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[64] 5′-Methylthioadenosine nucleosidase (Lupinus luteus seeds)
MTA NUCLEOSIDASE(L. luteus SEEDS)
[64]
365
[64] 5 ' - M e t h y l t h i o a d e n o s i n e N u c l e o s i d a s e (Lupinus luteus S e e d s )
By ANDRZEJ B. GURANOWSKI, PETER K. CHIANG, and GIULIO L. CANTONI 5'-Methylthioadenosine+ H20 ~ 5'-methylthioribose+ adenine Assay Method
Principle. Nucleosidase activity can be determined by measurement of radioactive adenine formation from 5'-methylthiol[adenine-U-14C] adenosine. Preparation of Radioactive 5'-Methylthioadenosine. 5'-Methylthio[adenine-U-14C]adenosine was prepared as follows. To the reaction mixture containing 50 mM HEPES 1 buffer, pH 7.6, 100 mM KC1, 5 mM MgCl2, l0 mM L-methionine, and 72 I.tM [adenine-U-14C]adenosine triphosphate, excess AdoMet I synthetase from yeast was added. (A purification procedure of the yeast AdoMet synthetase is described below.) After a 3-hr incubation at 37°, the mixture was heated at 100° to degrade the labeled AdoMet to MeSAdo, l which was next purified by chromatography on cellulose plates developed in distilled water. The labeled MeSAdo was :stored with l0 mM dithiothreitol. In the chromatographic system used, MeSAdo migrated as a sharp band (Rf = 0.45), and was readily separated from unconverted ATP and methionine, which migrated with the front, as well as from undegraded AdoMet (Rf = 0.03-0.08) and traces of adenine (Rf = 0.28). 5'-[methyl-14C]Methylthioadenosine was prepared in a similar way, and in this case the incubation mixture contained 5 mM ATP and 400 tzM L-[methyl-14C]methionine. Reagents and Chemicals Buffers A and B: 5% glycerol in l0 or 30 mM potassium phosphate (pH 6.8), respectively Buffer C: 50 mM BicinC-KOH (pH 8.3) and 5% glycerol Thin-layer chromatographic plates and aniline phthalate spray (E. Merck, Darmstadt, Federal Republic of Germany) ~Abbreviations: MeSAdo, 5'-deoxy-5'-methylthioadenosine;AdoHcy, S-adenosylhomocysteine; AdoMet, S-adenosylmethionine; HEPES, 4-(2-hydroxyethyl)-l-piperazineethanesulfonic acid; Bicine, N,N-bis(2-hydroxyethyl)glycine;DEAE, diethylaminoethyl; EDTA, ethylenediaminetetraaceticacid. METHODS IN ENZYMOLOGY, VOL. 94
Copyright © 1983 by Academic Press, Inc. All rights of reproduction in any form reserved. ISBN 0-12-181994-9
366
5'-METHYLTHIOADENOSINE AND 5-METHYLTHIORIBOSE
[64]
AdoHcy~-Sepharose prepared by the procedure of Kim et al. 2 Hydroxyapatite (BioGel HTP; Bio Rad Laboratories) Enzymes. AdoMet synthetase II was purified by a modification of the procedure described by Chiang and Cantoni. 3 The enzyme extract was processed essentially as described up to step 5, which was the first DEAE-cellulose I column, and then the fractions containing the highest specific activity of AdoMet synthetase II were concentrated to 5 ml by Aquacide III. The concentrated enzyme solution was applied onto a 400ml column (2.6 x 86 cm) of Sephadex G-100, equilibrated with 20 mM potassium phosphate (pH 6.8), 5 mM dithiothreitol, 0.5 mM EDTA.I The peak fractions from the Sephadex G-100 column were next adsorbed onto a 40-ml column (2.5 × 7.5 cm) of hydroxyapatite that had been equilibrated with 20 mM potassium phosphate (pH 6.8), 5 mM dithiothreitol, and 0.5 mM EDTA. The column was washed with the same buffer, then AdoMet synthetase II was eluted with a 500-ml linear gradient of 20 to 180 mM potassium phosphate (pH 6.8), containing 5 mM dithiothreitol and 0.5 mM EDTA. The fractions containing the highest enzyme activity were eluted at about 6.5 mmho. They were concentrated to 4 ml by Aquacide III, and applied onto a Sephadex G-150 column (2.6 × 60 cm) that had been equilibrated with 50 mM Tris (pH 7.8), 0.5 mM EDTA, 40 mM KC1, and 5 mM dithiothreitol. AdoMet synthetase was chromatographed with the same buffer. The specific activity of the AdoMet synthetase II purified at this stage was about 30 units/mg, each unit being as defined previously, 3 and the enzyme was homogeneous by polyacrylamide gel electrophoresis. Procedure. The standard incubation mixture contained, in a final volume of 50 /zl: 50 mM Bicine-KOH (pH 8.3), 100 txM radioactive MeSAdo, and the nucleosidase. Incubation at 37° was for 2-30 min depending on enzyme activity, and the reaction was stopped by heating at 100° for 1 min. Aliquots of the reaction mixture were applied to a cellulose paper, and chromatograms were developed with water. The spots containing radioactive adenine were cut out and immersed in 5 ml of aquasol; the radioactivity was determined. Definition of Unit. One unit of MeSAdo nucleosidase catalyzes the cleavage of I /zmol of substrate in 1 min at 37°. Purification The ionic strength of the extracting fluid strongly affects the solubility of lupin seed proteins. 4 Since MeSAdo nucleosidase can be extracted in good yields with low-ionic-strength buffers, it can be separated from the 2 S. Kim, S. Nochumson, W. Chin, and W. K. Paik, Anal. Biochem. 84, 415 (1978). 3 p. K. Chiang and G. L. Cantoni, J. Biol. Chem. 252, 4506 (1977). 4 H. Jakubowski and J. Pawelkiewicz, Acta Biochirn. Pol. 21, 271 (1974).
[64]
MTA NUCLEOSIDASE(L. luteus SEEDS)
367
bulk of other seed proteins that are insoluble under these conditions. After selective extraction, the four conventional steps described here yield a preparation about 20% pure with an 1860-fold purification. The last step utilizes adsorption on AdoHcy-Sepharose and desorption with MeSAdo. The purified MeSAdo nucleoside is a homogeneous preparation with a molecular weight of 62,000, representing an overall purification of 8900-fold and a yield of 12%. The enzyme consists of two subunits, each with a molecular weight of 31,000. The purified enzyme has a specific activity of 27 units per milligram of protein. The meal of yellow lupin seeds (670 g) was extracted with 2700 ml of buffer A for 30 min. The extract was next passed through cheesecloth and centrifuged at 30,000 g for 20 min. The MeSAdo nucleosidase was precipitated with ammonium sulfate (27-47% saturation) from the supernatant. The precipitate was dissolved in buffer B; after dialysis against this buffer and centrifugation, the supernatant was applied onto a DEAE-Sephacel column (5 x 33 cm) equilibrated with the same buffer. The column was washed with buffer B, 1 and a 3.5-1 linear gradient of 30 to 250 mM potassium phosphate (pH 6.8) and 5% glycerol was applied. The nucleosidase activity emerged at about 200 mM potassium phosphate. The enzyme was precipitated with ammonium sulfate (60% saturation), dissolved in a small volume of buffer B, and chromatographed on a Sephadex G-200 column (2.5 x 80 cm). It eluted at Ve/Vo = 1.8 and was concentrated by ammonium sulfate precipitation. The enzyme was dissolved in buffer A, dialyzed, and applied onto a hydroxyapatite column (1 x 10 cm) equilibrated with the same buffer. The column was washed with 30 ml of buffer A, and a 140-ml linear gradient of 10 to 80 mM potassium phosphate (pH 6.8) and 5% glycerol was applied. The active enzyme fractions, which eluted between 15 and 25 mM potassium phosphate, were pooled, concentrated with Aquacide I, dialyzed against buffer C, 1 and then applied onto an AdoHcy-Sepharose column (1 x 7 cm) equilibrated with the same buffer. The column was first washed with 30 ml of buffer C, and next with 20 ml of buffer C containing 200 mM KC1. Finally, MeSAdo nucleosidase was eluted from the affinity column with buffer C containing 200 mM KC1 and 10/.tM MeSAdo. The active fractions were pooled, concentrated with Aquacide I, dialyzed against buffer C, and stored frozen at -20 °. All the operations were carried out at about 4 °.
Properties Michaelis Constants. The Km value for MeSAdo estimated for lupin MeSAdo nucleosidase was 0.41 /xM, and is about the same as those of
368
5'-METHYLTHIOADENOSINE AND 5-METHYLTHIORIBOSE
[64]
MeSAdo phosphorylase from rat liver 5 and MeSAdo nucleosidase from Escherichia c o l i , 6 0.47 and 0.31 /zM, respectively. The MeSAdo Km values are 10.3 and 15/~M for MeSAdo nucleosidase from Vinca r o s e a 7 and tomato, 8 respectively. The MeSAdo Km values reported for MeSAdo phosphorylase from rat prostate and human placenta are 0.3 mM 9 and 43.5/zM, 10 respectively. Substrate Specificity. A number of thioether analogs of MeSAdo modified in the purine base, sugar, or aliphatic residues were checked as potential substrates for the lupin MeSAdo nucleosidase, lj The enzyme liberates the free base from most of them, and tolerates structural changes both in the nucleoside moiety and aliphatic residue of MeSAdo. The nucleosidase discriminates, however, between the analogs with and without an a-amino group in their aliphatic residues, since AdoHcy and Sadenosylcysteine are not degraded, in contrast to S-adenosylpropionic acid. Under our experimental conditions, MeSAdo and its adenosyl analogs are better substrates than their deaminated (inosyl) counterparts. The Vma~for MeSAdo is 50 times higher than that for 5'-methylthioinosine, the Km for the latter being 55/~M. 5'-Deoxyadenosine, a highly active substrate for the MeSAdo-cleaving enzyme of Sarcoma 180 cells, 12 is a very poor substrate for the lupin MeSAdo nucleosidase. Inhibition Studies. The following compounds were checked as potential effectors of the reaction catalyzed by the lupin MeSAdo nucleosidase: ribose, 5'-methylthioribose, adenine, 3-deazaadenine, hypoxanthine, cytosine, uracil, adenosine, 3-deazaadenosine, AMP, ATP, AdoHcy, putrescine, spermidine, spermine, 9-erythro-(2-hydroxyl-3-nonyl)adenine, sinefungin, decoyinine, (S)-9-(2,3-dihydroxypropyl)adenine and adenosine 5'-carboxamide, methionine, homocystein, dithiothreitol, and p-hydroxymercuribenzoate. Only some of them can exert an inhibitory effect. Adenine, a product of the nucleosidase activity, is a potent competitive inhibitor (Ki = 11/zM). In contrast, 5'-methylthioribose, the other product of the reaction, is a poor inhibitor ( g i = 1060/zM). The enzyme can be inhibited by 3-deazaadenine (Ki -- 19/xM), and 9-erythroA. J. Ferro, N. C. Wrobel, and J. A. Nicolette, Biochim. Biophys. Acta 570, 65 (1979). 6 A. J. Ferro, A. Barrett, and S. K. Shapiro, Biochim. Biophys. Acta 438, 487 (1976). 7 C. Baxter and C. J. Coscia, Biochem. Biophys. Res. Commun. 54, 147 (1973). 8 y. Yu, D. O. Adams, and S. F. Yang, Arch. Biochem. Biophys. 198, 280 (1979). 9 A. E. Pegg and H. G. Williams-Ashman, Biochem. J. 115, 241 (1969). ~0M, Carteni-Farina, F. Della Ragione, G. Ragosta, A. Oliva, and V. Zappia, FEBS Lett. 104, 266 (1979). See also this volume [60]. 11 A. B. Guranowski, P. K. Chiang, and G. L. Cantoni, Eur. J. Biochem. 114, 293 (1981). z~T. M. Savarese, G. W. Crabtree, and R. E. Parks, Jr., Biochem. Pharmacol. 28, 2227 (1979).
[64]
M T A NUCLEOSIDASE (L. luteus SEEDS)
369
(2-hydroxyl 3-nonyl)adenine (Ki = 37/zM), the latter being known chiefly as an inhibitor of adenosine deaminase. 13AdoHcy is a less effective inhibitor (Ki = 135 /zM). Sinefungin, t4,z5 decoyinine, ~6 (S)-9-(2,3-dihydroxypropyl)adenine (a known inhibitor of other enzymatic reactions17,18), and adenosine 5'-carboxamide (known as an effector of coronary dilatory action 19) can also inhibit the lupin MeSAdo nucleosidase. Their corresponding Ki values are 122, 133,638, and 482/zM, respectively. Neither dithiothreitol nor p-hydroxymercuribenzoate can affect the reaction of lupin MeSAdo nucleosidase, and this suggests that sulfhydryl groups are not essential for the enzyme activity. The other compounds have no apparent effect on lupin MeSAdo nucleosidase, and it also has been reported that adenosine, ATP, AdoHcy, and putrescine do not inhibit MeSAdo phosphorylase of Drosophila melanogaster. 2°
13 H. J. Schaeffer and C. F. Schwender, J. Med. Chem. 17, 6 (1974). 14R. W. Fuller, in "Transmethylation" (E. Usdin, R. T. Borchardt, and C. R. Creveling, eds.), p. 251. Elsevier/North-Holland, Amsterdam, 1979. 15 R. T. Borchardt and C. S. G. Pugh, in "Transmethylation" (E. Usdin, R. T. Borchardt, and C. R. Creveling, eds.), p. 197. Elsevier/North-Holland, Amsterdam, 1979. t6 R. J. Suhadolnik, "Nucleoside Antibiotics." Wiley-Interscience, New York, 1970. 17 H. J. Schaeffer, D. Vogel, and R. Vince, J. Med. Chem. 8, 502 0965). t8 E. DeClercq, J. Descamps, P. DeSomer, and A. Holy, Science 200, 563 (1978). 19 G. Raberger, W. Schutz, and O. Kraupp, Arch. Int. Pharmacodyn. Ther. 230, 140 (1977). 2o L. Shugart, M. Tancer, and J. Moore, Int. J. Biochem. 10, 901 (1979).
[64]
365
[64] 5 ' - M e t h y l t h i o a d e n o s i n e N u c l e o s i d a s e (Lupinus luteus S e e d s )
By ANDRZEJ B. GURANOWSKI, PETER K. CHIANG, and GIULIO L. CANTONI 5'-Methylthioadenosine+ H20 ~ 5'-methylthioribose+ adenine Assay Method
Principle. Nucleosidase activity can be determined by measurement of radioactive adenine formation from 5'-methylthiol[adenine-U-14C] adenosine. Preparation of Radioactive 5'-Methylthioadenosine. 5'-Methylthio[adenine-U-14C]adenosine was prepared as follows. To the reaction mixture containing 50 mM HEPES 1 buffer, pH 7.6, 100 mM KC1, 5 mM MgCl2, l0 mM L-methionine, and 72 I.tM [adenine-U-14C]adenosine triphosphate, excess AdoMet I synthetase from yeast was added. (A purification procedure of the yeast AdoMet synthetase is described below.) After a 3-hr incubation at 37°, the mixture was heated at 100° to degrade the labeled AdoMet to MeSAdo, l which was next purified by chromatography on cellulose plates developed in distilled water. The labeled MeSAdo was :stored with l0 mM dithiothreitol. In the chromatographic system used, MeSAdo migrated as a sharp band (Rf = 0.45), and was readily separated from unconverted ATP and methionine, which migrated with the front, as well as from undegraded AdoMet (Rf = 0.03-0.08) and traces of adenine (Rf = 0.28). 5'-[methyl-14C]Methylthioadenosine was prepared in a similar way, and in this case the incubation mixture contained 5 mM ATP and 400 tzM L-[methyl-14C]methionine. Reagents and Chemicals Buffers A and B: 5% glycerol in l0 or 30 mM potassium phosphate (pH 6.8), respectively Buffer C: 50 mM BicinC-KOH (pH 8.3) and 5% glycerol Thin-layer chromatographic plates and aniline phthalate spray (E. Merck, Darmstadt, Federal Republic of Germany) ~Abbreviations: MeSAdo, 5'-deoxy-5'-methylthioadenosine;AdoHcy, S-adenosylhomocysteine; AdoMet, S-adenosylmethionine; HEPES, 4-(2-hydroxyethyl)-l-piperazineethanesulfonic acid; Bicine, N,N-bis(2-hydroxyethyl)glycine;DEAE, diethylaminoethyl; EDTA, ethylenediaminetetraaceticacid. METHODS IN ENZYMOLOGY, VOL. 94
Copyright © 1983 by Academic Press, Inc. All rights of reproduction in any form reserved. ISBN 0-12-181994-9
366
5'-METHYLTHIOADENOSINE AND 5-METHYLTHIORIBOSE
[64]
AdoHcy~-Sepharose prepared by the procedure of Kim et al. 2 Hydroxyapatite (BioGel HTP; Bio Rad Laboratories) Enzymes. AdoMet synthetase II was purified by a modification of the procedure described by Chiang and Cantoni. 3 The enzyme extract was processed essentially as described up to step 5, which was the first DEAE-cellulose I column, and then the fractions containing the highest specific activity of AdoMet synthetase II were concentrated to 5 ml by Aquacide III. The concentrated enzyme solution was applied onto a 400ml column (2.6 x 86 cm) of Sephadex G-100, equilibrated with 20 mM potassium phosphate (pH 6.8), 5 mM dithiothreitol, 0.5 mM EDTA.I The peak fractions from the Sephadex G-100 column were next adsorbed onto a 40-ml column (2.5 × 7.5 cm) of hydroxyapatite that had been equilibrated with 20 mM potassium phosphate (pH 6.8), 5 mM dithiothreitol, and 0.5 mM EDTA. The column was washed with the same buffer, then AdoMet synthetase II was eluted with a 500-ml linear gradient of 20 to 180 mM potassium phosphate (pH 6.8), containing 5 mM dithiothreitol and 0.5 mM EDTA. The fractions containing the highest enzyme activity were eluted at about 6.5 mmho. They were concentrated to 4 ml by Aquacide III, and applied onto a Sephadex G-150 column (2.6 × 60 cm) that had been equilibrated with 50 mM Tris (pH 7.8), 0.5 mM EDTA, 40 mM KC1, and 5 mM dithiothreitol. AdoMet synthetase was chromatographed with the same buffer. The specific activity of the AdoMet synthetase II purified at this stage was about 30 units/mg, each unit being as defined previously, 3 and the enzyme was homogeneous by polyacrylamide gel electrophoresis. Procedure. The standard incubation mixture contained, in a final volume of 50 /zl: 50 mM Bicine-KOH (pH 8.3), 100 txM radioactive MeSAdo, and the nucleosidase. Incubation at 37° was for 2-30 min depending on enzyme activity, and the reaction was stopped by heating at 100° for 1 min. Aliquots of the reaction mixture were applied to a cellulose paper, and chromatograms were developed with water. The spots containing radioactive adenine were cut out and immersed in 5 ml of aquasol; the radioactivity was determined. Definition of Unit. One unit of MeSAdo nucleosidase catalyzes the cleavage of I /zmol of substrate in 1 min at 37°. Purification The ionic strength of the extracting fluid strongly affects the solubility of lupin seed proteins. 4 Since MeSAdo nucleosidase can be extracted in good yields with low-ionic-strength buffers, it can be separated from the 2 S. Kim, S. Nochumson, W. Chin, and W. K. Paik, Anal. Biochem. 84, 415 (1978). 3 p. K. Chiang and G. L. Cantoni, J. Biol. Chem. 252, 4506 (1977). 4 H. Jakubowski and J. Pawelkiewicz, Acta Biochirn. Pol. 21, 271 (1974).
[64]
MTA NUCLEOSIDASE(L. luteus SEEDS)
367
bulk of other seed proteins that are insoluble under these conditions. After selective extraction, the four conventional steps described here yield a preparation about 20% pure with an 1860-fold purification. The last step utilizes adsorption on AdoHcy-Sepharose and desorption with MeSAdo. The purified MeSAdo nucleoside is a homogeneous preparation with a molecular weight of 62,000, representing an overall purification of 8900-fold and a yield of 12%. The enzyme consists of two subunits, each with a molecular weight of 31,000. The purified enzyme has a specific activity of 27 units per milligram of protein. The meal of yellow lupin seeds (670 g) was extracted with 2700 ml of buffer A for 30 min. The extract was next passed through cheesecloth and centrifuged at 30,000 g for 20 min. The MeSAdo nucleosidase was precipitated with ammonium sulfate (27-47% saturation) from the supernatant. The precipitate was dissolved in buffer B; after dialysis against this buffer and centrifugation, the supernatant was applied onto a DEAE-Sephacel column (5 x 33 cm) equilibrated with the same buffer. The column was washed with buffer B, 1 and a 3.5-1 linear gradient of 30 to 250 mM potassium phosphate (pH 6.8) and 5% glycerol was applied. The nucleosidase activity emerged at about 200 mM potassium phosphate. The enzyme was precipitated with ammonium sulfate (60% saturation), dissolved in a small volume of buffer B, and chromatographed on a Sephadex G-200 column (2.5 x 80 cm). It eluted at Ve/Vo = 1.8 and was concentrated by ammonium sulfate precipitation. The enzyme was dissolved in buffer A, dialyzed, and applied onto a hydroxyapatite column (1 x 10 cm) equilibrated with the same buffer. The column was washed with 30 ml of buffer A, and a 140-ml linear gradient of 10 to 80 mM potassium phosphate (pH 6.8) and 5% glycerol was applied. The active enzyme fractions, which eluted between 15 and 25 mM potassium phosphate, were pooled, concentrated with Aquacide I, dialyzed against buffer C, 1 and then applied onto an AdoHcy-Sepharose column (1 x 7 cm) equilibrated with the same buffer. The column was first washed with 30 ml of buffer C, and next with 20 ml of buffer C containing 200 mM KC1. Finally, MeSAdo nucleosidase was eluted from the affinity column with buffer C containing 200 mM KC1 and 10/.tM MeSAdo. The active fractions were pooled, concentrated with Aquacide I, dialyzed against buffer C, and stored frozen at -20 °. All the operations were carried out at about 4 °.
Properties Michaelis Constants. The Km value for MeSAdo estimated for lupin MeSAdo nucleosidase was 0.41 /xM, and is about the same as those of
368
5'-METHYLTHIOADENOSINE AND 5-METHYLTHIORIBOSE
[64]
MeSAdo phosphorylase from rat liver 5 and MeSAdo nucleosidase from Escherichia c o l i , 6 0.47 and 0.31 /zM, respectively. The MeSAdo Km values are 10.3 and 15/~M for MeSAdo nucleosidase from Vinca r o s e a 7 and tomato, 8 respectively. The MeSAdo Km values reported for MeSAdo phosphorylase from rat prostate and human placenta are 0.3 mM 9 and 43.5/zM, 10 respectively. Substrate Specificity. A number of thioether analogs of MeSAdo modified in the purine base, sugar, or aliphatic residues were checked as potential substrates for the lupin MeSAdo nucleosidase, lj The enzyme liberates the free base from most of them, and tolerates structural changes both in the nucleoside moiety and aliphatic residue of MeSAdo. The nucleosidase discriminates, however, between the analogs with and without an a-amino group in their aliphatic residues, since AdoHcy and Sadenosylcysteine are not degraded, in contrast to S-adenosylpropionic acid. Under our experimental conditions, MeSAdo and its adenosyl analogs are better substrates than their deaminated (inosyl) counterparts. The Vma~for MeSAdo is 50 times higher than that for 5'-methylthioinosine, the Km for the latter being 55/~M. 5'-Deoxyadenosine, a highly active substrate for the MeSAdo-cleaving enzyme of Sarcoma 180 cells, 12 is a very poor substrate for the lupin MeSAdo nucleosidase. Inhibition Studies. The following compounds were checked as potential effectors of the reaction catalyzed by the lupin MeSAdo nucleosidase: ribose, 5'-methylthioribose, adenine, 3-deazaadenine, hypoxanthine, cytosine, uracil, adenosine, 3-deazaadenosine, AMP, ATP, AdoHcy, putrescine, spermidine, spermine, 9-erythro-(2-hydroxyl-3-nonyl)adenine, sinefungin, decoyinine, (S)-9-(2,3-dihydroxypropyl)adenine and adenosine 5'-carboxamide, methionine, homocystein, dithiothreitol, and p-hydroxymercuribenzoate. Only some of them can exert an inhibitory effect. Adenine, a product of the nucleosidase activity, is a potent competitive inhibitor (Ki = 11/zM). In contrast, 5'-methylthioribose, the other product of the reaction, is a poor inhibitor ( g i = 1060/zM). The enzyme can be inhibited by 3-deazaadenine (Ki -- 19/xM), and 9-erythroA. J. Ferro, N. C. Wrobel, and J. A. Nicolette, Biochim. Biophys. Acta 570, 65 (1979). 6 A. J. Ferro, A. Barrett, and S. K. Shapiro, Biochim. Biophys. Acta 438, 487 (1976). 7 C. Baxter and C. J. Coscia, Biochem. Biophys. Res. Commun. 54, 147 (1973). 8 y. Yu, D. O. Adams, and S. F. Yang, Arch. Biochem. Biophys. 198, 280 (1979). 9 A. E. Pegg and H. G. Williams-Ashman, Biochem. J. 115, 241 (1969). ~0M, Carteni-Farina, F. Della Ragione, G. Ragosta, A. Oliva, and V. Zappia, FEBS Lett. 104, 266 (1979). See also this volume [60]. 11 A. B. Guranowski, P. K. Chiang, and G. L. Cantoni, Eur. J. Biochem. 114, 293 (1981). z~T. M. Savarese, G. W. Crabtree, and R. E. Parks, Jr., Biochem. Pharmacol. 28, 2227 (1979).
[64]
M T A NUCLEOSIDASE (L. luteus SEEDS)
369
(2-hydroxyl 3-nonyl)adenine (Ki = 37/zM), the latter being known chiefly as an inhibitor of adenosine deaminase. 13AdoHcy is a less effective inhibitor (Ki = 135 /zM). Sinefungin, t4,z5 decoyinine, ~6 (S)-9-(2,3-dihydroxypropyl)adenine (a known inhibitor of other enzymatic reactions17,18), and adenosine 5'-carboxamide (known as an effector of coronary dilatory action 19) can also inhibit the lupin MeSAdo nucleosidase. Their corresponding Ki values are 122, 133,638, and 482/zM, respectively. Neither dithiothreitol nor p-hydroxymercuribenzoate can affect the reaction of lupin MeSAdo nucleosidase, and this suggests that sulfhydryl groups are not essential for the enzyme activity. The other compounds have no apparent effect on lupin MeSAdo nucleosidase, and it also has been reported that adenosine, ATP, AdoHcy, and putrescine do not inhibit MeSAdo phosphorylase of Drosophila melanogaster. 2°
13 H. J. Schaeffer and C. F. Schwender, J. Med. Chem. 17, 6 (1974). 14R. W. Fuller, in "Transmethylation" (E. Usdin, R. T. Borchardt, and C. R. Creveling, eds.), p. 251. Elsevier/North-Holland, Amsterdam, 1979. 15 R. T. Borchardt and C. S. G. Pugh, in "Transmethylation" (E. Usdin, R. T. Borchardt, and C. R. Creveling, eds.), p. 197. Elsevier/North-Holland, Amsterdam, 1979. t6 R. J. Suhadolnik, "Nucleoside Antibiotics." Wiley-Interscience, New York, 1970. 17 H. J. Schaeffer, D. Vogel, and R. Vince, J. Med. Chem. 8, 502 0965). t8 E. DeClercq, J. Descamps, P. DeSomer, and A. Holy, Science 200, 563 (1978). 19 G. Raberger, W. Schutz, and O. Kraupp, Arch. Int. Pharmacodyn. Ther. 230, 140 (1977). 2o L. Shugart, M. Tancer, and J. Moore, Int. J. Biochem. 10, 901 (1979).