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Soluble 5′-Nucleotidase from Thyroid Gland Partial Purification and Properties
BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS ARTICLE NO.
221, 471–476 (1996)
0619
Soluble 59-Nucleotidase from Thyroid Gland Partial Purification and Properties Janina Niedz´wiecka and Leokadia Jaroszewicz Department of Physical Chemistry, Medical Academy, 15-230 Bialystok, ul.Mickiewicza 2, Poland Received February 26, 1996 A soluble 59-nucleotidase from pig thyroid was purified over 110-fold by chromatography on phosphocellulose, (NH4)2SO4 precipitation and gel filtration on Sephadex G-150. The purified 5-nucleotidase was free of non-specific phosphatases. The enzyme had optimum pH at 6.5 and hydrolysed preferentially IMP and GMP. The Km values were 0.66 and 1.0 mM for IMP and GMP, respectively. The enzyme also hydrolysed other nucleotides and showed the following relative Vmax: IMP > GMP > CMP > AMP > UMP.Mg2+ was necessary for the enzyme activity. © 1996 Academic Press, Inc.
Purine nucleotide dephosphorylation is an obligatory step in nucleotide catabolism. 59Nucleotidase (EC 3.1.3.5) catalyzing the dephosphorylation of purine and pyrimidine rybo- and deoxyrybonucleoside monophosphates, has been described in various mammalian tissues (1–9) as well as in microorganisms (10,11). The membrane-bound ecto-59-nucleotidase has been known for many years and has been used extensively as a plasma membrane marker in subcellular fractionation techniques. Although its physiological function is not completely understood, it has been assumed that ecto-59-nucleotidase hydrolyses extracellular nucleotides into the membranepermeable nucleosides. One of this nucleosides, namely adenosine exerts different regulatory function in various organs (12). Besides the membrane-bound enzyme, the two types of soluble 59-nucleosidases have been described in mammals (3,23). One form, called “high Km” 59nucleotidase, found in rat and chicken liver (1,18), rat heart (19) and human placenta (3) has maximal activity in the presence of IMP and GMP, and is characterized by millimolar Km values, activation by ATP and inhibition by inorganic phosphate. The other soluble 59-nucleotidase “low Km” found in human placenta (23) shows substrate preference for AMP and pyrimidine nucleoside monophosphates, micromolar Km values and inhibition by ATP. Subcellular studies of bovine thyroid indicate that 59-nucleosidase is predominantly associated with plasma membranes (13). Matsuzaki et al.(14) showed that 59-nucleotidase activity was preferentially located in the pellet of bovine thyroid but approximately 27% of the total activity was recovered in the supernatant. The plasma-membrane bound 59-nucleotidase from thyroid gland has been purified and characterized (15). Neither the soluble enzyme from the thyroid has until now not been purified nor have its properties been studied. In this study we report the purification and some properties of soluble 59-nucleotidase from the pig thyroid gland. MATERIALS AND METHODS Materials. AMP, IMP, CMP, GMP, p-nitrophenyl phosphate (dicyclohexylammonium salt), ethylene-diamine-tetraacetic acid (EDTA) were obtained from Sigma Chemical Co., Sephadex G-150 from Farmacia Fine Chemicals, Tris (hydroxymethyl) amino methane, b-mercaptoethanol (Et-SH) from Fluka, phosphate cellulose from Whatman and other reagents of analytical grade from POCH Gliwice, Poland. The pig thyroid glands were obtained from the slaughterhouse immediately after killing the animals. Purification of soluble 58-nucleotidase. After removal of connective tissue and fat, the finely-chopped thyroid glands were homogenized in 0.05 M Tris-HCl buffer containing 1 mM EDTA and 10 mM Et-SH (1 g of tissue + 4 ml of buffer) in the Waring Blendor for 1 min (at high speed) four times. The homogenate was filtered through four-fold gauze and the filtrate was centrifuged at 10000 g for 15 min. The precipitate was suspended in the mentioned above buffer while the supernatant was centrifuged at 105000 g for 1 h. After centrifugation the supernatant was used for further purification. 471 0006-291X/96 $18.00 Copyright © 1996 by Academic Press, Inc. All rights of reproduction in any form reserved.
Vol. 221, No. 2, 1996
BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS TABLE 1 Purification of Soluble 59-Nucleotidase from Pig Thyroid
Step Supernatant Phosphocellulose eluate (NH4)2SO4 precipitate Sephadex G-150 filtrate
Specific activity (mmol/min/ mg protein)
Purification (fold)
Yield (%)
Volume (ml)
Protein (mg/ml)
Total activity (mmol/min)
315
41.4
48.25
0.0037
1
100.0
90
1.1
8.70
0.088
23.8
18.0
5
3.9
4.48
0.23
62.2
9.3
33
0.2
2.84
0.430
116.2
5.9
Phosphocellulose chromatography. Whatman P-11 cellulose phosphate (10g) was prepared as recommended by the manufacturer and was washed with 0.05 M Tris-HCl buffer of pH 7.4 containing 1 mM EDTA and 10 mM Et-SH. Cellulose phosphate suspension (140 ml) was mixed with supernatant (550 ml) prepared as described above, and was being mixed over night at 5°C. Then the suspension was filtered on the Büchner funnel and rinsed with 4 l of the same buffer. The column (2 × 40 cm) was then filled up with the filtrated suspension and the adsorbed protein was eluted by stepwise gradient of NaCl in the same buffer. (NH4)2SO4 precipitation. Fractions from cellulose phosphate column containing the 59-nucleotidase activity were pooled and precipitated between 40 and 60% of (NH4)2SO4 saturation. The precipitate was suspended in the buffer (about 10 ml) and centrifuged at 16000 g for 1 h to remove insoluble material. Gel filtration on Sephadex G-150. An active protein obtained after (NH4)2SO4 precipitation was applied on the Sephadex G-150 column (3.3 × 48 cm) previously equilibratated with 0.05 M Tris-HCl buffer of pH 7.4, containing 1 mM EDTA and 10 mM Et-SH. The same buffer was used for protein elution. Fractions with the highest activity of 59-nucleotidase were pooled and used for further investigations. 58-Nucleotidate activity. For routine assays, protein extracts were incubated for 30 min at 37°C in a reaction mixture containing 0.05 M Tris-HCl buffer of pH 6.5, 50 mM NaCl, 5mM MgCl2, 1 mM substrate solution and 0,02 - 0,05 mg protein in a total volume of 1 ml. After stopping the reaction by adding 1 ml 10% trichloroacetic acid, precipitated protein was removed by centrifugation and 0,25 ml of the supernatant was assayed for the release of inorganic phosphate according
FIG. 1. Sephadex G-150 gel filtration of 59-nucleotidase. The active 59-nucleotidase fraction obtained after (NH4)2SO4 precipitation (about 8 ml) was applied to the column (3,5 × 48 cm) equilibrated with 0.05 M Tris-HCl buffer, pH 7.4 containing 1 mM EDTA and 10 mM Et-SH. The enzyme was eluted with the same buffer and 3 ml fractions were collected. 472
Vol. 221, No. 2, 1996
BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS TABLE 2 Substrate Specificity of Purified Soluble 59Nucleotidase from Pig Thyroid
Substrate (1 mM)
Activities (nmol/min per mg of protein)
Relative activity (%)
440 416 90 212 60 0
100.0 93.8 20.7 48.3 13.8 0.0
59-IMP 59-GMP 59-AMP 59-CMP 59-UMP p-Nitrophenylphosphate
Experiments were performed under standard assay conditions in 0,05 M Tris-HCl buffer at pH 6,5.
to the method of Chen et al (16). All the experimental values were corrected by subtracting the respective control, i.e. the amount of inorganic phosphate present in parallel assays performed with acid precipitated samples. The specific activity was expressed in mmol of released inorganic phosphate × min−1 × mg of proteins−1. Non-specific phosphatase activity. The acid phosphatase activity and alkaline phosphatase activity were determined by using 4,8 mM p-nitrophenyl phosphate as a substrate in 0,2 M sodium citrate - citric acid buffer of pH 5 for acid phosphatase and glicine - MgCl2 - NaOH buffer of pH 10,3 for alkaline phosphatase. The reaction was started by adding the enzyme, then run at 37°C for 30 min, and stopped by adding 2 volumes of 1 m NaOH and optical density was read at 410 nm. Protein determination. Protein concentration was determined by the method of Lowry et al.(17) using bovine albumin as a standard. Proteins eluting from the columns were monitored by absorbance at 280 nm.
RESULTS The results of 59-nucleotidase purification at particular stages are summarized in Table I. By means of chromatography on phosphocellulose, (NH4)2SO4 precipitation and gel filtration on Sephadex G-150, over 110-fold purification of the enzyme with specific activity about 0.4 mmoles × min−1 × mg of protein, was obtained. On the step of phosphocelullose chromatography all non-specific phosphatase activity is removed. This non-phosphatase activity does not bind to phosphocelullose. 59-Nucleotidase adsorbed on phoshocelullose was eluted with the sodium chloride gradient from 0.4 to 0.6 M. The fractions containing 59-nucleotidase activity were pooled and fractionated with ammonium sulfatate between 0–30 %, 30–40% and 40–60% of saturation. The highest enzyme activity was found in the fraction
FIG. 2. Effect of pH on the activity of soluble thyroid 59-nucleotidase. Experiments were performed in 0,2 M Tris-HCl buffer, using 59 IMP as a substrate. 473
Vol. 221, No. 2, 1996
BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS
FIG. 3. Influence of substrate concentration on 59-nucleotidase activity. Activities were measured at pH 6,5 with 59 IMP and 59 GMP as substrates.
of 40–60% of saturation. The gel filtration on Sephadex G-150 showed a single peak of the 59-nucleotidase activity (Fig. 1). Properties of Enzyme Substrate specificity. The activity of soluble 59-nucleotidase towards various nucleoside monophosphates is shown in Table II. The preferred substrates for the purified enzyme were IMP and GMP. The enzyme also hydrolysed other nucleotides and showed the following relative Vmax values:IMP > GMP > CMP > AMP > UMP. p-Nitrophenyl phoshapte was not hydrolysed. Effect of pH. The optimum pH of soluble pig thyroid 59-nucleotidase was about 6.5 with IMP a substrate (Fig. 2). Kinetic properties. The soluble 59-nucleotidase showed Michaelis-Menten kinetics for both preferred substrates, IMP and GMP (Fig. 3). The Michaelis constant according to the LineweaverBurk plot was 660 mM for IMP and 1000 mM for GMP. Vmax was 0.48 and 0.416 mmoles/min per mg of protein for IMP and GMP, respectively (Fig. 4). Effect of MgCl2 and NaCl on enzyme activity. The 59-Nucleotidase activity was dependent on Mg2+ increasing with its concentration to 2 mM and remained unchanged at higher concentration (Fig. 5). Also the influence of NaCl on enzyme activity has been studied. 59-Nucleotidase showed maximal activity at the NaCl concentration of 10–40 mM (Fig. 6).
FIG. 4. Lineweaver-Burk plots for enzymatic dephosphorylation. 474
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BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS
FIG. 5. Effect of Mg2+ on activity of 59-nucleotidase. The enzyme activity was measured with 59 IMP under standard conditions except for the concentration of MgCl2 as indicated.
DISCUSSION In this study the soluble 59-nucleotidase from pig thyroid was purified over 110-fold to a specific activity of 0.43 mmol/min/mg. In the process of purification, the soluble 59-nucleotidase was separated from non-specific phosphatases during chromatography on phosphocellulose. The apparent almost 80% loss of 59-nucleotidase activity during this step may be related in part to the removal of non-specific phosphatases. 59-Nucleotidase from the pig thyroid shows several properties in common with the soluble enzyme from other tissues (1,3,18,21). The enzyme from the thyroid has an optimum at pH 6.5 and is Mg2+ dependent. IMP and GMP were the preferred substrates. The Km values were 0.66 mM for IMP and 1 mM for GMP. The properties of the presented 59-nucleotidase indicate that the thyroid enzyme belongs to the soluble “high Km” 59-nucleotidases (3). The thyroid enzyme is similar to those from rat liver (1,18,21), rat kidney (2), human placenta (“high Km”, 3) but different from bovine liver (22), human placenta (23) and human seminal plasma (4). Cellular 59-nucleotidases also include membrane-bound enzymes. Intracellular and membrane enzymes have distinctly different properties. The plasma membrane 59-nucleotidase from pig thyroid displayed an optimum at pH 7.5 and preferred AMP as a substrate (unpublished study). Plasma membrane 59-nucleotidase is still an enzyme in search for a function. The membrane-bound 59-nucleotidase as an ectoenzyme can provide a regulatory mechanism for local adenosine levels, which in turn may influence the intracellular levels of cAMP by interaction with adenyl cyclase (24). The soluble 59-nucleotidase may play a special role in the degradation of cytosolic nucleotides
FIG. 6. Effect of Na+ on activity of 59-nucleotidase. The enzyme activity was measured with 59 IMP under standard conditions except for the concentration of NaCl as indicated. 475
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BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS
and the maintenance of a stable pool for normal cell function (25). The soluble 59-nucleotidase in the thyroid prefers IMP as a substrate. It was shown that in pig thyroid also exists AMP deaminase catalyzing deamination of AMP to IMP (26). The activity of this enzyme is regulated by adenine and guanine nucleotides, inorganic phosphate and potassium ions. One could assume, that in the first step AMP is deaminated to IMP by deaminase and then IMP is dephosphorylated by soluble 59-nucleotidase. The two enzymes which participate in the AMP catabolic deamination and dephosphorylation in the thyroid, share common regulatory properties. REFERENCES 1. 2. 3. 4. 5. 6. 7. 8. 9. 10. 11. 12. 13. 14. 15. 16. 17. 18. 19. 20. 21. 22. 23. 24. 25. 26.
Itoh, R. (1981) Biochem. Biophys. Acta 657, 402–410. Le Hir, M. (1991) Biochem. J. 273, 795–798. Spycha.a, J., Madrit-Marina, V., and Fox, I. H. (1988) J. Biol. Chem. 263, 18759–18765. Minelli, A., Moroni, M., Fabiani, R., Miscetti, P., and Mezzasoma, I. (1991) Biochim. Biophys. Acta 1080, 252–258. Skladanowski, A. C., and Newby, A. C. (1990) Biochem. J. 268, 117–122. Truong, V. L., Collinson, A. R., and Lowenstein, J. M. (1988) Biochem. J. 253, 117–121. Le Hir, M., Gandhi, R., and Dubach, U. C. (1989) Enzyme 41, 87–93. Thompson, L. F., Ruedi, J. M., and Low, M. G. (1987) Biochem. Biophys. Res. Commun. 145, 118–125. Dornand, J., Bonnafous, J-C, and Mani, J-C. (1978) Eur. J. Biochem. 87, 459–465. Itami, H., Sakai, Y., Shimamoto, T., Hama, H., Tsuda, M., and Tsuchiya, T. (1989) J. Biochem. 105, 785–789. Ikura, Y., and Horikoshi, K. (1989) J. Ferment. Bioeng. 67, 210–211. Ohisalo, J. J. (1987) Med. Biol. 65, 181–191. De Wolf, M. J. S., Hilderson, H. J. J., Lagrou, A. R., and Dierick, W. S. H. (1978) Arch. Int. Physiol. Biochim. 86, 37–52. Matsuzaki, S., Pochet, R., and Schell-Frederick, E. (1973) Biochim. Biophys. Acta 313, 329–337. Peeters, C., de Wolf, M., Van Dessel, G., Lagrou, A., Hilderson, H., and Dierick, W. (1988) Int. J. Biochem. 20, 409–419. Chen, P. S., Toribara, T. Y., and Warner, H. (1956) Anal. Chem. 28, 1756–1758. Lowry, O. H., Rosebrough, N. H., Farr, A. L., and Randall, R. J. (1951) J. Biol. Chem. 193, 265–275. Van den Berge, G., Van Pottersberghe, Ch., and Hers, H-G. (1977) Biochem. J. 162, 611–616. Itoh, R., Oka, J., and Osaza, H. (1986) Biochem. J. 235, 847–851. Fritzon, P. (1969) Biochim. Biophys. Acta 178, 534–541. Itoh, R., Mitsui, A., and Tokushima, K. (1968) J. Biochem. 63, 165–169. Zekri, M., Harb, J., Bernard, S., and Meflah, K. (1988) Eur. J. Biochem. 172, 93–99. Madrit-Marina, V., and Fox, I. H. (1986) J. Biol. Chem. 261, 444–452. Wolff, J., Londos, C., and Cook, G. H. (1978) Archs. Biochem. Biophys. 191, 161–168. Edwards, N. L., Recker, D., Manfredi, J., and Rembecki, R. (1982) Am. J. Physiol. 243, C270–277. Stelmach, H., and Jaroszewicz, L. (1981) Biochim. Biophys. Res. Commun. 101, 144–152.
476
221, 471–476 (1996)
0619
Soluble 59-Nucleotidase from Thyroid Gland Partial Purification and Properties Janina Niedz´wiecka and Leokadia Jaroszewicz Department of Physical Chemistry, Medical Academy, 15-230 Bialystok, ul.Mickiewicza 2, Poland Received February 26, 1996 A soluble 59-nucleotidase from pig thyroid was purified over 110-fold by chromatography on phosphocellulose, (NH4)2SO4 precipitation and gel filtration on Sephadex G-150. The purified 5-nucleotidase was free of non-specific phosphatases. The enzyme had optimum pH at 6.5 and hydrolysed preferentially IMP and GMP. The Km values were 0.66 and 1.0 mM for IMP and GMP, respectively. The enzyme also hydrolysed other nucleotides and showed the following relative Vmax: IMP > GMP > CMP > AMP > UMP.Mg2+ was necessary for the enzyme activity. © 1996 Academic Press, Inc.
Purine nucleotide dephosphorylation is an obligatory step in nucleotide catabolism. 59Nucleotidase (EC 3.1.3.5) catalyzing the dephosphorylation of purine and pyrimidine rybo- and deoxyrybonucleoside monophosphates, has been described in various mammalian tissues (1–9) as well as in microorganisms (10,11). The membrane-bound ecto-59-nucleotidase has been known for many years and has been used extensively as a plasma membrane marker in subcellular fractionation techniques. Although its physiological function is not completely understood, it has been assumed that ecto-59-nucleotidase hydrolyses extracellular nucleotides into the membranepermeable nucleosides. One of this nucleosides, namely adenosine exerts different regulatory function in various organs (12). Besides the membrane-bound enzyme, the two types of soluble 59-nucleosidases have been described in mammals (3,23). One form, called “high Km” 59nucleotidase, found in rat and chicken liver (1,18), rat heart (19) and human placenta (3) has maximal activity in the presence of IMP and GMP, and is characterized by millimolar Km values, activation by ATP and inhibition by inorganic phosphate. The other soluble 59-nucleotidase “low Km” found in human placenta (23) shows substrate preference for AMP and pyrimidine nucleoside monophosphates, micromolar Km values and inhibition by ATP. Subcellular studies of bovine thyroid indicate that 59-nucleosidase is predominantly associated with plasma membranes (13). Matsuzaki et al.(14) showed that 59-nucleotidase activity was preferentially located in the pellet of bovine thyroid but approximately 27% of the total activity was recovered in the supernatant. The plasma-membrane bound 59-nucleotidase from thyroid gland has been purified and characterized (15). Neither the soluble enzyme from the thyroid has until now not been purified nor have its properties been studied. In this study we report the purification and some properties of soluble 59-nucleotidase from the pig thyroid gland. MATERIALS AND METHODS Materials. AMP, IMP, CMP, GMP, p-nitrophenyl phosphate (dicyclohexylammonium salt), ethylene-diamine-tetraacetic acid (EDTA) were obtained from Sigma Chemical Co., Sephadex G-150 from Farmacia Fine Chemicals, Tris (hydroxymethyl) amino methane, b-mercaptoethanol (Et-SH) from Fluka, phosphate cellulose from Whatman and other reagents of analytical grade from POCH Gliwice, Poland. The pig thyroid glands were obtained from the slaughterhouse immediately after killing the animals. Purification of soluble 58-nucleotidase. After removal of connective tissue and fat, the finely-chopped thyroid glands were homogenized in 0.05 M Tris-HCl buffer containing 1 mM EDTA and 10 mM Et-SH (1 g of tissue + 4 ml of buffer) in the Waring Blendor for 1 min (at high speed) four times. The homogenate was filtered through four-fold gauze and the filtrate was centrifuged at 10000 g for 15 min. The precipitate was suspended in the mentioned above buffer while the supernatant was centrifuged at 105000 g for 1 h. After centrifugation the supernatant was used for further purification. 471 0006-291X/96 $18.00 Copyright © 1996 by Academic Press, Inc. All rights of reproduction in any form reserved.
Vol. 221, No. 2, 1996
BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS TABLE 1 Purification of Soluble 59-Nucleotidase from Pig Thyroid
Step Supernatant Phosphocellulose eluate (NH4)2SO4 precipitate Sephadex G-150 filtrate
Specific activity (mmol/min/ mg protein)
Purification (fold)
Yield (%)
Volume (ml)
Protein (mg/ml)
Total activity (mmol/min)
315
41.4
48.25
0.0037
1
100.0
90
1.1
8.70
0.088
23.8
18.0
5
3.9
4.48
0.23
62.2
9.3
33
0.2
2.84
0.430
116.2
5.9
Phosphocellulose chromatography. Whatman P-11 cellulose phosphate (10g) was prepared as recommended by the manufacturer and was washed with 0.05 M Tris-HCl buffer of pH 7.4 containing 1 mM EDTA and 10 mM Et-SH. Cellulose phosphate suspension (140 ml) was mixed with supernatant (550 ml) prepared as described above, and was being mixed over night at 5°C. Then the suspension was filtered on the Büchner funnel and rinsed with 4 l of the same buffer. The column (2 × 40 cm) was then filled up with the filtrated suspension and the adsorbed protein was eluted by stepwise gradient of NaCl in the same buffer. (NH4)2SO4 precipitation. Fractions from cellulose phosphate column containing the 59-nucleotidase activity were pooled and precipitated between 40 and 60% of (NH4)2SO4 saturation. The precipitate was suspended in the buffer (about 10 ml) and centrifuged at 16000 g for 1 h to remove insoluble material. Gel filtration on Sephadex G-150. An active protein obtained after (NH4)2SO4 precipitation was applied on the Sephadex G-150 column (3.3 × 48 cm) previously equilibratated with 0.05 M Tris-HCl buffer of pH 7.4, containing 1 mM EDTA and 10 mM Et-SH. The same buffer was used for protein elution. Fractions with the highest activity of 59-nucleotidase were pooled and used for further investigations. 58-Nucleotidate activity. For routine assays, protein extracts were incubated for 30 min at 37°C in a reaction mixture containing 0.05 M Tris-HCl buffer of pH 6.5, 50 mM NaCl, 5mM MgCl2, 1 mM substrate solution and 0,02 - 0,05 mg protein in a total volume of 1 ml. After stopping the reaction by adding 1 ml 10% trichloroacetic acid, precipitated protein was removed by centrifugation and 0,25 ml of the supernatant was assayed for the release of inorganic phosphate according
FIG. 1. Sephadex G-150 gel filtration of 59-nucleotidase. The active 59-nucleotidase fraction obtained after (NH4)2SO4 precipitation (about 8 ml) was applied to the column (3,5 × 48 cm) equilibrated with 0.05 M Tris-HCl buffer, pH 7.4 containing 1 mM EDTA and 10 mM Et-SH. The enzyme was eluted with the same buffer and 3 ml fractions were collected. 472
Vol. 221, No. 2, 1996
BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS TABLE 2 Substrate Specificity of Purified Soluble 59Nucleotidase from Pig Thyroid
Substrate (1 mM)
Activities (nmol/min per mg of protein)
Relative activity (%)
440 416 90 212 60 0
100.0 93.8 20.7 48.3 13.8 0.0
59-IMP 59-GMP 59-AMP 59-CMP 59-UMP p-Nitrophenylphosphate
Experiments were performed under standard assay conditions in 0,05 M Tris-HCl buffer at pH 6,5.
to the method of Chen et al (16). All the experimental values were corrected by subtracting the respective control, i.e. the amount of inorganic phosphate present in parallel assays performed with acid precipitated samples. The specific activity was expressed in mmol of released inorganic phosphate × min−1 × mg of proteins−1. Non-specific phosphatase activity. The acid phosphatase activity and alkaline phosphatase activity were determined by using 4,8 mM p-nitrophenyl phosphate as a substrate in 0,2 M sodium citrate - citric acid buffer of pH 5 for acid phosphatase and glicine - MgCl2 - NaOH buffer of pH 10,3 for alkaline phosphatase. The reaction was started by adding the enzyme, then run at 37°C for 30 min, and stopped by adding 2 volumes of 1 m NaOH and optical density was read at 410 nm. Protein determination. Protein concentration was determined by the method of Lowry et al.(17) using bovine albumin as a standard. Proteins eluting from the columns were monitored by absorbance at 280 nm.
RESULTS The results of 59-nucleotidase purification at particular stages are summarized in Table I. By means of chromatography on phosphocellulose, (NH4)2SO4 precipitation and gel filtration on Sephadex G-150, over 110-fold purification of the enzyme with specific activity about 0.4 mmoles × min−1 × mg of protein, was obtained. On the step of phosphocelullose chromatography all non-specific phosphatase activity is removed. This non-phosphatase activity does not bind to phosphocelullose. 59-Nucleotidase adsorbed on phoshocelullose was eluted with the sodium chloride gradient from 0.4 to 0.6 M. The fractions containing 59-nucleotidase activity were pooled and fractionated with ammonium sulfatate between 0–30 %, 30–40% and 40–60% of saturation. The highest enzyme activity was found in the fraction
FIG. 2. Effect of pH on the activity of soluble thyroid 59-nucleotidase. Experiments were performed in 0,2 M Tris-HCl buffer, using 59 IMP as a substrate. 473
Vol. 221, No. 2, 1996
BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS
FIG. 3. Influence of substrate concentration on 59-nucleotidase activity. Activities were measured at pH 6,5 with 59 IMP and 59 GMP as substrates.
of 40–60% of saturation. The gel filtration on Sephadex G-150 showed a single peak of the 59-nucleotidase activity (Fig. 1). Properties of Enzyme Substrate specificity. The activity of soluble 59-nucleotidase towards various nucleoside monophosphates is shown in Table II. The preferred substrates for the purified enzyme were IMP and GMP. The enzyme also hydrolysed other nucleotides and showed the following relative Vmax values:IMP > GMP > CMP > AMP > UMP. p-Nitrophenyl phoshapte was not hydrolysed. Effect of pH. The optimum pH of soluble pig thyroid 59-nucleotidase was about 6.5 with IMP a substrate (Fig. 2). Kinetic properties. The soluble 59-nucleotidase showed Michaelis-Menten kinetics for both preferred substrates, IMP and GMP (Fig. 3). The Michaelis constant according to the LineweaverBurk plot was 660 mM for IMP and 1000 mM for GMP. Vmax was 0.48 and 0.416 mmoles/min per mg of protein for IMP and GMP, respectively (Fig. 4). Effect of MgCl2 and NaCl on enzyme activity. The 59-Nucleotidase activity was dependent on Mg2+ increasing with its concentration to 2 mM and remained unchanged at higher concentration (Fig. 5). Also the influence of NaCl on enzyme activity has been studied. 59-Nucleotidase showed maximal activity at the NaCl concentration of 10–40 mM (Fig. 6).
FIG. 4. Lineweaver-Burk plots for enzymatic dephosphorylation. 474
Vol. 221, No. 2, 1996
BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS
FIG. 5. Effect of Mg2+ on activity of 59-nucleotidase. The enzyme activity was measured with 59 IMP under standard conditions except for the concentration of MgCl2 as indicated.
DISCUSSION In this study the soluble 59-nucleotidase from pig thyroid was purified over 110-fold to a specific activity of 0.43 mmol/min/mg. In the process of purification, the soluble 59-nucleotidase was separated from non-specific phosphatases during chromatography on phosphocellulose. The apparent almost 80% loss of 59-nucleotidase activity during this step may be related in part to the removal of non-specific phosphatases. 59-Nucleotidase from the pig thyroid shows several properties in common with the soluble enzyme from other tissues (1,3,18,21). The enzyme from the thyroid has an optimum at pH 6.5 and is Mg2+ dependent. IMP and GMP were the preferred substrates. The Km values were 0.66 mM for IMP and 1 mM for GMP. The properties of the presented 59-nucleotidase indicate that the thyroid enzyme belongs to the soluble “high Km” 59-nucleotidases (3). The thyroid enzyme is similar to those from rat liver (1,18,21), rat kidney (2), human placenta (“high Km”, 3) but different from bovine liver (22), human placenta (23) and human seminal plasma (4). Cellular 59-nucleotidases also include membrane-bound enzymes. Intracellular and membrane enzymes have distinctly different properties. The plasma membrane 59-nucleotidase from pig thyroid displayed an optimum at pH 7.5 and preferred AMP as a substrate (unpublished study). Plasma membrane 59-nucleotidase is still an enzyme in search for a function. The membrane-bound 59-nucleotidase as an ectoenzyme can provide a regulatory mechanism for local adenosine levels, which in turn may influence the intracellular levels of cAMP by interaction with adenyl cyclase (24). The soluble 59-nucleotidase may play a special role in the degradation of cytosolic nucleotides
FIG. 6. Effect of Na+ on activity of 59-nucleotidase. The enzyme activity was measured with 59 IMP under standard conditions except for the concentration of NaCl as indicated. 475
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BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS
and the maintenance of a stable pool for normal cell function (25). The soluble 59-nucleotidase in the thyroid prefers IMP as a substrate. It was shown that in pig thyroid also exists AMP deaminase catalyzing deamination of AMP to IMP (26). The activity of this enzyme is regulated by adenine and guanine nucleotides, inorganic phosphate and potassium ions. One could assume, that in the first step AMP is deaminated to IMP by deaminase and then IMP is dephosphorylated by soluble 59-nucleotidase. The two enzymes which participate in the AMP catabolic deamination and dephosphorylation in the thyroid, share common regulatory properties. REFERENCES 1. 2. 3. 4. 5. 6. 7. 8. 9. 10. 11. 12. 13. 14. 15. 16. 17. 18. 19. 20. 21. 22. 23. 24. 25. 26.
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