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Characterization of Mg2+-ATPase from sheep kidney medulla: Purification
ARCHIVES OF BIOCHEMISTRY AND BIOPHYSICS Vol. 198, No. 1, November, pp. 263-267, 1979
Characterization
of Mg2+-ATPase from Sheep Kidney Medulla: Purification’ MARY
LOU GANTZER
AND CHARLES
Department of Chemistry, University of Virginia,
M. GRISHAM
Charlottesville,
Virginia
22901
Received June 6, 1979; revised July 20, 19’79 Magnesium-dependent adenosine triphosphatase has been purified from sheep kidney medulla plasma membranes. The purification, which is based on treatment of a kidney plasma membrane fraction with 0.5% digitonin in 3 mM MgCI,, effectively separates the Mg2+ATPase from (Na+ + KC)-ATPase present in the same tissue and yields the Mg2+-ATPase in soluble form. The purified enzyme is activated by a variety of divalent cations and trivalent cations, including MgZ+, Mn*+, Ca2+, Co2+, Fe*+, Zn2+, Eu3+, Gd3+, and VO’+. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis of the purified enzyme shows two bands with R, values corresponding to molecular weights of 150,000 and 77,000. The larger peptide is phosphorylated by [@*P]ATP, suggesting that this peptide may contain the active site of the MgZ+-ATPase. The Mg2+-ATPase activity is unaffected by the specific (Na+ + K+)ATPase inhibitor ouabain.
Our interest in membrane-bound ion transport systems has led us to attempt a purification of membrane-bound MgZ+ATPase.2 Mg2+-ATPase appears in all tissues containing the sodium and potassium transport enzyme, (Na+ + K+)-ATPase, yet little is known of its function or characteristics. At the present time, it is not known whether or not this membranebound ATPase is also a transport enzyme. In 1969, Somogyi et al. (1) described the effects of various detergents on the activity of the (Na+ + K+)-ATPase and the Mg*+ATPase from rat brain. Their results indicated an increase in the activity of the MgZ+-ATPase after incubation with the nonionic detergent digitonin. It has also been reported (2) that treatment of pig brain microsomes with 0.1% digitonin produces a 2.5fold increase in Mg2+-ATPase activity. These reports led us to examine * This work was supported by NIH Grant AM19419, ACS-PRF Grant 8757G4 administered by the American Chemical Society, and grants from the Research Corporation and the Muscular Dystrophy Association of America. 2 Abbreviations used: MgZ+-ATPase, magnesiumactivated adenosine triphosphatase; TMA, tetramethylammonium; TES, N-tris(hydroxymethyl)methyl-2-aminoethanesulfonic acid; SDS, sodium dodecyl sulfate. 263
the solubilizing effects of this detergent on the Me+-ATPase. The purification scheme which resulted from these studies effectively separates the My+-ATPase from the (Na+ + K+)-ATPase in sheep kidney. MATERIALS
AND METHODS
Materials. Frozen sheep kidneys were obtained from Pel-Freez Biologicals, Rogers, Arkansas or from the St. Louis Serum Company, East St. Louis, Illinois, and stored at -20°C until use. Sucrose was obtained from Calbiochem. Digitonin was purchased from either ICN Pharmaceuticals or Sigma Chemicals. Imidazole was obtained from Aldrich and recrystallized from benzene before use. [-Y-~*P]ATP was obtained from ICN Pharmaceuticals or New England Nuclear. All other reagents were the purest grade commercial products available. Plasma membranes were prepared from sheep kidney as previously described (3) and frozen at -20°C. Digitonin studies.Digitonin was prepared as a 5% (w/w) stock solution. Since digitonin is only sparingly soluble in water, the stock solution was heated until the digitonin was in solution, and the appropriate volume was then removed and added to the incubation mixture. The mixture was allowed to cool before the protein was added. In the initial studies of the solubilizing effects, 1 mg of membrane protein was incubated for 20 min at room temperature in 1 ml of solution with final concentrations of 6 mM MgCl,. 6H20 and concentrations of digitonin ranging from O-l%. At the end of the incubation, the solutions were centrifuged 1 h at 38,006 rpm in a Beckman 0003-9861/79/130263-05$02.00/O Copyright All rights
6 1979 by Academic Press, of reproduction in any form
Inc. reserved.
264
GANTZER
AND GRISHAM
Type 40 rotor at 0-4°C. The supernatant and pellet were separated, and the pellet resuspended in 1 ml 0.32 M sucrose, 1 mM EDTA, pH 7.0. Supernatant and resuspended pellet were then assayed for MgZ+ATPase activity and protein content. With the digitonin concentration held constant at 0.58, the other variables in the detergent incubation were examined. The protein concentration in the incubation solution was varied from 0.2 to 0.8 mg membrane protein/ml, and the supernatant material obtained after the 1 h centrifugation again assayed for enzymatic activity and protein content. This process was then sequentially repeated for the time of incubation, temperature of incubation, and optimal MgZ+ concentration, each time using the optimum conditions that had already been determined and varying only the variable being examined. Final pwijication procedure. In a typical purification, 12 ml of plasma membrane suspension (6.5 mgl ml protein concentration) was added to a solution containing 6.0 ml 0.1 M MgCl,.6H,O, 20 ml 5% (w/w) digitonin, and 162 ml H,O. This solution was incubated at room temperature for 20 min and then centrifuged for 1 h at 40,000 rpm (117,3OOg,,,) at O-XC in a Beckman 70 Ti rotor. The clear supernatant liquid was poured into clean tubes and centrifuged 7 h at 55,000 rpm (222,000gave) in the Beckman 70 Ti rotor at O&C. At the end of this centrifugation, there are two layers visible in the tube: a very small, dense reddish-gold liquid layer at the bottom of the tube, with a clear, colorless liquid layer above it. The clear layer was pipetted off and discarded. The dense reddish-gold layer, which contains the enzyme, was applied to a Sephadex G-25 column (8.5 x 2 cm), and 2-
ml fractions were collected using 20 mM TMA-TES, pH 7.2 as eluant. Fractions 4-7 were combined and left in a refrigerator overnight to allow precipitation of digitonin. The next morning the digitonin was removed by centrifugation of the enzyme solution at 18,000 rpm (25,4OOg,,,) for 20 min in a Beckman JA20 rotor. The clear supernatant was collected and concentrated to the desired protein concentration using a Millipore immersible molecular separator. Enzyme assays. Two methods were used to assay the MgZ+-ATPase. In the first method the enzyme was incubated for 10 min at 37°C in 0.5 ml of a solution containing: 3 mM ATP (or concentration shown), 3 mM MgCl, (or concentrations of other metals shown), and 10 mM imidazole, pH 7.0 at 37°C. The enzyme reaction was stopped by the addition of 2 ml of icecold 10% (w/v) trichloroacetic acid. Inorganic phosphate in the mixture was measured by the method of Chen et al. (4), with the modification that the color was developed at 45°C for 20 min and then read at 820 nm. The second method used was a radioisotope assay using modifications of the procedures of Siegel and Albers (5) and Martin and Doty (6). The amount of 32P liberated from [+Y-~~P]ATP was determined by quenching the reaction (0.5 ml) by the addition of 0.25 ml of 5% (w/v) ammonium molybdate in 4 N H,SO,. To this 0.1 ml of 0.1 M KH2P04 was added as carrier, followed by 1.0 ml isobutanol. The resulting mixture was vortexed for 15 s and then centrifuged for 5 min at lOOOg,,,. One-half milliliter of the isobutanol layer was counted in 5 ml of Yorktown Research ‘PI-21 liquid scintillation fluid. Protein content of the plasma membrane suspension was determined by the method of Lowry et al. (7) using crystalline bovine serum albumin as a standard. Protein content of the purified enzyme was determined using a dye-binding assay (8), since traces of digitonin would interfere with the Lowry assay. The protein composition of the enzyme preparations was analyzed by SDS-polyacrylamide gel electrophoresis using a 7.5% acrylamide gel (9). RESULTS
0
a2
0.4
0.6
DIGITONIN,
a6
1.0 %
FIG, 1. Activity of supernatant (e) and resuspended pellet (m) after incubation with digitonin and centrifugation as described under Materials and Methods.
AND DISCUSSION
In two earlier studies on brain membrane proteins (1, Z), no attempt was made to determine whether or not digitonin solubilized the membrane-bound Mg2+-ATPase as well as activating it. As shown in Fig. 1, we find that digitonin effectively solubilizes the Mg2+-ATPase from the membrane of sheep kidney medulla. The increase in the specific activity of the Mg2+-ATPase in the supernatant is sixfold when the digitonin concentration is increased from 0 to 0.5%; concentrations higher than 0.5% appear to be slightly inhibitory. A digitonin
SHEEP Me+-ATPase:
level of 0.5% was selected as the concentration to be used in all further purification steps. The mechanism of action of digitonin in this preparation is unknown. It has been known for many years, however, that digitonin forms an insoluble 1:l complex with cholesterol (10). One possibility for the mechanism of action of digitonin in this procedure, then, is the removal of cholesterol from the membrane fragments, with a resultant solubilization of the enzyme. With the digitonin concentration held constant at 0.5%, the protein concentration in the incubation mixture was varied from 0.2 to 0.8 mg membrane protein/ml. The supernatant material obtained after the 1 h centrifugation was assayed for enzymatic activity and protein content. As shown in Fig. 2, the optimal protein concentration was found to be 0.35 to 0.49 mg protein/ml of incubation solution. At this protein concentration, 16-B% of the total protein added is solubilized. Using a protein concentration of 0.37 mgl ml and a digitonin concentration of 0.5%, the time of the incubation was varied from O120 min. It was found that during this time period there was little variation in the specific activity of the enzyme with the variation in incubation time. This suggests that the digitonin acts very quickly once the membrane fragments are added to it. An incubation time of 20 min was routinely used in succeeding preparations. The dependence of the specific activity of the enzyme on the temperature of the deter-
FIG. 2. Effect of protein concentration during digitonin incubation on the solubilization and purification of MgZ+-ATPase. Incubations and assays were as described under Materials and Methods.
265
PURIFICATION
0
2
I [:+,B
1
I
I
I B
I
I lo
FIG. 3. Effect of MgCl, on solubilization of Mg2+ATPase by digitonin. Incubations and assays were as described under Materials and Methods.
gent incubation was also examined. It was found that carrying out the digitonin incubation at 25°C rather than 0 or 3’7°C would yield a preparation of slightly higher activity. The effect of Mg2+ ions on the detergent treatment is illustrated in Fig. 3. The enzyme exhibits its highest activity when the Mg2+ concentration in the detergent incubation is 3 mM; this concentration was used in all further purifications. The role the Mg2+ ions play during the purification procedure is unknown. It is possible that (a) Mg2+ ions bind to the enzyme active site, and thus help stabilize the enzyme during the purification procedure or (b) the Mg2+ ions bind to anionic protein and lipid sites on the membrane, affecting protein-protein, protein-lipid, or lipidlipid interactions. The initial specific activity of the Me+ATPase in the plasma membrane fragments is typically 0.010 pmol ATP hydrolyzed/ mg proteimmin (units/mg). At the end of the purification procedure the specific activity of the enzyme has risen to 0.23-0.5 unit/mg (using 3 mM Mg2+, 3 mM ATP, 20 mM TMATES, pH 7.5 at 25°C). Under a variety of conditions the enzyme activity is linear with protein concentration. The final yield is 710 mg protein from 74- 78 mg kidney medulla membrane protein. SDS-gel electrophoresis of the plasma membranes yields approximately 20-30 bands on the gel. When the purified enzyme is examined under the same conditions, two major bands are observed (Fig. 4). These two bands yield R, values corresponding to molecular
266
GANTZER
AND GRISHAM
2
1
FIG. 4. SDS-acrylamide gel of purified MgZ+ATPase (No. 2) and a blank gel (No. 1). Mobilities of the two bands in the purified enzyme correspond to molecular weights of 150,000 and 77,000.
weights of 150,000 and 77,000. Several lines of evidence argue that this enzyme is not a degraded or denatured form of (Na+ + K+)ATPase from the same membrane: The molecular weights of the two bands on the SDS-gels are much higher than those of the TABLE A~IVATION
Cation None W+ Zn2+ co*+ Ca*+ Mn*+ Fez+ vo*+ b Sr*+ La3+ Eu3+ Gd3+
I
OF Mg3+-ATPase BY DIVALENT TRIVALENT CATIONS”
AND
Specific activity Wlmg)
Percentage of maximal (Mg2+) activity
0.018.3 0.0809
22.6 100 38.7 87.1 87.1 77.5 48.5 38.7 54.9 38.7 45.2 61.3
0.0313 0.0705 0.0705 0.0627 0.0392 0.0313 0.0444 0.0313 0.0366 0.0496
a All assay solutions contained 0.5 mM cation (Cl- or SO:- salt), 0.5 mM ATP, 20 mM TMA-TES, pH 7.5 at 25°C. b To prevent oxidation 1 mM sodium dithionite was used.
(Na+ + K+)-ATPase, the combining weight from Mn2+ binding studies (11) is much larger (469,000 to 250,000), this enzyme demonstrates no ouabain sensitivity, is not activated to any degree by sodium ions, and shows a much broader response to multivalent ions than the (Na+ + K+)-ATPase (Table I, below). The insensitivity to ouabain also demonstrates that this enzyme preparation is not contaminated with (Na+ + K+)-ATPase. Similar gels to which were applied lo-fold higher amounts of enzyme protein showed no additional bands, suggesting that one or both of the bands observed represent the Mg2+-ATPase activity. Preliminary phosphorylation studies with [Y-~~P]ATP indicate that the larger peptide is phosphorylated under normal enzyme assay conditions, suggesting that this peptide may contain the active site of the M$+ATPase. Based on Mn2+ EPR binding studies (ll), we have estimated the molecular weight of this enzyme to be 469,000. An 02p2 complex of the two peptides observed on the gels would give a molecular weight of 454,000. In an attempt to determine whether multiple forms of the ATPase exist in the preparation used here, the enzyme preparation was subjected to electrophoresis at low temperature in acrylamide gels which did not contain sodium dodecyl sulfate or other denaturing agents. Following electrophoresis at 2 mA per gel, the gels were incubated at 3’7°C in a solution containing 3 mM ATP, 5 mM MgC12, 0.1 M TMATES, pH 7.7. Phosphate was then detected in the gels using the method of Abrams and Baron (12). As shown in Fig. 5, only a single phosphate band could be detected on these gels, even at high loadings of enzyme. Thus the data are consistent with the existence of a single ATPase in this preparation. The width of the phosphate band observed on the gel probably results from diffusion of the released phosphate within the gel. While it is possible that multiple ATPases could be located within this broad band, the temperature studies and the Mn2+ binding experiments in the following paper argue against a heterogeneity of ATPase active centers in this preparation.
SHEEP MgZ+-ATPase: PURIFICATION
267
specificity at the divalent cation sites. This fact may be relevant to the function of this enzyme in the plasma membrane. While other ATP-hydrolyzing enzymes have been implicated in the transport of Na+ and K+ (13) or Ca2+ (14) across membranes, the transport properties, if any, of this MgZ+ATPase are unknown. In the following paper, we examine the binding of divalent metals and ATP to this enzyme by kinetic and magnetic resonance methods (11). Additional studies aimed at characterizing the structure and function of this enzyme are currently underway in our laboratory. REFERENCES
1
234
5
FIG. 5. Localization of ATPase activity on poiyacrylamide gels. Gels, 7.5%, were prepared with Tris-glycine buffer, pH 8.3, which was also used as the buffer during electrophoresis. The gels were prernn for 30 mm at 2 mA/gel. Protein 25-100 pg, was layered on gels 1, 2, 3, and 5 and electrophoresis was carried out at 4°C at 2 mA/gel for 2 h. Protein was detected in gels 1 and 5 by staining with 0.4% amido black in 5:5:1 methanol: water: acetic acid. The position of the ATPase was determined on gels 2 and 3 by incubating the gel in 3 mM ATP, 5 mM MgZ+, 0.1 M TES-TMA, pH 7.5 at 37°C for 30 min. Inorganic phosphate released during the enzymatic reaction was then detected as described by Abrams and Baron (12). Gel 4 was a blank gel, run without protein and treated in the manner of gels 2 and 3.
As shown in Table I, this Mg2+-ATPase preparation is activated by a surprisingly large number of divalent and trivalent cations, with further activation observed upon addition of KCI. The activation by the vanadyl (VOz+) ion and by lanthanide ions is particularly noteworthy, since these ions do not normally activate enzymes in pIace of Mg2+ or Ca2+. The broad range of cations which can replace Mg2+ in the activation of this enzyme suggests a rather low degree of
1. SOMOGYI, J., BUDAI, M., NYIRO, L., KALUZA, G., NAGEL, W., AND WILLIG, F. (1969) Acta Biochim. Biophys. Acad. Sci. Hung. 4, 219236. 2. NAKAMARU, Y., ANDKONISHI, K. (1968) Biochim. Biophys. Acta 159, 206-208. 3. SCHWARTZ, A., BACHELAARD,H., ANDMCILWAIN, H. (1962) Biockem. J. 84, 626-637. 4. CHEN, P., TORIBARA, T., AND WARNER, H. (1956) Anal.
Chem.
28, 1756-1758.
5. SIEGEL, F., AND ALBERS, R. (1967)5. Biol. Ch,em. 242, 4972-4979. 6. MARTIN, J., AND DOTY, D. (1949) Anal. Chem. 21, 965-967. 7. LOWRY, O., ROSEBROUGH, N., FARR, A., AND RANDALL, R. (1951) J. Biol. Chem. 193, 265-275. 72, 2488. BRADFORD, M. (1976) Anal. Biochem. 254. 9. WEBER, K., AND OSBORN, M. (1969) J. Biol. Chm. 244, 4406-4412. 10. FIESER, L., AND FIESER, M. (1959) Steroids, Reinhold, New York. 11. GANTZER, M., AND GRISHAM, C. (1979) Arch. Biochem. Biophys. 198, 000-000. 12. ABRAMS, A., AND BARON, C. (1967) Biochemistry 6, 225. 36, 13. ALBERS, R. W. (1967) Annu. Rev. B&hem. 727. Chem. 14. HASSELBACH, W. (1964) Progr. Biophys, 14, 167-222.
Characterization
of Mg2+-ATPase from Sheep Kidney Medulla: Purification’ MARY
LOU GANTZER
AND CHARLES
Department of Chemistry, University of Virginia,
M. GRISHAM
Charlottesville,
Virginia
22901
Received June 6, 1979; revised July 20, 19’79 Magnesium-dependent adenosine triphosphatase has been purified from sheep kidney medulla plasma membranes. The purification, which is based on treatment of a kidney plasma membrane fraction with 0.5% digitonin in 3 mM MgCI,, effectively separates the Mg2+ATPase from (Na+ + KC)-ATPase present in the same tissue and yields the Mg2+-ATPase in soluble form. The purified enzyme is activated by a variety of divalent cations and trivalent cations, including MgZ+, Mn*+, Ca2+, Co2+, Fe*+, Zn2+, Eu3+, Gd3+, and VO’+. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis of the purified enzyme shows two bands with R, values corresponding to molecular weights of 150,000 and 77,000. The larger peptide is phosphorylated by [@*P]ATP, suggesting that this peptide may contain the active site of the MgZ+-ATPase. The Mg2+-ATPase activity is unaffected by the specific (Na+ + K+)ATPase inhibitor ouabain.
Our interest in membrane-bound ion transport systems has led us to attempt a purification of membrane-bound MgZ+ATPase.2 Mg2+-ATPase appears in all tissues containing the sodium and potassium transport enzyme, (Na+ + K+)-ATPase, yet little is known of its function or characteristics. At the present time, it is not known whether or not this membranebound ATPase is also a transport enzyme. In 1969, Somogyi et al. (1) described the effects of various detergents on the activity of the (Na+ + K+)-ATPase and the Mg*+ATPase from rat brain. Their results indicated an increase in the activity of the MgZ+-ATPase after incubation with the nonionic detergent digitonin. It has also been reported (2) that treatment of pig brain microsomes with 0.1% digitonin produces a 2.5fold increase in Mg2+-ATPase activity. These reports led us to examine * This work was supported by NIH Grant AM19419, ACS-PRF Grant 8757G4 administered by the American Chemical Society, and grants from the Research Corporation and the Muscular Dystrophy Association of America. 2 Abbreviations used: MgZ+-ATPase, magnesiumactivated adenosine triphosphatase; TMA, tetramethylammonium; TES, N-tris(hydroxymethyl)methyl-2-aminoethanesulfonic acid; SDS, sodium dodecyl sulfate. 263
the solubilizing effects of this detergent on the Me+-ATPase. The purification scheme which resulted from these studies effectively separates the My+-ATPase from the (Na+ + K+)-ATPase in sheep kidney. MATERIALS
AND METHODS
Materials. Frozen sheep kidneys were obtained from Pel-Freez Biologicals, Rogers, Arkansas or from the St. Louis Serum Company, East St. Louis, Illinois, and stored at -20°C until use. Sucrose was obtained from Calbiochem. Digitonin was purchased from either ICN Pharmaceuticals or Sigma Chemicals. Imidazole was obtained from Aldrich and recrystallized from benzene before use. [-Y-~*P]ATP was obtained from ICN Pharmaceuticals or New England Nuclear. All other reagents were the purest grade commercial products available. Plasma membranes were prepared from sheep kidney as previously described (3) and frozen at -20°C. Digitonin studies.Digitonin was prepared as a 5% (w/w) stock solution. Since digitonin is only sparingly soluble in water, the stock solution was heated until the digitonin was in solution, and the appropriate volume was then removed and added to the incubation mixture. The mixture was allowed to cool before the protein was added. In the initial studies of the solubilizing effects, 1 mg of membrane protein was incubated for 20 min at room temperature in 1 ml of solution with final concentrations of 6 mM MgCl,. 6H20 and concentrations of digitonin ranging from O-l%. At the end of the incubation, the solutions were centrifuged 1 h at 38,006 rpm in a Beckman 0003-9861/79/130263-05$02.00/O Copyright All rights
6 1979 by Academic Press, of reproduction in any form
Inc. reserved.
264
GANTZER
AND GRISHAM
Type 40 rotor at 0-4°C. The supernatant and pellet were separated, and the pellet resuspended in 1 ml 0.32 M sucrose, 1 mM EDTA, pH 7.0. Supernatant and resuspended pellet were then assayed for MgZ+ATPase activity and protein content. With the digitonin concentration held constant at 0.58, the other variables in the detergent incubation were examined. The protein concentration in the incubation solution was varied from 0.2 to 0.8 mg membrane protein/ml, and the supernatant material obtained after the 1 h centrifugation again assayed for enzymatic activity and protein content. This process was then sequentially repeated for the time of incubation, temperature of incubation, and optimal MgZ+ concentration, each time using the optimum conditions that had already been determined and varying only the variable being examined. Final pwijication procedure. In a typical purification, 12 ml of plasma membrane suspension (6.5 mgl ml protein concentration) was added to a solution containing 6.0 ml 0.1 M MgCl,.6H,O, 20 ml 5% (w/w) digitonin, and 162 ml H,O. This solution was incubated at room temperature for 20 min and then centrifuged for 1 h at 40,000 rpm (117,3OOg,,,) at O-XC in a Beckman 70 Ti rotor. The clear supernatant liquid was poured into clean tubes and centrifuged 7 h at 55,000 rpm (222,000gave) in the Beckman 70 Ti rotor at O&C. At the end of this centrifugation, there are two layers visible in the tube: a very small, dense reddish-gold liquid layer at the bottom of the tube, with a clear, colorless liquid layer above it. The clear layer was pipetted off and discarded. The dense reddish-gold layer, which contains the enzyme, was applied to a Sephadex G-25 column (8.5 x 2 cm), and 2-
ml fractions were collected using 20 mM TMA-TES, pH 7.2 as eluant. Fractions 4-7 were combined and left in a refrigerator overnight to allow precipitation of digitonin. The next morning the digitonin was removed by centrifugation of the enzyme solution at 18,000 rpm (25,4OOg,,,) for 20 min in a Beckman JA20 rotor. The clear supernatant was collected and concentrated to the desired protein concentration using a Millipore immersible molecular separator. Enzyme assays. Two methods were used to assay the MgZ+-ATPase. In the first method the enzyme was incubated for 10 min at 37°C in 0.5 ml of a solution containing: 3 mM ATP (or concentration shown), 3 mM MgCl, (or concentrations of other metals shown), and 10 mM imidazole, pH 7.0 at 37°C. The enzyme reaction was stopped by the addition of 2 ml of icecold 10% (w/v) trichloroacetic acid. Inorganic phosphate in the mixture was measured by the method of Chen et al. (4), with the modification that the color was developed at 45°C for 20 min and then read at 820 nm. The second method used was a radioisotope assay using modifications of the procedures of Siegel and Albers (5) and Martin and Doty (6). The amount of 32P liberated from [+Y-~~P]ATP was determined by quenching the reaction (0.5 ml) by the addition of 0.25 ml of 5% (w/v) ammonium molybdate in 4 N H,SO,. To this 0.1 ml of 0.1 M KH2P04 was added as carrier, followed by 1.0 ml isobutanol. The resulting mixture was vortexed for 15 s and then centrifuged for 5 min at lOOOg,,,. One-half milliliter of the isobutanol layer was counted in 5 ml of Yorktown Research ‘PI-21 liquid scintillation fluid. Protein content of the plasma membrane suspension was determined by the method of Lowry et al. (7) using crystalline bovine serum albumin as a standard. Protein content of the purified enzyme was determined using a dye-binding assay (8), since traces of digitonin would interfere with the Lowry assay. The protein composition of the enzyme preparations was analyzed by SDS-polyacrylamide gel electrophoresis using a 7.5% acrylamide gel (9). RESULTS
0
a2
0.4
0.6
DIGITONIN,
a6
1.0 %
FIG, 1. Activity of supernatant (e) and resuspended pellet (m) after incubation with digitonin and centrifugation as described under Materials and Methods.
AND DISCUSSION
In two earlier studies on brain membrane proteins (1, Z), no attempt was made to determine whether or not digitonin solubilized the membrane-bound Mg2+-ATPase as well as activating it. As shown in Fig. 1, we find that digitonin effectively solubilizes the Mg2+-ATPase from the membrane of sheep kidney medulla. The increase in the specific activity of the Mg2+-ATPase in the supernatant is sixfold when the digitonin concentration is increased from 0 to 0.5%; concentrations higher than 0.5% appear to be slightly inhibitory. A digitonin
SHEEP Me+-ATPase:
level of 0.5% was selected as the concentration to be used in all further purification steps. The mechanism of action of digitonin in this preparation is unknown. It has been known for many years, however, that digitonin forms an insoluble 1:l complex with cholesterol (10). One possibility for the mechanism of action of digitonin in this procedure, then, is the removal of cholesterol from the membrane fragments, with a resultant solubilization of the enzyme. With the digitonin concentration held constant at 0.5%, the protein concentration in the incubation mixture was varied from 0.2 to 0.8 mg membrane protein/ml. The supernatant material obtained after the 1 h centrifugation was assayed for enzymatic activity and protein content. As shown in Fig. 2, the optimal protein concentration was found to be 0.35 to 0.49 mg protein/ml of incubation solution. At this protein concentration, 16-B% of the total protein added is solubilized. Using a protein concentration of 0.37 mgl ml and a digitonin concentration of 0.5%, the time of the incubation was varied from O120 min. It was found that during this time period there was little variation in the specific activity of the enzyme with the variation in incubation time. This suggests that the digitonin acts very quickly once the membrane fragments are added to it. An incubation time of 20 min was routinely used in succeeding preparations. The dependence of the specific activity of the enzyme on the temperature of the deter-
FIG. 2. Effect of protein concentration during digitonin incubation on the solubilization and purification of MgZ+-ATPase. Incubations and assays were as described under Materials and Methods.
265
PURIFICATION
0
2
I [:+,B
1
I
I
I B
I
I lo
FIG. 3. Effect of MgCl, on solubilization of Mg2+ATPase by digitonin. Incubations and assays were as described under Materials and Methods.
gent incubation was also examined. It was found that carrying out the digitonin incubation at 25°C rather than 0 or 3’7°C would yield a preparation of slightly higher activity. The effect of Mg2+ ions on the detergent treatment is illustrated in Fig. 3. The enzyme exhibits its highest activity when the Mg2+ concentration in the detergent incubation is 3 mM; this concentration was used in all further purifications. The role the Mg2+ ions play during the purification procedure is unknown. It is possible that (a) Mg2+ ions bind to the enzyme active site, and thus help stabilize the enzyme during the purification procedure or (b) the Mg2+ ions bind to anionic protein and lipid sites on the membrane, affecting protein-protein, protein-lipid, or lipidlipid interactions. The initial specific activity of the Me+ATPase in the plasma membrane fragments is typically 0.010 pmol ATP hydrolyzed/ mg proteimmin (units/mg). At the end of the purification procedure the specific activity of the enzyme has risen to 0.23-0.5 unit/mg (using 3 mM Mg2+, 3 mM ATP, 20 mM TMATES, pH 7.5 at 25°C). Under a variety of conditions the enzyme activity is linear with protein concentration. The final yield is 710 mg protein from 74- 78 mg kidney medulla membrane protein. SDS-gel electrophoresis of the plasma membranes yields approximately 20-30 bands on the gel. When the purified enzyme is examined under the same conditions, two major bands are observed (Fig. 4). These two bands yield R, values corresponding to molecular
266
GANTZER
AND GRISHAM
2
1
FIG. 4. SDS-acrylamide gel of purified MgZ+ATPase (No. 2) and a blank gel (No. 1). Mobilities of the two bands in the purified enzyme correspond to molecular weights of 150,000 and 77,000.
weights of 150,000 and 77,000. Several lines of evidence argue that this enzyme is not a degraded or denatured form of (Na+ + K+)ATPase from the same membrane: The molecular weights of the two bands on the SDS-gels are much higher than those of the TABLE A~IVATION
Cation None W+ Zn2+ co*+ Ca*+ Mn*+ Fez+ vo*+ b Sr*+ La3+ Eu3+ Gd3+
I
OF Mg3+-ATPase BY DIVALENT TRIVALENT CATIONS”
AND
Specific activity Wlmg)
Percentage of maximal (Mg2+) activity
0.018.3 0.0809
22.6 100 38.7 87.1 87.1 77.5 48.5 38.7 54.9 38.7 45.2 61.3
0.0313 0.0705 0.0705 0.0627 0.0392 0.0313 0.0444 0.0313 0.0366 0.0496
a All assay solutions contained 0.5 mM cation (Cl- or SO:- salt), 0.5 mM ATP, 20 mM TMA-TES, pH 7.5 at 25°C. b To prevent oxidation 1 mM sodium dithionite was used.
(Na+ + K+)-ATPase, the combining weight from Mn2+ binding studies (11) is much larger (469,000 to 250,000), this enzyme demonstrates no ouabain sensitivity, is not activated to any degree by sodium ions, and shows a much broader response to multivalent ions than the (Na+ + K+)-ATPase (Table I, below). The insensitivity to ouabain also demonstrates that this enzyme preparation is not contaminated with (Na+ + K+)-ATPase. Similar gels to which were applied lo-fold higher amounts of enzyme protein showed no additional bands, suggesting that one or both of the bands observed represent the Mg2+-ATPase activity. Preliminary phosphorylation studies with [Y-~~P]ATP indicate that the larger peptide is phosphorylated under normal enzyme assay conditions, suggesting that this peptide may contain the active site of the M$+ATPase. Based on Mn2+ EPR binding studies (ll), we have estimated the molecular weight of this enzyme to be 469,000. An 02p2 complex of the two peptides observed on the gels would give a molecular weight of 454,000. In an attempt to determine whether multiple forms of the ATPase exist in the preparation used here, the enzyme preparation was subjected to electrophoresis at low temperature in acrylamide gels which did not contain sodium dodecyl sulfate or other denaturing agents. Following electrophoresis at 2 mA per gel, the gels were incubated at 3’7°C in a solution containing 3 mM ATP, 5 mM MgC12, 0.1 M TMATES, pH 7.7. Phosphate was then detected in the gels using the method of Abrams and Baron (12). As shown in Fig. 5, only a single phosphate band could be detected on these gels, even at high loadings of enzyme. Thus the data are consistent with the existence of a single ATPase in this preparation. The width of the phosphate band observed on the gel probably results from diffusion of the released phosphate within the gel. While it is possible that multiple ATPases could be located within this broad band, the temperature studies and the Mn2+ binding experiments in the following paper argue against a heterogeneity of ATPase active centers in this preparation.
SHEEP MgZ+-ATPase: PURIFICATION
267
specificity at the divalent cation sites. This fact may be relevant to the function of this enzyme in the plasma membrane. While other ATP-hydrolyzing enzymes have been implicated in the transport of Na+ and K+ (13) or Ca2+ (14) across membranes, the transport properties, if any, of this MgZ+ATPase are unknown. In the following paper, we examine the binding of divalent metals and ATP to this enzyme by kinetic and magnetic resonance methods (11). Additional studies aimed at characterizing the structure and function of this enzyme are currently underway in our laboratory. REFERENCES
1
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5
FIG. 5. Localization of ATPase activity on poiyacrylamide gels. Gels, 7.5%, were prepared with Tris-glycine buffer, pH 8.3, which was also used as the buffer during electrophoresis. The gels were prernn for 30 mm at 2 mA/gel. Protein 25-100 pg, was layered on gels 1, 2, 3, and 5 and electrophoresis was carried out at 4°C at 2 mA/gel for 2 h. Protein was detected in gels 1 and 5 by staining with 0.4% amido black in 5:5:1 methanol: water: acetic acid. The position of the ATPase was determined on gels 2 and 3 by incubating the gel in 3 mM ATP, 5 mM MgZ+, 0.1 M TES-TMA, pH 7.5 at 37°C for 30 min. Inorganic phosphate released during the enzymatic reaction was then detected as described by Abrams and Baron (12). Gel 4 was a blank gel, run without protein and treated in the manner of gels 2 and 3.
As shown in Table I, this Mg2+-ATPase preparation is activated by a surprisingly large number of divalent and trivalent cations, with further activation observed upon addition of KCI. The activation by the vanadyl (VOz+) ion and by lanthanide ions is particularly noteworthy, since these ions do not normally activate enzymes in pIace of Mg2+ or Ca2+. The broad range of cations which can replace Mg2+ in the activation of this enzyme suggests a rather low degree of
1. SOMOGYI, J., BUDAI, M., NYIRO, L., KALUZA, G., NAGEL, W., AND WILLIG, F. (1969) Acta Biochim. Biophys. Acad. Sci. Hung. 4, 219236. 2. NAKAMARU, Y., ANDKONISHI, K. (1968) Biochim. Biophys. Acta 159, 206-208. 3. SCHWARTZ, A., BACHELAARD,H., ANDMCILWAIN, H. (1962) Biockem. J. 84, 626-637. 4. CHEN, P., TORIBARA, T., AND WARNER, H. (1956) Anal.
Chem.
28, 1756-1758.
5. SIEGEL, F., AND ALBERS, R. (1967)5. Biol. Ch,em. 242, 4972-4979. 6. MARTIN, J., AND DOTY, D. (1949) Anal. Chem. 21, 965-967. 7. LOWRY, O., ROSEBROUGH, N., FARR, A., AND RANDALL, R. (1951) J. Biol. Chem. 193, 265-275. 72, 2488. BRADFORD, M. (1976) Anal. Biochem. 254. 9. WEBER, K., AND OSBORN, M. (1969) J. Biol. Chm. 244, 4406-4412. 10. FIESER, L., AND FIESER, M. (1959) Steroids, Reinhold, New York. 11. GANTZER, M., AND GRISHAM, C. (1979) Arch. Biochem. Biophys. 198, 000-000. 12. ABRAMS, A., AND BARON, C. (1967) Biochemistry 6, 225. 36, 13. ALBERS, R. W. (1967) Annu. Rev. B&hem. 727. Chem. 14. HASSELBACH, W. (1964) Progr. Biophys, 14, 167-222.