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NATHAN NELSON*, BARUCH I. KANNERt, AND DAVID L. GUTNICKt. * Department of ..... Butlin, J. D., Cox, G. B. & Gibson, F. (1971) Biochem. J. 124, 75-81. 4.
Proc. Nat. Acad. Sci. USA Vol. 71, No. 7, pp. 2720-2724, July 1974

Purification and Properties of Mg2+-Ca2+ Adenosinetriphosphatase from Escherichia coli (sulfhydryl reagent/catalytic subunits/antibody)

NATHAN NELSON*, BARUCH I. KANNERt, AND DAVID L. GUTNICKt *

Department of Biology, Technion, Haifa, Israel; and t Department of Microbiology, Tel-Aviv University, Ramat-Aviv, Israel

Communicated by Leon Heppel, April 17, 1974, ABSTRACT A procedure for the purification of Mg2+Ca2+ adenosinetriphosphatase (EC 3.6.1.3) from E. coli, yielding relatively large amounts of highly active enzyme, is described. The enzyme consists of four nonidentical subunits. Trypsin treatment of purified enzyme yields a preparation consisting exclusively of the two larger subunits, which are sufficient for ATPase activity. Purified enzyme is inhibited by 7-chloro-4-nitrobenzo-2-oxa-1,3diazole; this inhibition is reversed by dithiothreitol, and the diazole is found preferentially associated with the 13-subunit of the enzyme. Antibody prepared against the trypsin-treated enzyme inhibited various ATP-dependent reactions as well as membrane-bound ATPase itself.

Regular gel electrophoresis was performed as follows: 50 mM Tris-glycine buffer (pH 8.7) was used for both chambers. Preparation of the gels involved mixing of 11.1 ml of 0.1 M Tris-glycine (pH 8.7) 5 ml of H20 (for 5% gels), 5 ml of acrylamide solution (22.2 g of acrylamide and 0.6 g of methylenebisacrylamide in a final volume of 100 ml of H20), 1.1 ml of freshly prepared ammonium persulfate (15 mg/ml), and 30 Al of tetraethylmethylene diamine. The mixture was poured into 12 tubes (0.5 cm X 7 cm). It was covered with a few millimeters of water and allowed to polymerize for 2 hr. The tubes were first run at a constant current of 2 mA per tube for 1 hr, and the samples were run under the same conditions. The samples, up to 50 Ml, were added in 10% (w/v) sucrose or glycerol solutions. The gels were fixed, stained, and destained as described for the DodSO4- gels. 3H-Labeled 7-chloro-4-nitrobenzene-2-oxa-1,3-diazole (diazole) was a generous gift from Dr. D. W. Deters (Cornell University); the procedure for its preparation will be described t. The preparation of antibody and of DEAE-cellulose (Whatman DEII) was performed as described (18). Freund's Bacto adjuvant (complete form) was obtained from Difco Laboratories. The diazole was purchased from Pierce Chemical Co. Sodium dodecyl sulfate, acrylamide, bisacrylamide, ammonium persulfate, tetraethylmethylene diamine, and Biogel A 0.5m were obtained from Bio-Rad. All other materials were of the highest purity commercially available.

Recent studies using mutants of Escherichia coli K12, have demonstrated a role for the membrane Mg2+-Ca2+ adenosinetriphosphatase (EC 3.6.1.3) in energy-transducing processes (1-8). Bragg and Hou (9), Kobayashi and Anraku (10), and Hanson and Kennedy (11) have reported purification procedures for the enzyme from E. coli. The results of these studies, together with those using crude solubilized and membrane-bound enzyme (12, 13), indicate a similarity between the E. coli coupling factor and coupling factor 1 from chloroplasts or factor 1 from mitochondria. This communication describes a simple purification procedure, yielding relatively large quantities of a highly active preparation of the ATPase from E. coli. In addition, heretofore unreported properties of the ATPase, such as inhibition of the enzyme by 7-chloro-4-nitrobenzo-2-oxa-1,3-diazole and isolation of catalytically active subunits by treatment with trypsin, are reported.

RESULTS

amino-6-chloro-2-methoxyacridine (15) were assayed by published methods. Protein was determined by the procedure of Lowry (16), with bovine serum albumin as a standard. Gel electrophoresis in the presence of sodium dodecyl sulfate (DodSO4-) was performed according to Weber and Osborn (17). The procedures for fixation, staining, and destaining of the gels were described (18).

Purification and Characterization of the A TPase. About 280 g, wet weight, of E. coli K12, strain A428, were washed with 0.1 M Tricine * NaOH-0.1 M MgCl2-6 mM 2 mercaptoethanol60 mM NH4C1 (at pH 7.8) and resuspended in 800 ml of the same buffer. The cells were broken in a French pressure cell at 20,000 lbs./inch2, and the unbroken cells and debris were removed by centrifugation at 8000 X g for 10 min. The cell extract was centrifuged at 40,000 X g for 2 hr, and the pellet was homogenized in 175 ml of 0.4 M sucrose-10 mM Tricine10 mM NaCl(at pH 8.0) and stored at -70°. These frozen membranes were thawed and mixed with half the volume of a solution of protamine sulfate (2 mg/ml). After 20 min, the mixture was centrifuged at 40,000 X g for 50 min and the resulting pellet was resuspended in sucrose-Tricine-NaCl (100 ml). These steps were performed at 40; all subsequent steps

Abbreviations: Diazole, 7-chloro-4-nitrobenzo-2-oxa-1,3-diazole; DodSO4-, sodium dodecyl sulfate.

t D. W. Deters, E. Racker, H. Nelson, and N. aration.

MATERIALS AND METHODS E. coli K12, strain A428, was grown and harvested as described

(2). The growth medium consisted of Bacto Tryptone (1%), yeast extract (0.5%), and NaCl (1%) (all w/v). ATPase activities were measured with [y32-P]ATP as described (14). ATP-driven and respiration-driven transhydrogenase and ATP- and respiration-driven quenching of fluorescence of 9-

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in prep-

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/3

a

""T ".

b

TABLE 1. Purification of E. coli Mg2+-Caa2+ ATPase Specific

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a

activity Preparation

Protein Activity (units/mg (mg) (units*) of protein)

3.1 Starting membrane preparation 12,800 40,000 14.5 2,075 30,000 EDTA supernatant fractions 40 225 9,000 DEAE-cellulose peak fractions 50-70 113 6,500 Bio-Gel A 0.5m peak fractions 35 4,300 100-150 Sucrose gradient fractions *

One unit is defined is 1 jumole of Pi released per min.

were performed at room temperature. The suspension was diluted 20-fold with a solution containing 2 mM EDTA (pH 7.3), and after 10 min it was centrifuged at 35,000 X g for 20 min. The supernatant was saved and the pellet was resuspended in a minimal volume of sucrose-Tricine-NaCl, treated with EDTA, and centrifuged again. The two supernatant fractions were pooled. For each liter of supernatant, 25 ml of 2 M Tris HCl (pH 8), 10 ml of 0.1 M ATP, and glycerol (final concentration) 10% v/v were added. This solution was applied to a column of DEAE-cellulose (2 X 55 cm), previously equilibrated with 50 mM Tris HCl-2 mM EDTA-10% glycerol-1 mM ATP (at pH 7.8). After application, the column was washed with 500 ml of the above buffer and the ATPase was eluted by a gradient of 50-750 mM Tris HCl (pH 8) in a buffer containing 2 mM EDTA, 1 mM ATP, and 10% glycerol (400 ml in each chamber). The most active fractions were pooled, and 428 mg/ml of ammonium sulfate were added to a final concentration of 65% saturation. The resulting suspension was centrifuged immediately at 10,000 X g for 10 min, and the resulting precipitate dissolved in a minimum volume (5-10 ml) of 50 mM Tricine * NaOH-2 mM EDTA-10% glycerol-1 mM ATP (at pH 7.8). This suspension became turbid after a few minutes, and the insoluble material was removed by centrifugation at 10,000 X g for 10 min. The supernatant was applied to a column of BioGel A 0.5m (1.25 X 100 cm) equilibrated with Tricine-EDTAglycerol-ATP. The column was eluted with the same buffer at a rate of one drop per min, and fractions of 3 ml were collected. The most active fractions were applied to a sucrose gradient (1 ml of Bio-gel fraction per tube) of 5-15% in Tricine-EDTA-glycerol-ATP. The gradients were run at 150 in the SW-27 rotor at 96,000 X g for 20 hr. Fractions of 1.5 ml were collected, and those exhibiting specific activities exceeding 100 ,moles of Pi released per min/mg of protein were stored at -70°. The purified enzyme could be stored for as long as 4 months with no significant loss in activity. The purification of ATPase is summarized in Table 1. A 50-fold purification of the enzyme was achieved by this procedure. Gel electrophoresis (Fig. lc) indicates that the purified enzyme is nearly homogeneous, and as judged by electrophoresis in DodSO4- (Fig. la and b), the enzyme consists of four nonidentical subunits (a, f, y, and e). Based on the intensity scan of the DodSOc4 gels and the corresponding molecular weights of the subunits (11), the ratio of a:,B:y in this enzyme is about 2:2: 1. The specific activity of the purified enzyme varies as a function of both magnesium and ATP concentration (Fig. 2). The optimal ratio ATP: Mg2+ is about 2: 1, and the enzyme is inhibited when the concentration of magnesium is increased. -

...

-

"I

U

c

FIG. 1. (a) The DodSO4c gel electrophoresis pattern of purified ATPase. ATPase (30,g) was applied and electrophoresis was performed (17). After it was stained and destained, the gel was scanned at 600 nm in an attachment of the Gilford spectrophotometer. 7.5% gel was used. (b) The DodSO4- gel electrophoresis pattern of purified ATPase. (c) Regular gel electrophoresis of purified ATPase. ATPase (20 ug) was applied and electrophoresis was performed as described in Materials and Methods.

The Km for ATP, when for each ATP concentration the optimal magnesium concentration was used, was 0.25 mM. Inhibition of the enzyme activity by ADP is illustrated in Fig. 3. Similar results have been obtained by other authors (10-12). In addition, we have confirmed the inhibitory action on the enzyme activity by sodium azide and Pi (11, 12). Inhibition of ATPase by Diazole. The sulfhydryl reagent, 2-chloro-4-nitrobenzo-2-oxa-1,3-diazole, inhibits the ATPase activity of factor 1 and coupling factor 1 T. Two to three molecules of diazole per molecule of enzyme were sufficient to inhibit these coupling factors, and the inhibited enzyme could be reactivated by the inclusion of dithiothreitol during the assay. As illustrated in Fig. 4, ATPase from E. coli was sensitive to the diazole, and the inhibition was reversed by dithiothreitol. It was of interest to determine if preferential binding occurred to one of the subunits. Purified Mg2+-CaO+ ATPase was incubated with [3H]diazole and subjected to electrophoresis on DodSO4- gels (in the absence of mercaptoethanol). Subsequent counting of each of the bands demonstrated that most of the counts appeared in the , subunit (Table 2). Similar results were reported for factor 1 and coupling factor 1 1.

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200

0~~~~~~~

500

w

100

CD

E

01 0

* 2

4

50

C

0.1

0.2

I

0.5

2

4

6

mM MgCI,

FIG. 2. The effect of magnesium concentration on the ATPase activity of purified Mg2t-Ca2+ ATPase. The assay was performed as described in Materials and Methods. For each assay, 0.16 Mg of purified ATPase was used. (0) 4 mM ATP; (A) 2 mM ATP; (o) 1 mM ATP; (0) 0.5 mM ATP.

Preparation of the Subunits of A TPase Responsible for A TPActivity. Prolonged treatment of coupling factor 1 with trypsin, followed by chromatography on an agarose column, yielded a preparation of active ATPase consisting exclusively ase

100

0

6 8 WA DIAZOLE

10

12

FIG. 4. The inhibition of ATPase activity of Mg'+-Ca"+ ATPase by diazole and its reversal by dithiothreitol. A series of tubes containing 72 Mg of purified enzyme in 0.1 ml (0) was incubated with increasing concentrations of diazole (dissolved in dimethylsulfoxide). The tubes were incubated in the dark for 24 hr and subsequently assayed. Purified enzyme (0.7 Mg) was used for the assay described in Materials and Methods. An identical amount of enzyme was assayed in the presence of 1.5 mM dithiothreitol (0).

of the two largest subunits (a + ,B) . A similar preparation of a and ,B subunits was isolated after trypsin treatment of the purified Mg2+-Ca2+ ATPase (Fig. 5). This preparation retained most of the ATPase activity of the native enzyme, and was inhibited by ADP. Antibody that was prepared against the purified a and mixture inhibited the ATPase activities of the native enzyme, the membrane-bound Mg2+-Ca2+ ATPase (Fig. 6), as well as the ATPase activity of the trypsin-treated Mg2 +-Ca2+ ATPase. In addition, the ATP-driven transhydrogenase (Table 3) and ATP-driven quenching of fluorescence of 9-amino-6-chloro-2-methoxyacridine (not shown) were also

TABLE 2. Binding of 1PH]diazole to the subunits of Mg2+-Ca2+ ATPase

0

75

0

Subunit 0

a

0

16 -y

Total countst 25-

0.5

ID mM

1.5

ADP

2D

FIG. 3. The effect of ADP concentration on the ATPase activity of purified Mg2' -Ca2+ ATPase. The assay was performed as described in Materials and Methods. Specific activity of the controls: 0.5 mM ATP, 30 units/mg of protein; 1 mM ATP, 33 units/mg of protein; 2 mM ATP, 58 units/mg of protein; 4 mM ATP, 139 units/mg of protein. One unit is defined as 1 umole of Pi released per min. For each assay, 0.1ie ,g of purified enzyme was used. (0) 4 mM ATP; (A) 2 mM ATP; (0) 1 mM ATP; (0)0.5 mM ATP.

cpm* 29

~~~~~~83 7

840

Purified enzyme (72 Mg) was incubated in a volume of 0.1 ml with 11 ul of ['H]diazole dissolved in dimethylsulfoxide. The final concentration of [3H]diazole was 10 MAM. After 24 hr of preincubation, 20 ul of 10% DodSO4 were added and the incubation was continued for another 30 min. DodSO4 gels were run with samples containing 18 ,ug of protein. Gel electrophoresis was performed for 4 hr on a 7.5% gel. The gels were stained for 30 min and destained for 4 hr. The gels were sliced with a width of 3 mm, and the slices were incubated in 1 ml of Soluene-100 for 60 min at 600. The samples were subsequently dissolved in toluene scintillation fluid and counted in a liquid scintillation counter. * The background (slices cut from positions without bands) was 20 cpm and was the same irrespective of the position from which the gel was sliced. t cpm of the 20-M1 sample.

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a c

I-Q

0. Is

5

0

15

2

p1i of SERUM

-

i

..I.M M

FIG. 5. (a) The DodSOg4 gel electrophoresis pattern of +-Ca2+ ATPase. Protein (15 ug) was applied and processed as described in the legend of Fig. la, except that 10% gel was used. (b) The DodSO4- gel electrophoresis pattern of trypsin-treated enzyme.

FIG. 6. Effect of antibody against the catalytic subunits of E. coli ATPase (a and ,3) on the ATPase activity of membranebound Mg2t-Ca2+ ATPase. Membrane particles of E. coli K12, strain A428, were prepared as described (2) and assayed for ATPase activity. In this series of experiments, no [.y-32P]ATP was present and the released inorganic phosphate was assayed as described (2). During the assay, 2.4 mM phosphoenolpyruvate and 30 Aug of pyruvate kinase were present. For each assay, 190 ,ug of particle protein was used.

trypsin-treated Mg2

inhibited by antibody prepared against the and (3subunits. The respective respiration-driven reactions were unaffected. The procedure used was as follows: 6 ml of purified enzyme at 0.4 mg/ml in Tricine-EDTA-glycerol-ATP and about 10% sucrose was incubated at room temperature for 5 hr with 0.15 ml of TPCK-trypsin (5 mg/ml). This mixture was then diluted 1:1 with distilled water. Solid ammonium sulfate (428 mg/ml) was added to give 65% saturation, and the suspension was centrifuged at 20000 X g for 10 min, The pellet was dissolved in the buffer and applied to a BioGel AO.5m column (1.25 X 100 cm) equilibrated with the buffer. The column was run with the same buffer at a rate of one drop per min. The peak fractions were stored at -70°. a

and Kennedy (11) as well as the ATPase purified by our procedure. In this regard, the ATPase was not able to restore respiration- and ATP-driven quenching of aminochloromethoxyacridine fluorescence to suitable depleted particles, in which the activities were stimulated by the crude coupling factor (B. Kanner, unpublished observations). The sulfhydryl reagent, diazole, exerts a similar effect on factor 1, coupling factor 1, and Mg2+-Ca2+ ATPase. Only about 10% of the input radioactivity from the [3H]diazole was subsequently recovered in the denatured subunits of the enzyme. Nevertheless, this labeling appeared to be specific for the , TABLE 3. Effect of antibody against the catalytic subunits of E. coli A TPase (a and a) on the respiration- and ATP-driven transhydrogenase in membrane preparations of E. coli

DISCUSSION

Bragg and Hou (9) reported the purification of E. coli ATPase with a specific activity of 35 units per mg of protein, consisting of five different subunits. The preparation of Hanson and Kennedy (11) consisted of four different subunits on DodSO4gels, but with very low specific activity. Mg2t-Ca2+ ATPase

purified by the procedure described in this report has a specific activity of about 150 units per mg of protein, and also consists of four subunits. Trypsin treatment of the enzyme gives an active preparation consisting exclusively of subunits a and fB. These two subunits are therefore sufficient for catalytic activity of the enzyme. A similar property has been described for coupling factor 1 1. Bragg et al. (19) have recently shown that the elution of ATPase from a gel after electrophoresis yielded a preparation incapable of reconstituting respiration- and ATPdriven transhydrogenase in suitably depleted membrane particles from E. coli. It is of interest that this preparation was devoid of the a subunit, resembling the preparation of Hanson

Addition of serum (,l) None Control serum 50 100 Anti-(a and 0) 13 27 50 100

Transhydrogenase* RespirationATP-driven driven 25.4 27.9 26.5 25.2

25.A 25.4

27.9

7.6 3.8 2.5 0

24.1 24.1

26.4

Membrane particles of E. coli K12, strain A428, were prepared as described (2) and assayed for respiration- and ATPdriven transhydrogenase (20). For each assay, 0.95 mg of particle protein was used. The sera were included during the preincubation period. * Expressed as nmoles of NADPH formed per min/mg of protein.

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subunit, and may indicate that the catalytic site is located on the 3t subunit. Deters et al. t have discussed the implications of the effect of diazole on the ATPase activity of coupling factor 1.

A large body of evidence has now been accumulated emphasizing the striking similarity of the bacterial ATPase and coupling factors from subcellular organelles. It is interesting to note that both the subunit composition as well as the relative amounts of the subunits in the various enzymes are similar. The fact that E. coli. ATPase appears to require a minimum of four different cistrons leading to the synthesis of different numbers of subunit polypeptide molecules suggests an interesting question concerning the regulation of either synthesis or assembly of the enzyme. This work was supported by the D. Lou Harris Memorial Fund through the American Friends of Avihail Cultural Center, Inc. We thank Mr. F. J. Mallet for his support to this project. 1. Kanner, B. I. & Gutnick, D. L. (1972) J. Bacteriol. 111, 287-289. 2. Kanner, B. I. & Gutnick, D. L. (1972) FEBS Lett. 22, 197-199. 3. Butlin, J. D., Cox, G. B. & Gibson, F. (1971) Biochem. J. 124, 75-81. 4. Cox, G. B., Newton, N. A., Butlin, J. D. & Gibson, F. (1971) Biochem. J. 125, 484-493.

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5. Gutnick, D. L., Kanner, B. I. & Postma, P. W. (1972) Biochim. Biophys. Ada 283, 217-222. 6. Butlin, J. D., Cox, G. B. & Gibson, F. (1973) Biochim. Biophys. Ada 292, 366-375. 7. Schairer, H. U. & Haddock, B. A. (1972) Biochim. Biophys. Res. Commun. 48, 544-551. 8. Schairer, H. U. & Gruber, D. (1973) Eur. J. Biochem. 37, 282-286. 9. Bragg, P. D. & Hou, C. (1972) FEBS Lett. 28, 309-312. 10. Kobayashi, H. & Anraku, Y. (1972) J. Biochem. 71, 387-399. 11. Hanson, R. L. & Kennedy, E. P. (1973) J. Bacteriol. 114, 772-781. 12. Roisin, M. P. & Kepes, A. (1973) Biochim. Biophys. Ada 305, 249-259. 13. Giordano, G., Riviere, C. & Azoulay, E. (1973) Biochim. Biophys. Ada 307, 513-524. 14. Nelson, N., Nelson, H. & Racker, E. (1972) J. Biol. Chem. 247, 6506-6510. 15. Nieuwenhuis, F. J. R. M., Kanner, B. I., Gutnick, D. L., Postma, P. W. & Van Dam, K. (1973) Biochim. Biophys. Acta 325, 62-71. 16. Lowry, 0. H., Rosebrough, N. J., Farr, A. L. & Randall, R. J. (1951) J. Biol. Chem. 193, 265-275. 17. Weber, K. & Osborn, M. (1969) J. Biol. Chem. 244, 44064412. 18. Nelson, N., Deters, D. W., Nelson, H. & Racker, E. (1973) J. Biol. Chem. 248, 2049-2055. 19. Bragg, P. D., Davies, P. L. & Hou, C. (1973) Arch. Biochem. Biophys. 159, 664-670. 20. Fisher, R. J. & Sanadi, D. R. (1971) Biochim. Biophys. Ada 245, 34-41.