/ . Biochem., 81, 477-483 (1977)
Purification and Characterization of Inorganic Pyrophosphatase from Thiobacillus thiooxidans Noriko TOMTNAGA* and Takeshi MORI** •Biological Institute, Faculty of Science, Nagoya University, Chikusa-ku, Nagoya, Aichi 464, and **Japan University of Social Welfare, Showa-ku, Nagoya, Aichi 464 Received for publication, July 9, 1976
An inorganic pyrophosphatase [EC 3.6.1.1] was isolated from Thiobacillus thiooxidans and purified 975-fold to a state of apparent homogeneity. The enzyme catalyzed the hydrolysis of inorganic pyrophosphate and no activity was found with a variety of other phosphate esters. The cation Mgt+ was required for maximum activity; Co3+ and Mn1+ supported 25 per cent and 10.6 per cent of the activity with Mg t+ , respectively. The pH optimum was 8.8. The molecular weight was estimated to be 88,000 by gel filtration and SDS gel electrophoresis, and the enzyme consisted of four identical subunits. The isoelectric point was found to be 5.05. The enzyme was exceptionally heat-stable in the presence of 0.01 M Mgt+.
Thiobacillus thiooxidans, a gram-negative and chemolithotrophic bacterium, uses carbon dioxide as a carbon source and derives energy from the oxidation of elemental sulfur, making the culture medium extremely acidic (below pH 1.0). Enzymes relatively specific for the hydrolysis of inorganic pyrophosphate are widely distributed in nature. While extensive investigations of inorganic pyrophosphatase [EC 3.6.1.1] have been carried out with heterotrophs, yeast {1-10) and Escherichia coli (11-13), the isolation and purification of this enzyme from a chemolithotrophic bacterium was firstly reported with Ferrobacillus ferrooxidans (14). The occurrence of activity hydrolyzing PPi was also reported in our T. thiooxidans strain (15). The purification and some properties of inorganic pyrophosphatase of T. thiooxidans are described here.
Vol. 81, No. 2, 1977
477
MATERIALS AND METHODS Microorganism—T. thiooxidans was cultured and harvested as described previously (76). Chemicals—Tetrasodium pyrophosphate was purchased from Hayashi Pure Chemicals. ATP and ADP were obtained from Kojin Co. AMP, bovine serum albumin, and ovalbumin were from Sigma Co. ^-Globulin was purchased from Katayama Chemical Industries Co. Glyceraldehyde phosphate dehydrogenase [EC 1.2.1.12] and aldolase [EC 4.1.2.13] were obtained from Boehringer Mannheim Co. Crystalline cryptocytochrome c from Pseudomonas denitrificans was kindly supplied by Dr. Iwasaki of our laboratory. Measurement of Enzyme Activity—Inorganic pyrophosphatase assays were conducted for 20 min at 30°; the assay system contained 4 [imo\ of Na4P,O7, 100 (imo\ of glycine-NaOH buffer (pH 8.8), 10 /*mol of MgCl,, and enzyme in a total
478
volume of 2 ml. The reaction was stopped by the addition of 0.5 ml of 30% trichloroacetic acid. Any precipitate formed was removed by centrifugation and then the supernatant was used for the determination of Pi. A unit of pyrophosphatase activity was defined as the quantity of enzyme catalyzing the liberation of 1 ^mol of Pj per min under the conditions specified. Specific activity was defined as enzyme units per mg protein. Analytical Methods—Pi was determined colorimetrically by the method of Allen (77) as modified by Nakamura (18). Protein was determined by the method of Lowry et al. (19), using bovine serum albumin as a standard. The molecular weight of the enzyme was estimated by the gel filtration technique of Andrews (20). A Sephadex G-150 column (2.4 x90 cm) was equilibrated with 0.025 M Tris-HCl buffer (pH 7.5). Protein was eluted with the same buffer at a flow rate of 25 ml/h. The isoelectric point of pyrophosphatase was determined by isoelectric focusing with carrier ampholyte giving a pH gradient between 3 and 10 (LKB Produkter AB, Sweden) at a mean concentration of 1 %. Focusing was carried out at 4° in a special vertical electrophoresis column (2 x 24 cm). Stabilization against convection was achieved with a sucrose density gradient of 0-50 %. After focusing, the column was drained and fractions of 2 ml were collected. The method of Davis (2/) was used for polyacrylamide gel electrophoresis and the power supply was adjusted to 3 mA per tube. The protein bands were stained with 0.05% Coomassie blue in 12.5% TCA. For histochemical analysis of the pyrophosphatase activity in polyacrylamide gel, the gel was incubated for 10 min at 30° in the reaction mixture and rinsed with water. The color of liberated Pi was developed by applying Nakamura's reagents. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis was performed according to the method of Weber and Osborn (22), using 10% gels.
N. TOMINAGA and T. MORI Pyrophosphatase activity was easily extracted by sonication of T. thiooxidans cells with 0.1 M Tris-HCl buffer (pH 7.5), and more than 90 % of the activity of cell-free extract was found in the supernatant fluid after centrifugation for 1 h at 105,000 X g. The cell-free extract showed more pyrophosphatase activity than did intact cells, which exhibited only about 5 % of the activity of the extract. Lyophilized cells (18 g dry wt.) were suspended in 720 ml of 0.1 M Tris-HCl buffer (pH 7.5) and disrupted for 15 min in a 10 kc sonic oscillator (Kubota KMS 100) at 0°. After centrifugation for 30 min at 21,000 xg, the precipitate was resuspended in 350 ml of the same buffer and sonicated again. The combined supernatant was used as cell-free extract. The fraction precipitated between 55 and 90% ammonium sulfate saturation was dissolved in 100 ml of distilled water and dialyzed overnight against 10 liters of distilled water. The dialysate was applied to a DEAE-cellulose column (4.2 x 5.5 cm) previously equilibrated with 0.05 M Tris-HCl buffer (pH 7.5) (buffer A). After addition of the sample, the column was washed with buffer A (approx. 200 ml); this treatment removed pigments containing soluble cytochrome c (23). The enzyme was then eluted with a linear gradient of ammonium sulfate (0-0.3 M) in .buffer A. The active fractions were collected, diluted with 3 volumes of buffer A and again fractionated on a DEAE-cellulose column. The conditions were the same as those of the first chromatography except for column size (3x5 cm). The active fractions were diluted with an equal volume of buffer A and loaded on a hydroxylapatite column (4.2x3 cm) equilibrated with buffer A. The column was washed with 100 ml of buffer A containing 0.25 M ammonium sulfate and then developed with a linear gradient of ammonium sulfate (0.25-0.75 M) in buffer A.
Fractions containing enzyme activity were diluted with 2 volumes of buffer A and again applied to a hydroxylapatite column (4 x 1 cm) equilibrated with buffer A. After washing with 50 ml of buffer A containing 0.25 M ammonium sulfate, the enzyme was eluted with buffer A containing 0.75 M ammonium sulfate and dialyzed overnight against 3 liters RESULTS of 0.025 M Tris-HCl buffer (pH 7.5). Purification of Pyrophosphatase—Unless otherThe enzyme was further purified by isoelectric wise indicated, all procedures were carried out in a focusing. The elution profile is illustrated in Fig. 1. cold room at 4-5°. Fraction number 12 was dialyzed overnight against /. Biochem.
INORGANIC PYROPHOSPHATASE FROM T. thiooxidans
479
12
-.400 c-
300
200
100 I Q.
O tr 20 30 40 FRACTION NUMBER
10
50
60
Fig. 1. Isoelectric fractionation of inorganic pyrophosphatase The active fractions (2.7 mg protein) obtained from the second hydroxylapatite column chromatography were dialyzed against 0.025 M Tris-HCl buffer (pH 7 5) then subjected to isoelectric focusing at 900 volts for 49 h at 4°, as described in "MATERIALS AND METHODS." , optical density at 280nm; - - O - - , pyrophosphatase activity; , pH. TABLE I. Purification of inorganic pyrophosphatase of T. thiooxidans. Total volume (ml) Crude extract Ammonium sulfate DEAE-cellulose I DEAE-cellulose II Hydroxylapatite I Hydroxylapatite II Electrofocusing (No. 12)
1,000
Total activity1 (units)
31
6,108 5,450 5,316 4,561 4,433 1,244
7
652
140 135 104 130
Total protein (mg) 7,400 1,232 297 149
38.35 5.425 0.812
Specific activity (units/mg protein) 0.825 4.424 17 90 30.62 115.6 229.4 804.1
Yield (%) 100
89.2 87.0 74.7 72.6 20.4 10.7
Assay conditions are described in "MATERIALS AND METHODS."
2 liters of 0.025 M Tris-HCl buffer (pH 7.5) and stored at 0°. The process of purification, in which the enzyme was purified about 975-fold with a recovery of 10.7 % over the crude extract activity, is summarized in Table I. The enzyme preparation gave a single protein band on polyacrylamide gel electrophoresis at pH 9.4 and the locus of inorganic pyrophosphatase activity coincided exactly with the protein band (Fig. 2A). Vol. 81, No. 2, 1977
The purified enzyme was stable at 0° for more than nine months. Properties of Inorganic Pyrophosphatase—The molecular weight of the enzyme was estimated to be approximately 88,000 by gel filtration (Fig. 3). The subunit molecular weight of the enzyme was estimated by SDS-polyacrylamide gel electrophoresis with SDS-denatured ^-globulin, bovine serum albumin, ovalbumin, aldolase, and glyceraldehyde phosphate dehydrogenase as reference standards. The inorganic pyrophosphatase was
N. TOM1NAGA and T. MORI
480
50 Bovine serum albumin LU
345
7-Globulin H chain Ovalbumin Aldolase tf Glyceraldehyde phosphate dehydrogenase /-Globulin L chain Inorganic pyrophosphatase
CD O
Fig. 2. Disc gel electrophoresis of the purified inorganic pyrophosphatase. (A) Electrophoresis was carried out for 50 mm at 3mA per tube on 7.5% polyacrylamide gel using 1 fig (a) and 0.25 pg (b) of protein, (a) The gel was stained with Coomassie blue, (b) The gel was incubated in the reaction mixture for pyrophosphatase assay and inorganic phosphate released was detected with Nakamura's reagents (7S). (B) Electrophoresis of 1 fig of the enzyme was earned out for 4 h at 8 mA per tube on 10% polyacrylamide gel containing 0.1% SDS. The protein band was stained with Coomassie blue.
300
250 z o
02
04
0.6
0.8
10
MOBILITY
Fig. 4. Estimation of subunit molecular weight by SDS-polyacrylamide gel electrophoresis. Electrophoresis was earned out on 10% polyacrylamide gel containing 0.1% SDS according to Weber and Osborn (22).
Cryptocytochrome c (Ps. denrtnftcans) j Ovalbumin ^Bovine serum albumin Inorganic pyrophosphatase
2
200
45 50 LOG MOLECULAR WEIGHT
4
6
8
10
CONCENTRATION OF M g 2 + (mM)
Serum albumin dimer 150 40
40
55
Fig. 3. Molecular weight estimation of inorganic pyrophosphatase by Sephadex G-150 gel filtration. A Sephadex G-150 column (2.4x90 cm) was equilibrated with 0.025 M Tris-HCl buffer (pH 7.5). Cryptocytochrome c, ovalbumin, and serum albumin (molecular weights, 26,400, 45,000, and 67,000, respectively) were cluted with the same buffer at a flow rate of 25 ml/h. found to be composed of only one species of polypeptide (Fig. 2B), the molecular weight of which was estimated to be approximately 20,000 (Fig. 4). Thus, the inorganic pyrophosphatase may consist of four identical subunits. The isoelectric point of the enzyme, as determined by isoelectric focusing, was found to be 5.05 (Fig. 1).
Fig. 5. Effect of magnesium on inorganic pyrophosphatase. Assays were performed using the following incubation mixture (2.0 ml); 2 mM sodium pyrophosphate, 50 mM glycine-NaOH buffer (pH 8.8), magnesium chloride and purified enzyme (0.08 fig protein). For measurement of the pH dependence of activity, Tris-maleate buffer was used from pH 5.9 to 8.25 and glycine-NaOH buffer from pH 8.2 to 9.5 at a concentration of 0.05 M. The enzyme had a pH optimum of 8.8 and the activities were 4.5 % and 72.2% of optimum at pH 5.9 and 9.5, respectively. There was no detectable activity in the absence of divalent cations. Of several chloride salts (5 mM) tested, Mg1+ was required for maximum activity; Co1+ and Mn t+ supported 25 % and 10.6% of the activity with Mg t+ , respectively. Ca l+ , Zn1+, Cu1+, and Hg1+ were not effective. As can be seen in Fig. 5, the optimal concentration of Mg1+ J. Biochem.
481
INORGANIC PYROPHOSPHATASE FROM T. thiooxidans TABLE n . Substrate specificity of inorganic pyrophosphatase. The substrate concentration was 2 nw. The enzyme activities were calculated from the amounts of Pi liberated from various substrates under the assay conditions. Relative activities are expressed as percentages of those for PPi. Relative rate of hydrolysis
Substrates PPi
100 4.2 2.6
ATP ADP AMP ar-Glycerophosphate p-Nitrophenyl phosphate Glucose 6-phosphate Thiamine pyrophosphate
0 0 0.4 0 0
50 70 90 100 TEMPERATURE C )
TABLE III. Effects of inhibitors. The standard assays described in "MATERIALS AND METHODS" were carried out with addition of the inhibitor. Concentration % Inhibition (HIM) NaF KCN Iodoacetamide N-Ethylmaleimide p-Chloromercuribenzoate NaN,
1.25 5 5 5 1 1
Guanidine-HCl
5
81.3 20.9 14.8 10.1 3 11.6 22.9
was approximately the same as that of the substrate (2 ITIM), suggesting that the metal ion was required in a stoichiometnc relationship with respect to PPi anion. The initial rates of hydrolysis of various phosphate esters by the purified enzyme are summarized in Table II. Under the conditions employed, the enzyme was found to be highly specific for inorganic pyrophosphate. The effects of various compounds on enzyme activity are summarized in Table III. The activity was strongly inhibited by NaF. KCN, azide, guanidine-HCl, and sulfhydryl reagents caused less than 25 % inhibition. Figure 6 shows the thermal inactivation profile of pyrophosphatase; the enzyme was heated at Vol. 81, No. 2, 1977
Fig. 6. Thermal inactivation of inorganic pyrophosphatase. The enzyme obtained from the second hydroxylapatite column chromatography (0.07 mg protein) was incubated in either 0.025 M Tris-HCl buffer (pH 7.5) ( • ) or 0.025 M Tris-HCl buffer (pH 7 5) containing 0.01 M MgCli (O) for 10 min at the indicated temperatures. After cooling on ice, the enzyme solution was diluted 25-fold with distilled water and pyrophosphatase activity was measured as described in "MATERIALS AND METHODS." various temperatures for 10 min. The enzyme was quite resistant to heat treatment. For example, in the presence of 0.01 M Mg 1+ , 99% of the activity remained after 10 min at 80° and 4 0 % at 100°. Although Marunouchi and Mori (15) reported, that sulfite slightly stimulated the pyrophosphatase activity, the purified enzyme was not stimulated by sulfite ions. The following salts had slightly inhibitory effects (2-12%) at a concentration of 20 ITIM; NaCl, KC1, NaHCO,, Na a SO,, Na,SO 4 , Na s SeO,, and K,CrO 4 . DISCUSSION Inorganic pyrophosphatase from T. thiooxidans has been purified to a state of apparent homogeneity. The findings that the intact cells exhibit only a small fraction of the total activity obtained in the cell-free extract and that 90 % of the enzyme activity of crude extract was found in the 105,000 x g supernatant suggest that this enzyme is present in the cell cytoplasm, as are those of E. coli (11) and F. ferrooxidans (14).
482
N. TOMINAGA and T. MORI
The molecular weight of the enzyme was estimated to be approximately 80,000-88,000. This value is different from molecular weights determined for pyrophosphatases from E. coli (13) and Bacillus stearothermophilis (24) (about 120,000) as well as those from yeast (3, 5, 6) and Bacillus subtilis (25) (about 70,000). Electrophoresis on SDS-polyacrylamide gel indicated that the inorganic pyrophosphatase from T. thiooxidans consists of four identical subunits with a molecular weight of approximately 20,000. The subunit structure of this enzyme was different from those reported for other microorganisms. The yeast enzyme consisted of two identical subunits with a molecular weight of 33,000-35,000 (3, 5), while the B. stearothermophilis enzyme also consisted of two subunits but its molecular weight was 70,000 (24). On the other hand, the enzyme from E. coli had six identical subunits with a molecular weight of 20,000 (7).
acid phosphatase [EC 3.1.3.2] which also hydrolyzes PPi at a considerable rate in the presence of sulfate ions and in the absence of Mg1+ at acidic pH (27). Although the metabolic roles of these PPihydrolyzing activities are not certain, it has been argued that they may regulate the equilibria of several synthetic reactions in the anabolic cycle producing inorganic pyrophosphate (28). It is noteworthy that the pH optimum of inorganic pyrophosphatase lies in a quite alkaline region (pH 8.8) and the enzymic activity is manifested only partially in intact bacterial cells that are known to be very acidophilic. In the cell-free system of T. thiooxidans, the oxidation of sulfur and sulfite (16, 29), CO, fixation (30), and ATPase [EC 3.6.1.3] activities (15) have pH optima much higher than that of the growth environment, which is extremely acidic (below pH 1.0). Although an enzyme should not always be considered to exert its physiological The enzyme required Mg1+ for full activity, and role at the optimum conditions, these findings acother cations, Co l+ and Mn!+, were shown to be count for the internal localization of inorganic partially effective. This requirement is similar to pyrophosphatase, in marked contrast to the acid that reported for the enzyme from the thermophilic phosphatase previously reported (27). The carbacterium B. stearothermophilis (24). Unlike the boxylating enzyme in COj fixation is also thought to enzymes from yeast (1), E. coli (11), and B. subtilis be an internal enzyme and its activity under acidic (25), Zn2+ had no effect on the activity of the en- condition may depend on the permeability of COS. zyme. Josse (12) and Moe and Butler (4) extensively studied the relationship of pyrophosphate and The authors are indebted to Mr. Y Yokota for performmagnesium concentrations relative to pyrophos- ing SDS-polyacrylamide gel electrophoresis. phatase activity and proposed that a stoichiometric complex, MgPPr, was the actual substrate. The REFERENCES present results (Fig. 5) support this proposal. 1. Kumtz, M. (1952) /. Gen. Physiol. 35, 423-450 The findings that the enzyme from T. thiooxi2. Cooperman, B S., Chiu, N.Y , Bruckmann, R H., dans was inhibited by NaF and not affected by Bunick, G.J..& McKenna, G P. (1973) Biochemistry sulf hydryl reagents are the same as those for the E. 12, 1665-1669 coli (11), F. ferrooxidans (14), and B. subtilis (25) 3. Ridlington, J.W., Yang, Y., & Butler, L.G. (1972) enzymes. Arch. Biochem. Biophys. 153, 714-725 The enzymes obtained from a variety of bac4. Moe, O.A. & Butler, L.G. (1972) J. Biol. Chem. 141, terial species have some similar properties (neutral 7308-7314 or alkaline pH optima and requirement for Mg1+ or 5. Heinrikson, R.L., Sterner, R., & Yoyes, C. (1973) / . Biol. Chem. 248, 2521-2528 some cation), and have been classified according to 6. Negi, T. & Ine, M. (1971) / . Biochem. 70, 165-168 heat stability and inducibihty by PPi by Blumenthal 7. Negi, T., Samejima, T., & Ine, M. (1971) /. Biochem. et al. (26). The heat stability of the enzyme from 70, 359-363 T. thiooxidans (Fig. 6) supports their finding that all 8. Negi, T., Samejima, T.,& Ine, M (1912) J. Biochem. gram-negative organisms have a heat-stable enzyme. 71, 29-37 There are two PPi-hydrolyzing activities in T. 9. Yano, Y., Negi, T., & Ine, M. (L973) /. Biochem. 1A, thiooxidans. One is the inorganic pyrophosphatase 67-76 described in this report, which is operative at 10. Yano, Y. & Ine, M. (1975) /. Biochem. 78,1001-1011 alkaline pH values and is specific for PPi. The 11. Josse, J. (1966) /. Biol. Chem. 241, 1938-1947 other is a membrane-bound and sulfate-dependent 12. Josse, J. (1966) /. Biol. Chem. 241, 1948-1957 J. Biochem.
INORGANIC PYROPHOSPHATASE FROM T. thlooxidans 13 Wong, S.C.K., Hall, D.C., & Josse, J. (1970) /. Biol.
14 15. 16. 17. 18. 19. 20. 21. 22.
Chem. 245, 4335-4345 Howard, A. & Lundgren, D.G. (1970) Can. J. Biochem. 4», 1302-1307 Marunouchi, T. & Mori, T. (1967) J. Biochem. 62, 401^107 Kodama, A. & Mori, T. (1967) Plant & Cell Physiol. 9, 709-723 Allen, R.J.L. (1940) Biochem. J. 34, 858-865 Nakamura, M. (1950) Nippon Nogeikagakukaishi (in Japanese) 24, 1-5 Lowry, D.H., Rosenbrough, NJ., Farr, L., & Randall, R J . (1951) /. Biol. Chem. 193, 265-275 Andrews, P. (1964) Biochem. J. 91, 222-233 Davis, B. (1964) Ann. New York Acad. Set 121, 404427 Weber, K. & Osborn, M. (1969) /. Biol. Chem. 244, 440(5-4412
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23. Takakuwa, S. (1975) J. Biochem. 78, 181-185 24. Hachimori, A., Takeda, A., Kaibuchi, M., Ohkawara, N., & Samejima, T. (1975) /. Biochem. TJ, 1177-1183 25. Tono, H. & Kornberg, A. (1967) /. Biol. Chem. 242, 2375-2382 26. Blumenthal, B.I., Johnson, M.K., & Johnson, E.J. (1967) Can. J. Microbiol 13, 1695-1699 27. Tominaga, N. & Mori, T. (1974) /. Biochem. 76, 397-408 28. Kornberg, A. (1962) in Horizones in Biochemistry pp. 251-264, Academic Press, Inc., New York 29. Kodama, A. (1969) Plant & Cell Physiol. 10,645-655 30. Iwatsuka, H., Kuno, M., & Oura, E. (1962) Plant & Cell Physiol. 3, 157-166
Purification and Characterization of Inorganic Pyrophosphatase from Thiobacillus thiooxidans Noriko TOMTNAGA* and Takeshi MORI** •Biological Institute, Faculty of Science, Nagoya University, Chikusa-ku, Nagoya, Aichi 464, and **Japan University of Social Welfare, Showa-ku, Nagoya, Aichi 464 Received for publication, July 9, 1976
An inorganic pyrophosphatase [EC 3.6.1.1] was isolated from Thiobacillus thiooxidans and purified 975-fold to a state of apparent homogeneity. The enzyme catalyzed the hydrolysis of inorganic pyrophosphate and no activity was found with a variety of other phosphate esters. The cation Mgt+ was required for maximum activity; Co3+ and Mn1+ supported 25 per cent and 10.6 per cent of the activity with Mg t+ , respectively. The pH optimum was 8.8. The molecular weight was estimated to be 88,000 by gel filtration and SDS gel electrophoresis, and the enzyme consisted of four identical subunits. The isoelectric point was found to be 5.05. The enzyme was exceptionally heat-stable in the presence of 0.01 M Mgt+.
Thiobacillus thiooxidans, a gram-negative and chemolithotrophic bacterium, uses carbon dioxide as a carbon source and derives energy from the oxidation of elemental sulfur, making the culture medium extremely acidic (below pH 1.0). Enzymes relatively specific for the hydrolysis of inorganic pyrophosphate are widely distributed in nature. While extensive investigations of inorganic pyrophosphatase [EC 3.6.1.1] have been carried out with heterotrophs, yeast {1-10) and Escherichia coli (11-13), the isolation and purification of this enzyme from a chemolithotrophic bacterium was firstly reported with Ferrobacillus ferrooxidans (14). The occurrence of activity hydrolyzing PPi was also reported in our T. thiooxidans strain (15). The purification and some properties of inorganic pyrophosphatase of T. thiooxidans are described here.
Vol. 81, No. 2, 1977
477
MATERIALS AND METHODS Microorganism—T. thiooxidans was cultured and harvested as described previously (76). Chemicals—Tetrasodium pyrophosphate was purchased from Hayashi Pure Chemicals. ATP and ADP were obtained from Kojin Co. AMP, bovine serum albumin, and ovalbumin were from Sigma Co. ^-Globulin was purchased from Katayama Chemical Industries Co. Glyceraldehyde phosphate dehydrogenase [EC 1.2.1.12] and aldolase [EC 4.1.2.13] were obtained from Boehringer Mannheim Co. Crystalline cryptocytochrome c from Pseudomonas denitrificans was kindly supplied by Dr. Iwasaki of our laboratory. Measurement of Enzyme Activity—Inorganic pyrophosphatase assays were conducted for 20 min at 30°; the assay system contained 4 [imo\ of Na4P,O7, 100 (imo\ of glycine-NaOH buffer (pH 8.8), 10 /*mol of MgCl,, and enzyme in a total
478
volume of 2 ml. The reaction was stopped by the addition of 0.5 ml of 30% trichloroacetic acid. Any precipitate formed was removed by centrifugation and then the supernatant was used for the determination of Pi. A unit of pyrophosphatase activity was defined as the quantity of enzyme catalyzing the liberation of 1 ^mol of Pj per min under the conditions specified. Specific activity was defined as enzyme units per mg protein. Analytical Methods—Pi was determined colorimetrically by the method of Allen (77) as modified by Nakamura (18). Protein was determined by the method of Lowry et al. (19), using bovine serum albumin as a standard. The molecular weight of the enzyme was estimated by the gel filtration technique of Andrews (20). A Sephadex G-150 column (2.4 x90 cm) was equilibrated with 0.025 M Tris-HCl buffer (pH 7.5). Protein was eluted with the same buffer at a flow rate of 25 ml/h. The isoelectric point of pyrophosphatase was determined by isoelectric focusing with carrier ampholyte giving a pH gradient between 3 and 10 (LKB Produkter AB, Sweden) at a mean concentration of 1 %. Focusing was carried out at 4° in a special vertical electrophoresis column (2 x 24 cm). Stabilization against convection was achieved with a sucrose density gradient of 0-50 %. After focusing, the column was drained and fractions of 2 ml were collected. The method of Davis (2/) was used for polyacrylamide gel electrophoresis and the power supply was adjusted to 3 mA per tube. The protein bands were stained with 0.05% Coomassie blue in 12.5% TCA. For histochemical analysis of the pyrophosphatase activity in polyacrylamide gel, the gel was incubated for 10 min at 30° in the reaction mixture and rinsed with water. The color of liberated Pi was developed by applying Nakamura's reagents. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis was performed according to the method of Weber and Osborn (22), using 10% gels.
N. TOMINAGA and T. MORI Pyrophosphatase activity was easily extracted by sonication of T. thiooxidans cells with 0.1 M Tris-HCl buffer (pH 7.5), and more than 90 % of the activity of cell-free extract was found in the supernatant fluid after centrifugation for 1 h at 105,000 X g. The cell-free extract showed more pyrophosphatase activity than did intact cells, which exhibited only about 5 % of the activity of the extract. Lyophilized cells (18 g dry wt.) were suspended in 720 ml of 0.1 M Tris-HCl buffer (pH 7.5) and disrupted for 15 min in a 10 kc sonic oscillator (Kubota KMS 100) at 0°. After centrifugation for 30 min at 21,000 xg, the precipitate was resuspended in 350 ml of the same buffer and sonicated again. The combined supernatant was used as cell-free extract. The fraction precipitated between 55 and 90% ammonium sulfate saturation was dissolved in 100 ml of distilled water and dialyzed overnight against 10 liters of distilled water. The dialysate was applied to a DEAE-cellulose column (4.2 x 5.5 cm) previously equilibrated with 0.05 M Tris-HCl buffer (pH 7.5) (buffer A). After addition of the sample, the column was washed with buffer A (approx. 200 ml); this treatment removed pigments containing soluble cytochrome c (23). The enzyme was then eluted with a linear gradient of ammonium sulfate (0-0.3 M) in .buffer A. The active fractions were collected, diluted with 3 volumes of buffer A and again fractionated on a DEAE-cellulose column. The conditions were the same as those of the first chromatography except for column size (3x5 cm). The active fractions were diluted with an equal volume of buffer A and loaded on a hydroxylapatite column (4.2x3 cm) equilibrated with buffer A. The column was washed with 100 ml of buffer A containing 0.25 M ammonium sulfate and then developed with a linear gradient of ammonium sulfate (0.25-0.75 M) in buffer A.
Fractions containing enzyme activity were diluted with 2 volumes of buffer A and again applied to a hydroxylapatite column (4 x 1 cm) equilibrated with buffer A. After washing with 50 ml of buffer A containing 0.25 M ammonium sulfate, the enzyme was eluted with buffer A containing 0.75 M ammonium sulfate and dialyzed overnight against 3 liters RESULTS of 0.025 M Tris-HCl buffer (pH 7.5). Purification of Pyrophosphatase—Unless otherThe enzyme was further purified by isoelectric wise indicated, all procedures were carried out in a focusing. The elution profile is illustrated in Fig. 1. cold room at 4-5°. Fraction number 12 was dialyzed overnight against /. Biochem.
INORGANIC PYROPHOSPHATASE FROM T. thiooxidans
479
12
-.400 c-
300
200
100 I Q.
O tr 20 30 40 FRACTION NUMBER
10
50
60
Fig. 1. Isoelectric fractionation of inorganic pyrophosphatase The active fractions (2.7 mg protein) obtained from the second hydroxylapatite column chromatography were dialyzed against 0.025 M Tris-HCl buffer (pH 7 5) then subjected to isoelectric focusing at 900 volts for 49 h at 4°, as described in "MATERIALS AND METHODS." , optical density at 280nm; - - O - - , pyrophosphatase activity; , pH. TABLE I. Purification of inorganic pyrophosphatase of T. thiooxidans. Total volume (ml) Crude extract Ammonium sulfate DEAE-cellulose I DEAE-cellulose II Hydroxylapatite I Hydroxylapatite II Electrofocusing (No. 12)
1,000
Total activity1 (units)
31
6,108 5,450 5,316 4,561 4,433 1,244
7
652
140 135 104 130
Total protein (mg) 7,400 1,232 297 149
38.35 5.425 0.812
Specific activity (units/mg protein) 0.825 4.424 17 90 30.62 115.6 229.4 804.1
Yield (%) 100
89.2 87.0 74.7 72.6 20.4 10.7
Assay conditions are described in "MATERIALS AND METHODS."
2 liters of 0.025 M Tris-HCl buffer (pH 7.5) and stored at 0°. The process of purification, in which the enzyme was purified about 975-fold with a recovery of 10.7 % over the crude extract activity, is summarized in Table I. The enzyme preparation gave a single protein band on polyacrylamide gel electrophoresis at pH 9.4 and the locus of inorganic pyrophosphatase activity coincided exactly with the protein band (Fig. 2A). Vol. 81, No. 2, 1977
The purified enzyme was stable at 0° for more than nine months. Properties of Inorganic Pyrophosphatase—The molecular weight of the enzyme was estimated to be approximately 88,000 by gel filtration (Fig. 3). The subunit molecular weight of the enzyme was estimated by SDS-polyacrylamide gel electrophoresis with SDS-denatured ^-globulin, bovine serum albumin, ovalbumin, aldolase, and glyceraldehyde phosphate dehydrogenase as reference standards. The inorganic pyrophosphatase was
N. TOM1NAGA and T. MORI
480
50 Bovine serum albumin LU
345
7-Globulin H chain Ovalbumin Aldolase tf Glyceraldehyde phosphate dehydrogenase /-Globulin L chain Inorganic pyrophosphatase
CD O
Fig. 2. Disc gel electrophoresis of the purified inorganic pyrophosphatase. (A) Electrophoresis was carried out for 50 mm at 3mA per tube on 7.5% polyacrylamide gel using 1 fig (a) and 0.25 pg (b) of protein, (a) The gel was stained with Coomassie blue, (b) The gel was incubated in the reaction mixture for pyrophosphatase assay and inorganic phosphate released was detected with Nakamura's reagents (7S). (B) Electrophoresis of 1 fig of the enzyme was earned out for 4 h at 8 mA per tube on 10% polyacrylamide gel containing 0.1% SDS. The protein band was stained with Coomassie blue.
300
250 z o
02
04
0.6
0.8
10
MOBILITY
Fig. 4. Estimation of subunit molecular weight by SDS-polyacrylamide gel electrophoresis. Electrophoresis was earned out on 10% polyacrylamide gel containing 0.1% SDS according to Weber and Osborn (22).
Cryptocytochrome c (Ps. denrtnftcans) j Ovalbumin ^Bovine serum albumin Inorganic pyrophosphatase
2
200
45 50 LOG MOLECULAR WEIGHT
4
6
8
10
CONCENTRATION OF M g 2 + (mM)
Serum albumin dimer 150 40
40
55
Fig. 3. Molecular weight estimation of inorganic pyrophosphatase by Sephadex G-150 gel filtration. A Sephadex G-150 column (2.4x90 cm) was equilibrated with 0.025 M Tris-HCl buffer (pH 7.5). Cryptocytochrome c, ovalbumin, and serum albumin (molecular weights, 26,400, 45,000, and 67,000, respectively) were cluted with the same buffer at a flow rate of 25 ml/h. found to be composed of only one species of polypeptide (Fig. 2B), the molecular weight of which was estimated to be approximately 20,000 (Fig. 4). Thus, the inorganic pyrophosphatase may consist of four identical subunits. The isoelectric point of the enzyme, as determined by isoelectric focusing, was found to be 5.05 (Fig. 1).
Fig. 5. Effect of magnesium on inorganic pyrophosphatase. Assays were performed using the following incubation mixture (2.0 ml); 2 mM sodium pyrophosphate, 50 mM glycine-NaOH buffer (pH 8.8), magnesium chloride and purified enzyme (0.08 fig protein). For measurement of the pH dependence of activity, Tris-maleate buffer was used from pH 5.9 to 8.25 and glycine-NaOH buffer from pH 8.2 to 9.5 at a concentration of 0.05 M. The enzyme had a pH optimum of 8.8 and the activities were 4.5 % and 72.2% of optimum at pH 5.9 and 9.5, respectively. There was no detectable activity in the absence of divalent cations. Of several chloride salts (5 mM) tested, Mg1+ was required for maximum activity; Co1+ and Mn t+ supported 25 % and 10.6% of the activity with Mg t+ , respectively. Ca l+ , Zn1+, Cu1+, and Hg1+ were not effective. As can be seen in Fig. 5, the optimal concentration of Mg1+ J. Biochem.
481
INORGANIC PYROPHOSPHATASE FROM T. thiooxidans TABLE n . Substrate specificity of inorganic pyrophosphatase. The substrate concentration was 2 nw. The enzyme activities were calculated from the amounts of Pi liberated from various substrates under the assay conditions. Relative activities are expressed as percentages of those for PPi. Relative rate of hydrolysis
Substrates PPi
100 4.2 2.6
ATP ADP AMP ar-Glycerophosphate p-Nitrophenyl phosphate Glucose 6-phosphate Thiamine pyrophosphate
0 0 0.4 0 0
50 70 90 100 TEMPERATURE C )
TABLE III. Effects of inhibitors. The standard assays described in "MATERIALS AND METHODS" were carried out with addition of the inhibitor. Concentration % Inhibition (HIM) NaF KCN Iodoacetamide N-Ethylmaleimide p-Chloromercuribenzoate NaN,
1.25 5 5 5 1 1
Guanidine-HCl
5
81.3 20.9 14.8 10.1 3 11.6 22.9
was approximately the same as that of the substrate (2 ITIM), suggesting that the metal ion was required in a stoichiometnc relationship with respect to PPi anion. The initial rates of hydrolysis of various phosphate esters by the purified enzyme are summarized in Table II. Under the conditions employed, the enzyme was found to be highly specific for inorganic pyrophosphate. The effects of various compounds on enzyme activity are summarized in Table III. The activity was strongly inhibited by NaF. KCN, azide, guanidine-HCl, and sulfhydryl reagents caused less than 25 % inhibition. Figure 6 shows the thermal inactivation profile of pyrophosphatase; the enzyme was heated at Vol. 81, No. 2, 1977
Fig. 6. Thermal inactivation of inorganic pyrophosphatase. The enzyme obtained from the second hydroxylapatite column chromatography (0.07 mg protein) was incubated in either 0.025 M Tris-HCl buffer (pH 7.5) ( • ) or 0.025 M Tris-HCl buffer (pH 7 5) containing 0.01 M MgCli (O) for 10 min at the indicated temperatures. After cooling on ice, the enzyme solution was diluted 25-fold with distilled water and pyrophosphatase activity was measured as described in "MATERIALS AND METHODS." various temperatures for 10 min. The enzyme was quite resistant to heat treatment. For example, in the presence of 0.01 M Mg 1+ , 99% of the activity remained after 10 min at 80° and 4 0 % at 100°. Although Marunouchi and Mori (15) reported, that sulfite slightly stimulated the pyrophosphatase activity, the purified enzyme was not stimulated by sulfite ions. The following salts had slightly inhibitory effects (2-12%) at a concentration of 20 ITIM; NaCl, KC1, NaHCO,, Na a SO,, Na,SO 4 , Na s SeO,, and K,CrO 4 . DISCUSSION Inorganic pyrophosphatase from T. thiooxidans has been purified to a state of apparent homogeneity. The findings that the intact cells exhibit only a small fraction of the total activity obtained in the cell-free extract and that 90 % of the enzyme activity of crude extract was found in the 105,000 x g supernatant suggest that this enzyme is present in the cell cytoplasm, as are those of E. coli (11) and F. ferrooxidans (14).
482
N. TOMINAGA and T. MORI
The molecular weight of the enzyme was estimated to be approximately 80,000-88,000. This value is different from molecular weights determined for pyrophosphatases from E. coli (13) and Bacillus stearothermophilis (24) (about 120,000) as well as those from yeast (3, 5, 6) and Bacillus subtilis (25) (about 70,000). Electrophoresis on SDS-polyacrylamide gel indicated that the inorganic pyrophosphatase from T. thiooxidans consists of four identical subunits with a molecular weight of approximately 20,000. The subunit structure of this enzyme was different from those reported for other microorganisms. The yeast enzyme consisted of two identical subunits with a molecular weight of 33,000-35,000 (3, 5), while the B. stearothermophilis enzyme also consisted of two subunits but its molecular weight was 70,000 (24). On the other hand, the enzyme from E. coli had six identical subunits with a molecular weight of 20,000 (7).
acid phosphatase [EC 3.1.3.2] which also hydrolyzes PPi at a considerable rate in the presence of sulfate ions and in the absence of Mg1+ at acidic pH (27). Although the metabolic roles of these PPihydrolyzing activities are not certain, it has been argued that they may regulate the equilibria of several synthetic reactions in the anabolic cycle producing inorganic pyrophosphate (28). It is noteworthy that the pH optimum of inorganic pyrophosphatase lies in a quite alkaline region (pH 8.8) and the enzymic activity is manifested only partially in intact bacterial cells that are known to be very acidophilic. In the cell-free system of T. thiooxidans, the oxidation of sulfur and sulfite (16, 29), CO, fixation (30), and ATPase [EC 3.6.1.3] activities (15) have pH optima much higher than that of the growth environment, which is extremely acidic (below pH 1.0). Although an enzyme should not always be considered to exert its physiological The enzyme required Mg1+ for full activity, and role at the optimum conditions, these findings acother cations, Co l+ and Mn!+, were shown to be count for the internal localization of inorganic partially effective. This requirement is similar to pyrophosphatase, in marked contrast to the acid that reported for the enzyme from the thermophilic phosphatase previously reported (27). The carbacterium B. stearothermophilis (24). Unlike the boxylating enzyme in COj fixation is also thought to enzymes from yeast (1), E. coli (11), and B. subtilis be an internal enzyme and its activity under acidic (25), Zn2+ had no effect on the activity of the en- condition may depend on the permeability of COS. zyme. Josse (12) and Moe and Butler (4) extensively studied the relationship of pyrophosphate and The authors are indebted to Mr. Y Yokota for performmagnesium concentrations relative to pyrophos- ing SDS-polyacrylamide gel electrophoresis. phatase activity and proposed that a stoichiometric complex, MgPPr, was the actual substrate. The REFERENCES present results (Fig. 5) support this proposal. 1. Kumtz, M. (1952) /. Gen. Physiol. 35, 423-450 The findings that the enzyme from T. thiooxi2. Cooperman, B S., Chiu, N.Y , Bruckmann, R H., dans was inhibited by NaF and not affected by Bunick, G.J..& McKenna, G P. (1973) Biochemistry sulf hydryl reagents are the same as those for the E. 12, 1665-1669 coli (11), F. ferrooxidans (14), and B. subtilis (25) 3. Ridlington, J.W., Yang, Y., & Butler, L.G. (1972) enzymes. Arch. Biochem. Biophys. 153, 714-725 The enzymes obtained from a variety of bac4. Moe, O.A. & Butler, L.G. (1972) J. Biol. Chem. 141, terial species have some similar properties (neutral 7308-7314 or alkaline pH optima and requirement for Mg1+ or 5. Heinrikson, R.L., Sterner, R., & Yoyes, C. (1973) / . Biol. Chem. 248, 2521-2528 some cation), and have been classified according to 6. Negi, T. & Ine, M. (1971) / . Biochem. 70, 165-168 heat stability and inducibihty by PPi by Blumenthal 7. Negi, T., Samejima, T., & Ine, M. (1971) /. Biochem. et al. (26). The heat stability of the enzyme from 70, 359-363 T. thiooxidans (Fig. 6) supports their finding that all 8. Negi, T., Samejima, T.,& Ine, M (1912) J. Biochem. gram-negative organisms have a heat-stable enzyme. 71, 29-37 There are two PPi-hydrolyzing activities in T. 9. Yano, Y., Negi, T., & Ine, M. (L973) /. Biochem. 1A, thiooxidans. One is the inorganic pyrophosphatase 67-76 described in this report, which is operative at 10. Yano, Y. & Ine, M. (1975) /. Biochem. 78,1001-1011 alkaline pH values and is specific for PPi. The 11. Josse, J. (1966) /. Biol. Chem. 241, 1938-1947 other is a membrane-bound and sulfate-dependent 12. Josse, J. (1966) /. Biol. Chem. 241, 1948-1957 J. Biochem.
INORGANIC PYROPHOSPHATASE FROM T. thlooxidans 13 Wong, S.C.K., Hall, D.C., & Josse, J. (1970) /. Biol.
14 15. 16. 17. 18. 19. 20. 21. 22.
Chem. 245, 4335-4345 Howard, A. & Lundgren, D.G. (1970) Can. J. Biochem. 4», 1302-1307 Marunouchi, T. & Mori, T. (1967) J. Biochem. 62, 401^107 Kodama, A. & Mori, T. (1967) Plant & Cell Physiol. 9, 709-723 Allen, R.J.L. (1940) Biochem. J. 34, 858-865 Nakamura, M. (1950) Nippon Nogeikagakukaishi (in Japanese) 24, 1-5 Lowry, D.H., Rosenbrough, NJ., Farr, L., & Randall, R J . (1951) /. Biol. Chem. 193, 265-275 Andrews, P. (1964) Biochem. J. 91, 222-233 Davis, B. (1964) Ann. New York Acad. Set 121, 404427 Weber, K. & Osborn, M. (1969) /. Biol. Chem. 244, 440(5-4412
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23. Takakuwa, S. (1975) J. Biochem. 78, 181-185 24. Hachimori, A., Takeda, A., Kaibuchi, M., Ohkawara, N., & Samejima, T. (1975) /. Biochem. TJ, 1177-1183 25. Tono, H. & Kornberg, A. (1967) /. Biol. Chem. 242, 2375-2382 26. Blumenthal, B.I., Johnson, M.K., & Johnson, E.J. (1967) Can. J. Microbiol 13, 1695-1699 27. Tominaga, N. & Mori, T. (1974) /. Biochem. 76, 397-408 28. Kornberg, A. (1962) in Horizones in Biochemistry pp. 251-264, Academic Press, Inc., New York 29. Kodama, A. (1969) Plant & Cell Physiol. 10,645-655 30. Iwatsuka, H., Kuno, M., & Oura, E. (1962) Plant & Cell Physiol. 3, 157-166
