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Acetylated polyamines as substrates for human pregnancy serum diamine oxidase
Life Sciences, Vol. 29, pp. 2177-2179 Printed in the U.S.A.
Pergamon Press
ACETYLATED POLYAMINES AS SUBSTRATES FOR HUMAN PREGNANCY SERUM DIAMINE OXIDASE William A. Gahl and Henry C. Pitot Departments of Oncology, Pathology, and Pediatrics University of Wisconsin Center for Health Sciences Madison, Wisconsin 53706 (Received in final form September 22, 1981) Summary Human pregnancy serum diamine oxidase was purified 50 fold and tested for activity with a variety of subst~ates. Putrescine, spermi~ine, spermine, N-acetylputrescine, N -acetylspermidine, and N -acetylspermidine were acceptable substrates ifor the enzyme, which exhibited greatest activity against N -acetylspermidine. During human pregnancy, diamine oxidase (EC 1.4.3.6), which degrades both histamine (i) and putrescine (2), circulates in maternal serum. In this report, we present further information on the substrate specificity of pregnancy serum diamine oxidase. Methods Diamine Oxidase Assay. A m ~ i f i c a t i o n (3) of the radioactive method of Okuyama and Kobayashi (4), using C-putrescine as substrate, was employed for the measurement of diamine oxidase activity in fractions eluted from a chromatography column. When different amines were compared as substrates for diamine oxidase, the o-dianisidine/peroxidase method of Gunther and Glick (5) was used. Acetylated Polyamines. N-acetylputrescine, N l - a c e t y l s p e r m i d i n e , and NS-acetyl spermidine were synthesized as described by Tabor et al. (6). The acetylated compounds w~re identified by melting points and by paper chromatography using authentic N - and N -acetylspermidine kindly provided ~y Dr. M.M. Abdel-Monem (University of Minnesota, Minneapolis, MN). N -acetylspermidine was contaminated with 0.5% spermidine. Polyamines were separated by paper chromatography on Whatman No. 1 paper using both propan- 2-ol/85% (v/v) formic a~id/water 8 (8:1:1, by vol.) (7) and n-propanol/HCl/water (3:1:1, by vol.) (8). N - and N -acetylspermidine were separated on Schleicher and Schuell No. 507 paper using n-propanol/triethylamine/water (85:3:15, vol.) (8). Partial Purification of Diamine Oxidase. Pooled serum (100 ml) from women in the second and third trimesters of pregnancy was diluted to 500 ml with 0.01 M potassium phosphate, pH 8.0, and placed on a 3 x 7 cm column of cadaverineSepharose (9). A linear 500 ml gradient (0-0.5 M NaCI) in 0.05 M sodium phosphate, pH 7.5, was applied at a rate of 200 ml/h, and aliquots of 7.6 ml fractions were assayed for diamine oxidase. Active fractions (0.10-0.35 M NaCI) were pooled and dialyzed. 0024-3205/81/212177-03502.00/0 Copyright (c) 1981 Pergamon Press Ltd.
2178
Pregnancy Serum Diamine Oxidase
Protein Determination.
Vol. 29, No. 21, 1981
The method of Lowry et al.
(i0) was used.
Results A partially purified preparation of human pregnancy serum diamine oxidase was examined for activity against various substrates. A 1.2 ml aliquot of the enzyme produced 1.5 nanomoles of hydrogen peroxide per hour with spermidine as substrate, and the product formed from other amines was compared with this (TABLE I). Spermine, as well as 3 to ~ carbon aliphatic diamines, proved good substrates for the enzyme. So did N--acet{Ispermidine and N-acetylputrescine. However, the best substrate tested was N -acetylspermidine, which repeatedly formed over one and one-half times the hydrogen peroxide per hour produced by spermidine.
TABLE I Substrate Preferences of Human Pregnancy Serum Diamine Oxidase Substrate
Activity relative to spermidine
Spermidine Spermine Putrescine N=acetylputrescine I N_-acetylspermidine N -acetylspermldine Benzylamine Histamine 1,3-Diaminopropane 1,6-Diaminohexane 1,7-Diaminoheptane Methylamine Ethylamine Cadaverine Arginine Ethylenediamine
1.00 1.02 1.12 1.35 1.57 1.01 0.46 0.58 1.40 0.94 0.82 0 0.34 0.88 0 0
Pregnancy serum diamine oxidase was purified 50-fold as described in Methods. A 1.2 ml aliquot (120 g protein) was incubated for 5 h at 37°C in the presence of 1 mM substrate. Production of hydrogen peroxide was measured by the o-dianisidine/peroxidase assay. Results are means of duplicate determinations.
Discussion Diamine oxidase levels rise in maternal serum as pregnancy progresses, reaching a plateau at approximately 20 weeks' gestation (2). Several investigators have purified and characterized diamine oxidase from pregnancy plasma (9) and amniotic fluid (ii), and we have shown that amine oxidase activity against both putrescine and spermidine are contained within a single enzyme protein in human pregnancy serum (Biochem. J., submitted for publication). We report here that the pregnancy serum enzyme acts on a broad range of substrates, of which Nl_acetylspermidin e is the best. The pregnancy serum enzyme was initially called histaminase, reflecting the early use of histamine as substrate (i). Later, the enzyme was identified as
Vol. 29, No. 21, 1981
Pregnancy Serum Diamine Oxidase
2179
diamine oxidase, since putrescine was employed as substrate in a convenient radioactive assay (24). In view of the range of substrates degraded by the enzyme (TABLE I), the names histaminase and diamine oxidase may reflect currently available assays for amine oxidase activity rather than the enzyme's functional importance. Elevated levels of an enzyme with activity against spermidine, spermine, and monoacetylated spermidines may be an important accompaniment of the elevated levels of polyamines previously reported in pregnancy (12). Of greatest interest is {hat the pregnancy serum amine oxidase has as its most effective substrate, N -acetylspermidine, which is also the optimal substrate for rat liver polyamine oxidase (13) and an intermediate in spermidine degradation to putrescine (14). In analogy to the rat liver polyamine oxidase, o~e might expect the pregnancy serum enzyme to produce 3-acetamidopropanal from N -acetylspermidine (14) and 3-aminopropionaldehyde from spermidine (13). This would be in sharp contrast to the production of acrolein by bovine serum amine oxidase (15), and may reflect the importance of protecting a growing organism from the toxic effects of acrolein (16). Whatever the products, however, the finding that Nl-acetylspermidine is t~e most effective substrate for the pregnancy serum amine oxidase suggests that N -acetylspermidine may be a major polyaminelmetabolite in human pregnancy. It remains to be determined what portion of N -acetylspermidine is destined for urinary excretion and what portion is recycled to replenish the polyamine pool. Acknowledgements This work was supported in part by a grant from the Cystic Fibrosis Foundation for the study of polyamine-degrading enzymes, and by National Cancer Institute grant CA 22484. References i. 2. 3. 4. 5. 6. 7. 8. 9. 10. ii. 12. 13. 14. 15. 16.
H. SWANBERG, Acta Physiol. Scand 23 (Supp. 79), 1-69 (1950). E.R.CARRINGTON, G.J. FRISHMUTH, M.J. OESTERLING, F.M. ADAMS, and S.E. COX, Obstet. & Gynecol. 39, 426-430 (1972). W.A. GAHL, A.M. VALE, and H.C. PITOT, Biochem. J. 187, 197-204 (1980). T. OKUYAMA and Y. KOBAYASHI, Arch. Biochem. Biophys. 95, 242-250 (1961). R.E. GUNTHER and D. GLICK, J. Histochem. Cytochem. 15, 431-435 (1967). H. TABOR, C.W. TABOR, and L. DE MEIS, Method in Enzymology, Vol. 17, Part B, pp. 829-833, Academic Press, New York (1971). M. MENASHE, J. FABER, and U. BACHRACH, Biochem. J. 188, 263-267 (1980). D.T. DUBIN and S.M. ROSENTHAL, J. Biol. Chem. 235, 776-782 (1960). S.B. BAYLIN and S. MARGOLIS'" Biochim. Biophys. Acta 397, 294-306 (1975). O.H. LOWRY, N.J. ROSEBROUGH, A.L. FARR, and R.J. RANDALL, J. Biol. Chem. 193, 265-275 (1951). G. TUFVESSON, Scand. J. Clin. Lab. Invest. 38, 436-572 (1978). D.H. RUSSELL, H.R. GILES, C.D. CHRISTIAN, and J.L. CAMPBELL, Am. J. Obstet. & Gynecol. 132, 649-652 (1978). E. HOLTTA, Biochemistry 16, 91-100 (1977). I. MATSUI, L. WIEGAND, and A.E. PEGG, J. Biol. Chem. 256, 2454-2459 (1981). R.A. ALARCON, Arch. Biochem. Biophys. 137, 365-372 (1970). C. IZARD and C. LIBERMANN, Mutation Res. 47, 115-138 (1978).
Pergamon Press
ACETYLATED POLYAMINES AS SUBSTRATES FOR HUMAN PREGNANCY SERUM DIAMINE OXIDASE William A. Gahl and Henry C. Pitot Departments of Oncology, Pathology, and Pediatrics University of Wisconsin Center for Health Sciences Madison, Wisconsin 53706 (Received in final form September 22, 1981) Summary Human pregnancy serum diamine oxidase was purified 50 fold and tested for activity with a variety of subst~ates. Putrescine, spermi~ine, spermine, N-acetylputrescine, N -acetylspermidine, and N -acetylspermidine were acceptable substrates ifor the enzyme, which exhibited greatest activity against N -acetylspermidine. During human pregnancy, diamine oxidase (EC 1.4.3.6), which degrades both histamine (i) and putrescine (2), circulates in maternal serum. In this report, we present further information on the substrate specificity of pregnancy serum diamine oxidase. Methods Diamine Oxidase Assay. A m ~ i f i c a t i o n (3) of the radioactive method of Okuyama and Kobayashi (4), using C-putrescine as substrate, was employed for the measurement of diamine oxidase activity in fractions eluted from a chromatography column. When different amines were compared as substrates for diamine oxidase, the o-dianisidine/peroxidase method of Gunther and Glick (5) was used. Acetylated Polyamines. N-acetylputrescine, N l - a c e t y l s p e r m i d i n e , and NS-acetyl spermidine were synthesized as described by Tabor et al. (6). The acetylated compounds w~re identified by melting points and by paper chromatography using authentic N - and N -acetylspermidine kindly provided ~y Dr. M.M. Abdel-Monem (University of Minnesota, Minneapolis, MN). N -acetylspermidine was contaminated with 0.5% spermidine. Polyamines were separated by paper chromatography on Whatman No. 1 paper using both propan- 2-ol/85% (v/v) formic a~id/water 8 (8:1:1, by vol.) (7) and n-propanol/HCl/water (3:1:1, by vol.) (8). N - and N -acetylspermidine were separated on Schleicher and Schuell No. 507 paper using n-propanol/triethylamine/water (85:3:15, vol.) (8). Partial Purification of Diamine Oxidase. Pooled serum (100 ml) from women in the second and third trimesters of pregnancy was diluted to 500 ml with 0.01 M potassium phosphate, pH 8.0, and placed on a 3 x 7 cm column of cadaverineSepharose (9). A linear 500 ml gradient (0-0.5 M NaCI) in 0.05 M sodium phosphate, pH 7.5, was applied at a rate of 200 ml/h, and aliquots of 7.6 ml fractions were assayed for diamine oxidase. Active fractions (0.10-0.35 M NaCI) were pooled and dialyzed. 0024-3205/81/212177-03502.00/0 Copyright (c) 1981 Pergamon Press Ltd.
2178
Pregnancy Serum Diamine Oxidase
Protein Determination.
Vol. 29, No. 21, 1981
The method of Lowry et al.
(i0) was used.
Results A partially purified preparation of human pregnancy serum diamine oxidase was examined for activity against various substrates. A 1.2 ml aliquot of the enzyme produced 1.5 nanomoles of hydrogen peroxide per hour with spermidine as substrate, and the product formed from other amines was compared with this (TABLE I). Spermine, as well as 3 to ~ carbon aliphatic diamines, proved good substrates for the enzyme. So did N--acet{Ispermidine and N-acetylputrescine. However, the best substrate tested was N -acetylspermidine, which repeatedly formed over one and one-half times the hydrogen peroxide per hour produced by spermidine.
TABLE I Substrate Preferences of Human Pregnancy Serum Diamine Oxidase Substrate
Activity relative to spermidine
Spermidine Spermine Putrescine N=acetylputrescine I N_-acetylspermidine N -acetylspermldine Benzylamine Histamine 1,3-Diaminopropane 1,6-Diaminohexane 1,7-Diaminoheptane Methylamine Ethylamine Cadaverine Arginine Ethylenediamine
1.00 1.02 1.12 1.35 1.57 1.01 0.46 0.58 1.40 0.94 0.82 0 0.34 0.88 0 0
Pregnancy serum diamine oxidase was purified 50-fold as described in Methods. A 1.2 ml aliquot (120 g protein) was incubated for 5 h at 37°C in the presence of 1 mM substrate. Production of hydrogen peroxide was measured by the o-dianisidine/peroxidase assay. Results are means of duplicate determinations.
Discussion Diamine oxidase levels rise in maternal serum as pregnancy progresses, reaching a plateau at approximately 20 weeks' gestation (2). Several investigators have purified and characterized diamine oxidase from pregnancy plasma (9) and amniotic fluid (ii), and we have shown that amine oxidase activity against both putrescine and spermidine are contained within a single enzyme protein in human pregnancy serum (Biochem. J., submitted for publication). We report here that the pregnancy serum enzyme acts on a broad range of substrates, of which Nl_acetylspermidin e is the best. The pregnancy serum enzyme was initially called histaminase, reflecting the early use of histamine as substrate (i). Later, the enzyme was identified as
Vol. 29, No. 21, 1981
Pregnancy Serum Diamine Oxidase
2179
diamine oxidase, since putrescine was employed as substrate in a convenient radioactive assay (24). In view of the range of substrates degraded by the enzyme (TABLE I), the names histaminase and diamine oxidase may reflect currently available assays for amine oxidase activity rather than the enzyme's functional importance. Elevated levels of an enzyme with activity against spermidine, spermine, and monoacetylated spermidines may be an important accompaniment of the elevated levels of polyamines previously reported in pregnancy (12). Of greatest interest is {hat the pregnancy serum amine oxidase has as its most effective substrate, N -acetylspermidine, which is also the optimal substrate for rat liver polyamine oxidase (13) and an intermediate in spermidine degradation to putrescine (14). In analogy to the rat liver polyamine oxidase, o~e might expect the pregnancy serum enzyme to produce 3-acetamidopropanal from N -acetylspermidine (14) and 3-aminopropionaldehyde from spermidine (13). This would be in sharp contrast to the production of acrolein by bovine serum amine oxidase (15), and may reflect the importance of protecting a growing organism from the toxic effects of acrolein (16). Whatever the products, however, the finding that Nl-acetylspermidine is t~e most effective substrate for the pregnancy serum amine oxidase suggests that N -acetylspermidine may be a major polyaminelmetabolite in human pregnancy. It remains to be determined what portion of N -acetylspermidine is destined for urinary excretion and what portion is recycled to replenish the polyamine pool. Acknowledgements This work was supported in part by a grant from the Cystic Fibrosis Foundation for the study of polyamine-degrading enzymes, and by National Cancer Institute grant CA 22484. References i. 2. 3. 4. 5. 6. 7. 8. 9. 10. ii. 12. 13. 14. 15. 16.
H. SWANBERG, Acta Physiol. Scand 23 (Supp. 79), 1-69 (1950). E.R.CARRINGTON, G.J. FRISHMUTH, M.J. OESTERLING, F.M. ADAMS, and S.E. COX, Obstet. & Gynecol. 39, 426-430 (1972). W.A. GAHL, A.M. VALE, and H.C. PITOT, Biochem. J. 187, 197-204 (1980). T. OKUYAMA and Y. KOBAYASHI, Arch. Biochem. Biophys. 95, 242-250 (1961). R.E. GUNTHER and D. GLICK, J. Histochem. Cytochem. 15, 431-435 (1967). H. TABOR, C.W. TABOR, and L. DE MEIS, Method in Enzymology, Vol. 17, Part B, pp. 829-833, Academic Press, New York (1971). M. MENASHE, J. FABER, and U. BACHRACH, Biochem. J. 188, 263-267 (1980). D.T. DUBIN and S.M. ROSENTHAL, J. Biol. Chem. 235, 776-782 (1960). S.B. BAYLIN and S. MARGOLIS'" Biochim. Biophys. Acta 397, 294-306 (1975). O.H. LOWRY, N.J. ROSEBROUGH, A.L. FARR, and R.J. RANDALL, J. Biol. Chem. 193, 265-275 (1951). G. TUFVESSON, Scand. J. Clin. Lab. Invest. 38, 436-572 (1978). D.H. RUSSELL, H.R. GILES, C.D. CHRISTIAN, and J.L. CAMPBELL, Am. J. Obstet. & Gynecol. 132, 649-652 (1978). E. HOLTTA, Biochemistry 16, 91-100 (1977). I. MATSUI, L. WIEGAND, and A.E. PEGG, J. Biol. Chem. 256, 2454-2459 (1981). R.A. ALARCON, Arch. Biochem. Biophys. 137, 365-372 (1970). C. IZARD and C. LIBERMANN, Mutation Res. 47, 115-138 (1978).