrose was recovered, diluted with 3 vol of KCl/Mops, and then centrifuged at 95,OOOg for 30 min. The pellet was resuspended in a
284
PURIFICATION
OF
(Na+,K+)-ATPase
solution containing 0.25 M sucrose, 25 mM imidazole (pH 7), and 1 mM EDTA and kept frozen at -70°C until use. Preparation of SDS-treated enzyme. Sarcolemma1 vesicles (1.4 mg/ml) were treated with 0.58 mg/ml of SDS in the presence of 2 mM ATP according to the method of Jsrgensen (2). The SDS-treated sample was layered on a sucrose gradient of 10 to 30% in 25 mM imidazole (pH 7.5) and 1 mM EDTA and centrifuged at 140,OOOg for 40 min in a Dupont-Servall vertical rotor, TV-865. Fractions were collected from the top of the centrifuged tube. Active fractions (at around 25% sucrose) were collected and diluted with 4 vol of cold imidazole-EDTA buffer and centrifuged at 95,000g for 30 min. The pellets were resuspended in the imidazole-EDTA buffer and stored in ice until use. Purification of (Na+,K’)-ATPase by Chaps. The SDS-treated sarcolemma (1 mg/ml) in the imidazole-EDTA buffer was solubilized with 3 mg/ml of Chaps for 30 min at 4°C and was then centrifuged at 480,OOOg for 30 min on Beckman TL- 100 ultracentrifuge. When native sarcolemmal vesicles were used instead of SDS-treated sarcolemmal vesicles (Fig. 2) the concentrations of protein and Chaps are 2 and 4 mg/ml, respectively. The supernatant obtained was diluted with an equal volume of a solution (0°C) containing 25 mM imidazole (pH 7.5) 1 mM EDTA, and 0.2 M NaCl and was left overnight at 4°C. The precipitate formed was collected by centrifugation and suspended in 25 mM imidazole (pH 7.5) and 1 mM EDTA, and the sample was stored in ice until use. (Na+,K+)-ATPase from rat brain and kidney were prepared according to the methods of Sweadner (3) and Jorgensen (2) respectively. (Na+,K’)-ATPase assays. The standard assay mixture (a final volume of 0.1 ml) contained 3 mM ATP, 5 mM MgCl,, 1 mM EDTA-Tris, 50 mM Tris-HCl (pH 7.5) 120 mM NaCl, and 10 mM KC1 with or without 1 mM ouabain. The reaction which was started by the enzyme addition was carried out at 37°C. Liberated orthophosphate was detcrmined by the method of Lin and Morales
FROM
RAT
285
HEART
( 10). One unit of specific activity was defined as 1 pmole of inorganic phosphate liberated per mg protein per hour, after subtraction of inorganic phosphate liberated in the presence of 1 mM ouabain. The concentration dependence of ouabain inhibition of the ATPase activity (Fig. 4) was determined after preincubating the enzyme (2-5 pg) in the standard assay mixture with various amounts of ouabain for 10 min before starting the reaction with ATP. The specific activities of (Na+, K+)-ATPase from rat brain and kidney were 80-340 and 130-320 pmol Pi/mg/h, respectively. Miscellaneous. Discontinuous SDS-polyacrylamide gel electrophoresis was performed according to Laemmli (11). The acrylamide concentration in the running gel was 7.5% (w/v). Protein concentrations were determined by the method of Lowry et al. (12) in the presence of 0.5% SDS with bovine serum albumin as a standard. The sources of materials used in this work were as follows: Chaps, from Doojin Chemical Co.; ATP, from Kyowa Hakko Co.; Ouabain, Tris, and molecular weight standards, from Sigma Chemical Co.; SDS, acrylamide, bisacrylamide, and Temed, from Nakarai Chemical Co. RESULTS
AND
DISCUSSION
Purification procedures. The early procedures (7,8) for purification of (Na+,K+)-ATPase from bovine cardiac muscle included solubilization of NaI-treated microsomes with deoxycholate and subsequent precipitation with glycerol or modification of each step. Schwalb et al. (13) prepared rat cardiac (Na+,K+)-ATPase by the method of Pitts and Schwartz (8) and the specific activity they obtained was low (8.4 wmol Pi/mg/h). In order to obtain highly active (Na+,K+)-ATPase, we used sarcolemmal preparations (see Materials and Methods) as the starting material instead of microsome. The specific activity of (Na+,K+)-ATPase in rat sarcolemmal vesicles was usually 20-40 pmol Pi/mg/h and this ATPase activity is about 10% of the total ATPase activity of sarcolemmal vesicles. We
286
WATANABE,
TAWADA,
TABLE 1 PURIFICATION FROM
OF (Na+,K+)-ATPase RAT HEART Specific
Protein Rat heart Sarcolemma SDS-enzyme Chaps-enzyme
9g 3w 550 PlI 57.2 pg
b-1
activity Pi/w&)
25 (1)” 94 (3.7)# 417(16.7)’
Total Olmol9
activity PI
75.0 (1oo)b 51.7 (69)b 23.9 (32)b
“Numbers in parentheses show activities relative to those detected in sarcolemmal vesicles. b Numbers in parentheses show percentage recovery of activities.
found that the SDS treatment method of Jsrgensen (2), which was successfully used in purification of (Na+,K+)-ATPase from mammalian kidney microsomes, did not improve the purity of the cardiac enzyme preparation to a similar extent; when sarcolemmal vesicles from rat heart were treated with SDS, the specific activity obtained was around 100 pmol Pdmg/h and the ouabain sensitivity was found to be about 50% (cf. Table 1). We tested several other detergents for their abilities to solubilize (Na+,K+)-ATPase activity from rat sarcolemmal membranes and found that Chaps, cholate, and CIZEg were effective. We used Chaps to solubilize (Na+,K+)-ATPase activity from SDS-treated or nontreated sarcolemmal membranes. We found then that (Na+,K+)-ATPase was solubilized at lower concentrations of Chaps in the absence of NaCl than in the presence of NaCl. As shown in Fig. 1, the maximum (Na+,K+)-ATPase activity was solubilized from the SDStreated sarcolemmal membranes at around 2 mg Chaps/ml in the absence of NaCl, whereas in the presence of NaCI, only a little activity was solubilized at this concentration of Chaps. We also found that simple addition of NaCl(O.1 M) to the supematant, which was obtained by centrifugation of the solubilized membranes in the absence of NaCl, precipitated (Na+,K+)-ATPase activity. Reduction of the Chaps concentration to one-half also precipitated (Na+,K+)-ATPase activity. KC1 was also found to be as effective as NaCl.
AND SHIGEKAWA
Figure 2 describes the purification procedures used in this study. Procedure 1 includes the SDS treatment step, whereas in Procedure 2, sarcolemmal vesicles (2 mgjml) were directly treated with Chaps (4 mg/ml). Table 1 shows the results obtained for a typical preparation. The specific activity of (Na+, K+)-ATPase after treatment with SDS and Chaps was 4 17 pmol P-Jmg/h and 1 mM ouabain inhibited more than 90% of the total ATPase activity. The specific activity was increased 16.7-fold over that in native sarcolemmal vesicles with 32% recovery of the activity. By this procedure, 60 pg of the enzyme (from 10 g of rat ventricular muscle) with specific activity ranging 200 to 600 pmol P-Jmg/h was obtained; the specific activity measured in five independent preparations was 480 + 57 pmol PJmg/h. Purity of the starting sarcolemmal vesicles and the conditions for the SDS treatment seem to affect the specific activity of the enzyme obtained in the
0
2
4 CHAPS
6
(mglmll
PIG. 1. The effect of NaCl on the extent of solubilization of rat cardiac (Na+,K+)-ATPase. SDS-treated sarcolemmal vesicles ( 1 mg/ml) were treated with indicated amounts of Chaps in the presence (0) and absence (0) of 0.1 M NaCl and then centrifuged at 480 kg for 15 min. Ouabain-sensitive ATPase activity in the supematant was measured as described under Materials and Methods.
PURIFICATION
OF (Na+,K+)-ATPase
287
FROM RAT HEART
Procedure-l 1.4mglml.
sarcolemma 0.58mg/ml. SDS sucrose gradient
srJs-,r,,~~rd~Aps
Procedure-2
2m~‘~s~~~;~m~HAps
sup Dilute (0.2M
with NaCI.
4-C
overnight
equal vol. of buffer 25mM imidazole. 1mM EDTA. pH7.5) 0 -lOglouabalni
r ppt (CHAPS-enzyme)
FIG. 2. Flow sheet of purification procedures for rat heart (Na+,K+)-ATPase.
final preparation. Procedure 2 is simple but the specific activity of the enzyme was 12 1 + 57 pmol Pi/mg/h (n = 4). The preparations obtained by these procedures were stable to such an extent that more than 80% of the enzyme activity was retained after 1 week of storage at 4°C. Figure 3 shows a densitometric scan of SDS-Chaps enzyme. The major protein on the gel is a 95,000-Da band, which is presumably the a-subunit of (Na+,K’)-ATPase because this band was phosphorylated in the presence of Naf, Mg2+ and [+Y-~*P]ATP (not shown). It is difficult to identify the glycoprotein subunit of (Na+,K+)-ATPase on coomassie brilliant blue-stained gel because there are several protein bands in the low molecular mass region. By densitometric scan, the area under the 95,000-Da band was esti-
B 2 6 0.5 0” -l
h 205K
116K 97K
66K
45K
FIG. 3. Sodium dodecyl sulfate gel electrophoresis of SDS-Chaps-treated enzyme. Acrylamide concentration was 7.5% and protein was stained by Coomassie brilliant blue.
IMi
FIG. 4. Ouabain inhibition ofthe activities of rat brain, kidney, and heart (Na+,K’)-ATPase. The kidney and brain enzymes were prepared by the method of Jsrgensen and Sweadner (2,3), respectively. The ATPax reaction in the presence of various concentrations of ouabain was carried out as described under Materials and Methods.
mated to be 35.8% of the total area. If we simply assume that the stain for the glycoprotein subunit is equivalent to 50% that for the 95,000-Da band, the enzyme would account for 50% of the stain on the gel. This estimate of the purity of the enzyme preparation is rather high compared to that deduced from the enzyme activity. This may be due partly to inactivation of the enzyme during detergent treatment. Dose-response curve of (Na’,lEft)-ATPase activity to ouabain. The enzymes prepared from rat heart, kidney, and brain showed different ouabain sensitivity as shown in Fig. 4. (Na+,K’)-ATPase activity of rat heart was inhibited by ouabain biphasically. About 20% of its activity was inhibited at low concentrations of ouabain ( - 3 X 1O-’ M) and remaining 80% was inhibited by higher concentrations of ouabain (- 1Oe4 M). This result is consistent with the results reported by Noel and Godfraind ( 14), who measured ouabainsensitivity of (Nat&+)-ATPase activity in rat sarcolemma membranes, and by Lee et al. (15), who measured both ouabain binding and enzyme activity. Mansier and Lelievre (16) also reported biphasic inhibition of (Na+,K+)-ATPase activity by ouabain with sarcolemma prepared after perfusion of rat
288
WATANABE,
TAWADA,
heart with calcium free buffer. Recently Charlemagne et al. (17) showed that the CYsubunit of rat heart enzyme could be separated into two bands on SDS gel electrophoresis after reduction and subsequent alkylation of sulfhydryl groups of the enzyme with iodoacetamide. Sweadner (23) recently also detected two a-subunits in rat heart immunologically after electrophoresis on SDS-polyacrylamide gel and transfer to nitrocellulose. Furthermore, Shull et al. (24) and Herrera et al. (25) isolate cDNA clones for three isoforms of a-subunit from rat brain and/or liver libraries. These results suggest that there may be more than one species of (Na+,K+)ATPase in rat heart as in brain ( 18), adipose tissue (19), skeletal muscle (19), dog heart (20,21), and ferret heart (22). The existance of more than one (Na+,K+)-ATPase species in rat heart may provide an explanation for the apparent discrepancy between the ouabain concentrations that induce inotropy and that induce (Na+,K+)-ATPase inhibition (26-29). Charlemagne et al. (30) reported that the property of (Na+,K+)-ATPase of rat heart changes during development and in cardiac hypertrophy. The purification method described here is rather simple and would thus enable us to study the isozymic change of (Na+,K+)-ATPase from hearts of small experimental animals.
AND
7. Matsui, H., and Schwartz, A. (1966) Biochim. Biophys. Acta 128,380-390. 8. Pitts, B. J. R., and Schwartz, A. (1975) B&him. Biophys. Acta401, 184-195. 9. Pitts, B. J. R. (1979) J. Biol. Chem. 254,6232-6235. 10. Lin, T., and Morales, M. (1977) Anal. Biochem. 77, 10-17. 11. Laemmli, U. K. (1970) Nature (London) 227,680685.
12. Lowry, 0. H., Rosebrough, N. J., Farr, A. L., and Randall, R. J. (1951) J. Biol. Chem. 193, 265275.
13. Schwalb, H., Dickstein, Y., and Heller, M. (1982) Biochim. Biophys. Acta 689,24 l-248. 14. Noel, F., and Godfraind, T. (1984) Biochem. Pharmacol. 33,47-53. 15. Lee, S.-W., Schwartz, A., Adams, R. J., Yamori, Y., Whitmer, K., Lane, L. K., and Wallick, E. T. (1983) Hypertension 5,682-688. 16. Mansier, P., and Lelievre, L. G. (1982) Nature (London) 300,535-537. 17. Charlemagne, D., Mayoux, E., Poyard, M., Oliviero, P., and Geering K. (1987) J. Biol. Chem. 262, 8941-8943. 18. Sweadner, K. J. (1979) J. Biol. Chem. 254, 60606067.
19. Lytton, J., Lin, J. C., and Guidotti, G. (1985) J. Biol. Chem. 260,1177-l 184. 20. Matsuda, T., Iwata, H., and Cooper, J. R. (1984) J. Biol. Chem. 259,3858-3863. 2 1. Maixent, J. M., Charlemagne, D., de la Chapelle, B., and Lelievre, L. G. (1987) i Biol. Chem. 262, 6842-6848. 22.
23. 24. 25.
REFERENCES 1. Lane, L. K., Copenhaver, J. H., Jr., Lindenmayer, G. E., and Schwartz, A. (1973) J. Biol. Chem. 248, 7 197-7200. 2.
Jorgensen, P. L. (1974) Biochim. Biophys. Acta 356,
3.
Sweadner, K. J. (1978) Biochim. Biophys. Acta 508,
26. 27.
36-52. 486-499.
Hokin, L. E., Dahl, J. L., Deupree, J. D., Dixon, J. F., Hackney, J. F., and Perdue, J. F. (1973) J. Biol. Chem. 248,2593-2606. 5. Dixon, J. F., and Hokin, L. E. (1974) Arch. Biochem. Biophys. 163,749-758. 6. Peterson, G. L., and Hokin, L. E. (1980) Biochem. J. 192,107-l 18.
4.
SHIGEKAWA
Ng, Y.-C., and Akera, T. (1987) Amer. J. Physiol. 252, H1016-H1022. Sweadner, K. J., and Farshi, S. K. (1987) Proc. Natl. Acad. Sci. USA 84,8404-8407. Shull, G. E., Greeb, J., and Lingrel, J. B. (1986) Biochemistry 25,8 125-8 132. Herrera, V. L. M., Emanuel, J. R., Ruiz-Opazo, N., Levenson, R., and Nadal-Ginard, B. (1987) J. Cell. Biol. 105, 1855-1865. Langer, G. A., Brady, A. J., Tan, S. T., and Serena, D. (1975) Circ. Res. 36,744-752. Adams, R. J., Schwartz, A., Grupp, G., Grupp, I., Lee, S.-W., Wallick, E. T., Powell, T., Twist, V. W., and Gathiram, P. (1982) Nature (London) 296,167-169.
Erdman, E., Philipp, G., and Scholz, H. (1980) Biothem. Pharmacol. 29,32 19-3229. 29. Werdan, K., Wagenknecht, B., Zwissler, B., Brown, L., Krawietz, W., and Erdmann, E. (1984) Biothem. Pharmacol. 33,1873-l 886. 30. Charlemagne, D., Maixent, J.-M., Preteseille, M., and Lelievre, L. G. (1986) J. Biol. Chem. 261, 185-189. 28.
BIOCHEMISTRY
175,284-288
Purification
( 1988)
of Cardiac (Na+,K+)-Activated Triphosphatase from Rat’
Adenosine
TAKAHIDE WATANABE, YUKO TAWADA, AND MUNEKAZU
SHIGEKAWA
Department ofMolecular Physiology, National Cardiovascular Center Research Institute, Suita, Osaka 565. Japan Received May 26,1988 A procedure is described for preparation of highly active (Na+,K+)-ATPax from rat heart which has a specific activity of 200-600 amol PJmg/h. The procedure is simple and can be applied to small amounts of heart muscle. (- 1 g). The ATPase activity was more than 90% sensitive to ouahain (at concentrations up to 1 mM). The ouabain sensitivity is biphasic with about 20% of the ATPase activity being inhibited at -3 X lo-’ M ouabain. Q 1988 Academic Pits,
Inc.
WORDS: protein/enzyme purification; microsomes; membrane-bound brane solubilization; detergents (surfactants). KEY
proteins; mem-
Purification and characterization of (Na+, K+)-ATPase (ATP phosphohydrolase, EC 3.6.1.3) from cardiac tissue are important because of its probable role as the receptor for cardiac glycosides in cardiac muscle. This enzyme has been highly purified from mammalian kidney ( 1,2) and brain (3) and from nonmammalian tissues such as the rectal gland of dog fish shark (4), electric organ of the electric eel (5) and brine shrimp (6) and has been reported to have specific activities ranging from 400 to 2200 pmol PJmglh. Despite its pharmacological importance, (Na+,K+)-ATPase has been only partially purified from cardiac muscle. Matsui and Schwartz (7) reported a purification procedure using the deoxycholate and NaI treatments which yielded a preparation with a specific activity of around 30 pmol Pi/mg/h. Pitts and Schwartz (8) later improved the purification method and obtained an enzyme preparation with a specific activity of up to 400 pmol Pi/mg/h from bovine heart. These procedures, however, are rather complex and difficult to apply to the
hearts from small animals. We report here a simple purification method by which the enzyme with specific activities of 200-600 pmol Pi/mg/h could easily be obtained from a small amount of heart muscle.
‘This work was supported by a Research Grant from Cardiovascular Diseases (61-2) from the Ministry of Health and Welfare and a Grant-in-Aid from Science and Technology Agency.
*Abbreviations used: Chaps, 3-[3-(chlolamidopropyl)dimethylammoniol-I-propanesulfonate; Mops, 4-morpholinepropansulfonic acid: SDS, sodium dodecyl sulfate.
0003-2697188 $3.00 Copyright 0 1988 by Academic Press. Inc. All rights of reproduction in any form reserved.
MATERIALS
AND METHODS
Preparation of sarcolemmal vesicles. Ventricles were dissected from hearts of Wistar rats (250-300 g). Sarcolemmal vesicles were prepared according to the method of Pitts (9) with a slight modification. The microsomal fraction was suspended in 30% sucrose solution containing 0.3 M KCl, 50 mM sodium pyrophosphate, and 0.1 M T&-Cl (pH 8.3). A solution containing 160 mM KC1 and 20 mM Mops2 (pH 7) (KCl/Mops) was layered over the top of this suspension and centrifuged at 95,OOOg for 60 min. The white band at the interface between KCl/Mops and 30% sucrose was recovered, diluted with 3 vol of KCl/Mops, and then centrifuged at 95,OOOg for 30 min. The pellet was resuspended in a
284
PURIFICATION
OF
(Na+,K+)-ATPase
solution containing 0.25 M sucrose, 25 mM imidazole (pH 7), and 1 mM EDTA and kept frozen at -70°C until use. Preparation of SDS-treated enzyme. Sarcolemma1 vesicles (1.4 mg/ml) were treated with 0.58 mg/ml of SDS in the presence of 2 mM ATP according to the method of Jsrgensen (2). The SDS-treated sample was layered on a sucrose gradient of 10 to 30% in 25 mM imidazole (pH 7.5) and 1 mM EDTA and centrifuged at 140,OOOg for 40 min in a Dupont-Servall vertical rotor, TV-865. Fractions were collected from the top of the centrifuged tube. Active fractions (at around 25% sucrose) were collected and diluted with 4 vol of cold imidazole-EDTA buffer and centrifuged at 95,000g for 30 min. The pellets were resuspended in the imidazole-EDTA buffer and stored in ice until use. Purification of (Na+,K’)-ATPase by Chaps. The SDS-treated sarcolemma (1 mg/ml) in the imidazole-EDTA buffer was solubilized with 3 mg/ml of Chaps for 30 min at 4°C and was then centrifuged at 480,OOOg for 30 min on Beckman TL- 100 ultracentrifuge. When native sarcolemmal vesicles were used instead of SDS-treated sarcolemmal vesicles (Fig. 2) the concentrations of protein and Chaps are 2 and 4 mg/ml, respectively. The supernatant obtained was diluted with an equal volume of a solution (0°C) containing 25 mM imidazole (pH 7.5) 1 mM EDTA, and 0.2 M NaCl and was left overnight at 4°C. The precipitate formed was collected by centrifugation and suspended in 25 mM imidazole (pH 7.5) and 1 mM EDTA, and the sample was stored in ice until use. (Na+,K+)-ATPase from rat brain and kidney were prepared according to the methods of Sweadner (3) and Jorgensen (2) respectively. (Na+,K’)-ATPase assays. The standard assay mixture (a final volume of 0.1 ml) contained 3 mM ATP, 5 mM MgCl,, 1 mM EDTA-Tris, 50 mM Tris-HCl (pH 7.5) 120 mM NaCl, and 10 mM KC1 with or without 1 mM ouabain. The reaction which was started by the enzyme addition was carried out at 37°C. Liberated orthophosphate was detcrmined by the method of Lin and Morales
FROM
RAT
285
HEART
( 10). One unit of specific activity was defined as 1 pmole of inorganic phosphate liberated per mg protein per hour, after subtraction of inorganic phosphate liberated in the presence of 1 mM ouabain. The concentration dependence of ouabain inhibition of the ATPase activity (Fig. 4) was determined after preincubating the enzyme (2-5 pg) in the standard assay mixture with various amounts of ouabain for 10 min before starting the reaction with ATP. The specific activities of (Na+, K+)-ATPase from rat brain and kidney were 80-340 and 130-320 pmol Pi/mg/h, respectively. Miscellaneous. Discontinuous SDS-polyacrylamide gel electrophoresis was performed according to Laemmli (11). The acrylamide concentration in the running gel was 7.5% (w/v). Protein concentrations were determined by the method of Lowry et al. (12) in the presence of 0.5% SDS with bovine serum albumin as a standard. The sources of materials used in this work were as follows: Chaps, from Doojin Chemical Co.; ATP, from Kyowa Hakko Co.; Ouabain, Tris, and molecular weight standards, from Sigma Chemical Co.; SDS, acrylamide, bisacrylamide, and Temed, from Nakarai Chemical Co. RESULTS
AND
DISCUSSION
Purification procedures. The early procedures (7,8) for purification of (Na+,K+)-ATPase from bovine cardiac muscle included solubilization of NaI-treated microsomes with deoxycholate and subsequent precipitation with glycerol or modification of each step. Schwalb et al. (13) prepared rat cardiac (Na+,K+)-ATPase by the method of Pitts and Schwartz (8) and the specific activity they obtained was low (8.4 wmol Pi/mg/h). In order to obtain highly active (Na+,K+)-ATPase, we used sarcolemmal preparations (see Materials and Methods) as the starting material instead of microsome. The specific activity of (Na+,K+)-ATPase in rat sarcolemmal vesicles was usually 20-40 pmol Pi/mg/h and this ATPase activity is about 10% of the total ATPase activity of sarcolemmal vesicles. We
286
WATANABE,
TAWADA,
TABLE 1 PURIFICATION FROM
OF (Na+,K+)-ATPase RAT HEART Specific
Protein Rat heart Sarcolemma SDS-enzyme Chaps-enzyme
9g 3w 550 PlI 57.2 pg
b-1
activity Pi/w&)
25 (1)” 94 (3.7)# 417(16.7)’
Total Olmol9
activity PI
75.0 (1oo)b 51.7 (69)b 23.9 (32)b
“Numbers in parentheses show activities relative to those detected in sarcolemmal vesicles. b Numbers in parentheses show percentage recovery of activities.
found that the SDS treatment method of Jsrgensen (2), which was successfully used in purification of (Na+,K+)-ATPase from mammalian kidney microsomes, did not improve the purity of the cardiac enzyme preparation to a similar extent; when sarcolemmal vesicles from rat heart were treated with SDS, the specific activity obtained was around 100 pmol Pdmg/h and the ouabain sensitivity was found to be about 50% (cf. Table 1). We tested several other detergents for their abilities to solubilize (Na+,K+)-ATPase activity from rat sarcolemmal membranes and found that Chaps, cholate, and CIZEg were effective. We used Chaps to solubilize (Na+,K+)-ATPase activity from SDS-treated or nontreated sarcolemmal membranes. We found then that (Na+,K+)-ATPase was solubilized at lower concentrations of Chaps in the absence of NaCl than in the presence of NaCl. As shown in Fig. 1, the maximum (Na+,K+)-ATPase activity was solubilized from the SDStreated sarcolemmal membranes at around 2 mg Chaps/ml in the absence of NaCl, whereas in the presence of NaCI, only a little activity was solubilized at this concentration of Chaps. We also found that simple addition of NaCl(O.1 M) to the supematant, which was obtained by centrifugation of the solubilized membranes in the absence of NaCl, precipitated (Na+,K+)-ATPase activity. Reduction of the Chaps concentration to one-half also precipitated (Na+,K+)-ATPase activity. KC1 was also found to be as effective as NaCl.
AND SHIGEKAWA
Figure 2 describes the purification procedures used in this study. Procedure 1 includes the SDS treatment step, whereas in Procedure 2, sarcolemmal vesicles (2 mgjml) were directly treated with Chaps (4 mg/ml). Table 1 shows the results obtained for a typical preparation. The specific activity of (Na+, K+)-ATPase after treatment with SDS and Chaps was 4 17 pmol P-Jmg/h and 1 mM ouabain inhibited more than 90% of the total ATPase activity. The specific activity was increased 16.7-fold over that in native sarcolemmal vesicles with 32% recovery of the activity. By this procedure, 60 pg of the enzyme (from 10 g of rat ventricular muscle) with specific activity ranging 200 to 600 pmol P-Jmg/h was obtained; the specific activity measured in five independent preparations was 480 + 57 pmol PJmg/h. Purity of the starting sarcolemmal vesicles and the conditions for the SDS treatment seem to affect the specific activity of the enzyme obtained in the
0
2
4 CHAPS
6
(mglmll
PIG. 1. The effect of NaCl on the extent of solubilization of rat cardiac (Na+,K+)-ATPase. SDS-treated sarcolemmal vesicles ( 1 mg/ml) were treated with indicated amounts of Chaps in the presence (0) and absence (0) of 0.1 M NaCl and then centrifuged at 480 kg for 15 min. Ouabain-sensitive ATPase activity in the supematant was measured as described under Materials and Methods.
PURIFICATION
OF (Na+,K+)-ATPase
287
FROM RAT HEART
Procedure-l 1.4mglml.
sarcolemma 0.58mg/ml. SDS sucrose gradient
srJs-,r,,~~rd~Aps
Procedure-2
2m~‘~s~~~;~m~HAps
sup Dilute (0.2M
with NaCI.
4-C
overnight
equal vol. of buffer 25mM imidazole. 1mM EDTA. pH7.5) 0 -lOglouabalni
r ppt (CHAPS-enzyme)
FIG. 2. Flow sheet of purification procedures for rat heart (Na+,K+)-ATPase.
final preparation. Procedure 2 is simple but the specific activity of the enzyme was 12 1 + 57 pmol Pi/mg/h (n = 4). The preparations obtained by these procedures were stable to such an extent that more than 80% of the enzyme activity was retained after 1 week of storage at 4°C. Figure 3 shows a densitometric scan of SDS-Chaps enzyme. The major protein on the gel is a 95,000-Da band, which is presumably the a-subunit of (Na+,K’)-ATPase because this band was phosphorylated in the presence of Naf, Mg2+ and [+Y-~*P]ATP (not shown). It is difficult to identify the glycoprotein subunit of (Na+,K+)-ATPase on coomassie brilliant blue-stained gel because there are several protein bands in the low molecular mass region. By densitometric scan, the area under the 95,000-Da band was esti-
B 2 6 0.5 0” -l
h 205K
116K 97K
66K
45K
FIG. 3. Sodium dodecyl sulfate gel electrophoresis of SDS-Chaps-treated enzyme. Acrylamide concentration was 7.5% and protein was stained by Coomassie brilliant blue.
IMi
FIG. 4. Ouabain inhibition ofthe activities of rat brain, kidney, and heart (Na+,K’)-ATPase. The kidney and brain enzymes were prepared by the method of Jsrgensen and Sweadner (2,3), respectively. The ATPax reaction in the presence of various concentrations of ouabain was carried out as described under Materials and Methods.
mated to be 35.8% of the total area. If we simply assume that the stain for the glycoprotein subunit is equivalent to 50% that for the 95,000-Da band, the enzyme would account for 50% of the stain on the gel. This estimate of the purity of the enzyme preparation is rather high compared to that deduced from the enzyme activity. This may be due partly to inactivation of the enzyme during detergent treatment. Dose-response curve of (Na’,lEft)-ATPase activity to ouabain. The enzymes prepared from rat heart, kidney, and brain showed different ouabain sensitivity as shown in Fig. 4. (Na+,K’)-ATPase activity of rat heart was inhibited by ouabain biphasically. About 20% of its activity was inhibited at low concentrations of ouabain ( - 3 X 1O-’ M) and remaining 80% was inhibited by higher concentrations of ouabain (- 1Oe4 M). This result is consistent with the results reported by Noel and Godfraind ( 14), who measured ouabainsensitivity of (Nat&+)-ATPase activity in rat sarcolemma membranes, and by Lee et al. (15), who measured both ouabain binding and enzyme activity. Mansier and Lelievre (16) also reported biphasic inhibition of (Na+,K+)-ATPase activity by ouabain with sarcolemma prepared after perfusion of rat
288
WATANABE,
TAWADA,
heart with calcium free buffer. Recently Charlemagne et al. (17) showed that the CYsubunit of rat heart enzyme could be separated into two bands on SDS gel electrophoresis after reduction and subsequent alkylation of sulfhydryl groups of the enzyme with iodoacetamide. Sweadner (23) recently also detected two a-subunits in rat heart immunologically after electrophoresis on SDS-polyacrylamide gel and transfer to nitrocellulose. Furthermore, Shull et al. (24) and Herrera et al. (25) isolate cDNA clones for three isoforms of a-subunit from rat brain and/or liver libraries. These results suggest that there may be more than one species of (Na+,K+)ATPase in rat heart as in brain ( 18), adipose tissue (19), skeletal muscle (19), dog heart (20,21), and ferret heart (22). The existance of more than one (Na+,K+)-ATPase species in rat heart may provide an explanation for the apparent discrepancy between the ouabain concentrations that induce inotropy and that induce (Na+,K+)-ATPase inhibition (26-29). Charlemagne et al. (30) reported that the property of (Na+,K+)-ATPase of rat heart changes during development and in cardiac hypertrophy. The purification method described here is rather simple and would thus enable us to study the isozymic change of (Na+,K+)-ATPase from hearts of small experimental animals.
AND
7. Matsui, H., and Schwartz, A. (1966) Biochim. Biophys. Acta 128,380-390. 8. Pitts, B. J. R., and Schwartz, A. (1975) B&him. Biophys. Acta401, 184-195. 9. Pitts, B. J. R. (1979) J. Biol. Chem. 254,6232-6235. 10. Lin, T., and Morales, M. (1977) Anal. Biochem. 77, 10-17. 11. Laemmli, U. K. (1970) Nature (London) 227,680685.
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