Skip to main content
NCBI home page
Biochemical Journal logoLink to Biochemical Journal
. 1990 Apr 1;267(1):75–78. doi: 10.1042/bj2670075

Monoclonal antibodies that inhibit the enzyme activity of NAD(+)-dependent 15-hydroxyprostaglandin dehydrogenase.

C L Tai 1, O T Mak 1, T Arai 1, H H Tai 1
PMCID: PMC1131246  PMID: 1691637

Abstract

Three hybridoma cell lines secreting antibodies against human placental NAD(+)-dependent 15-hydroxyprostaglandin dehydrogenase (15-OH-PGDH) were produced. Purified IgG2b from these cell lines recognized a distinct band of Mr 28,000 on SDS/PAGE from the purified enzyme as well as a band of Mr 56,000 from the crude enzyme preparation. These three monoclonal antibodies inhibited 15-OH-PGDH activity to different degrees. Inhibition of the enzyme activity could be prevented by prior incubation of the enzyme with NAD+ but not with prostaglandin E2 (PGE2) or NADP+. Inhibition by monoclonal antibodies appears to be non-competitive with respect to NAD+ and PGE2. An increased concentration of antibodies alters the apparent Km for NAD+ but not for PGE2, further supporting the notion that the antibodies bind to the coenzyme-binding site. The availability of these monoclonal antibodies should be valuable for probing the structure of the active site.

Full text

PDF
75

Images in this article

77Fig. 3.
on p.77

Selected References

These references are in PubMed. This may not be the complete list of references from this article.

  1. Alam N. A., Russell P. T., Tabor M. W., Moulton B. C. Progesterone and estrogen control of uterine prostaglandin dehydrogenase activity during deciduomal growth. Endocrinology. 1976 Apr;98(4):859–863. doi: 10.1210/endo-98-4-859. [DOI] [PubMed] [Google Scholar]
  2. Anggård E., Larsson C., Samuelsson B. The distribution of 15-hydroxy prostaglandin dehydrogenase and prostaglandin-delta 13-reductase in tissues of the swine. Acta Physiol Scand. 1971 Mar;81(3):396–404. doi: 10.1111/j.1748-1716.1971.tb04914.x. [DOI] [PubMed] [Google Scholar]
  3. Anggård E. The biological activities of three metabolites of prostaglandin E 1. Acta Physiol Scand. 1966 Apr;66(4):509–510. doi: 10.1111/j.1748-1716.1966.tb03231.x. [DOI] [PubMed] [Google Scholar]
  4. Bedwani J. R., Marley P. B. Enhanced inactivation of prostaglandin E2 by the rabbit lung during pregnancy or progesterone treatment. Br J Pharmacol. 1975 Apr;53(4):547–554. doi: 10.1111/j.1476-5381.1975.tb07393.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  5. Bergholte J. M., Okita R. T. Isolation and properties of lung 15-hydroxyprostaglandin dehydrogenase from pregnant rabbits. Arch Biochem Biophys. 1986 Mar;245(2):308–315. doi: 10.1016/0003-9861(86)90221-3. [DOI] [PubMed] [Google Scholar]
  6. Ey P. L., Prowse S. J., Jenkin C. R. Isolation of pure IgG1, IgG2a and IgG2b immunoglobulins from mouse serum using protein A-sepharose. Immunochemistry. 1978 Jul;15(7):429–436. doi: 10.1016/0161-5890(78)90070-6. [DOI] [PubMed] [Google Scholar]
  7. Jarabak J., Watkins J. D. Purification of the human placental NAD-linked 15-hydroxyprostaglandin dehydrogenase. Prostaglandins. 1988 Aug;36(2):205–213. doi: 10.1016/0090-6980(88)90307-3. [DOI] [PubMed] [Google Scholar]
  8. Knecht D. A., Dimond R. L. Visualization of antigenic proteins on Western blots. Anal Biochem. 1984 Jan;136(1):180–184. doi: 10.1016/0003-2697(84)90321-x. [DOI] [PubMed] [Google Scholar]
  9. Kung-Chao D. T., Tai H. H. NAD+-dependent 15-hgydroxyprostaglandin dehydrogenase from porcine kidney. II. Kinetic studies. Biochim Biophys Acta. 1980 Jul 10;614(1):14–24. doi: 10.1016/0005-2744(80)90162-x. [DOI] [PubMed] [Google Scholar]
  10. Laemmli U. K. Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature. 1970 Aug 15;227(5259):680–685. doi: 10.1038/227680a0. [DOI] [PubMed] [Google Scholar]
  11. Mak O. T., Jörnvall H., Jeffery J. The primary prostaglandin-inactivating enzyme of human placenta is a dimeric short-chain dehydrogenase. Biosci Rep. 1982 Jul;2(7):503–508. doi: 10.1007/BF01115248. [DOI] [PubMed] [Google Scholar]
  12. Moore P. K., Hoult J. R. Anti-inflammatory steroids reduce tissue PG synthetase activity and enhance PG breakdown. Nature. 1980 Nov 20;288(5788):269–270. doi: 10.1038/288269a0. [DOI] [PubMed] [Google Scholar]
  13. Pace-Asciak C. Activity profiles of prostaglandin 15- and 9-hydroxydehydrogenase and 13-reductase in the developing rat kidney. J Biol Chem. 1975 Apr 25;250(8):2795–2800. [PubMed] [Google Scholar]
  14. SAMUELSSON B. PROSTAGLANDINS AND RELATED FACTORS. 28. METABOLISM OF PROSTAGLANDIN E1 IN GUINEA PIG LUNG: THE STRUCTURES OF TWO METABOLITES. J Biol Chem. 1964 Dec;239:4097–4102. [PubMed] [Google Scholar]
  15. Shen R. F., Tai H. H. Monoclonal antibodies to thromboxane synthase from porcine lung. Production and application to development of a tandem immunoradiometric assay. J Biol Chem. 1986 Sep 5;261(25):11585–11591. [PubMed] [Google Scholar]
  16. Tai H. H. Enzymatic synthesis of (15s)-[15-3h]prostaglandins and their use in the development of a simple and sensitive assay for 15-hydroxyprostaglandin dehydrogenase. Biochemistry. 1976 Oct 19;15(21):4586–4592. doi: 10.1021/bi00666a007. [DOI] [PubMed] [Google Scholar]
  17. Tanaka T., Kotani T., Ohtaki S., Nagai K., Tsuruta K., Mori N. Labile oligomeric structure of human placental 15-hydroxyprostaglandin dehydrogenase. Biochem Biophys Res Commun. 1986 Mar 28;135(3):1058–1063. doi: 10.1016/0006-291x(86)91035-1. [DOI] [PubMed] [Google Scholar]
  18. Towbin H., Staehelin T., Gordon J. Electrophoretic transfer of proteins from polyacrylamide gels to nitrocellulose sheets: procedure and some applications. Proc Natl Acad Sci U S A. 1979 Sep;76(9):4350–4354. doi: 10.1073/pnas.76.9.4350. [DOI] [PMC free article] [PubMed] [Google Scholar]

Articles from Biochemical Journal are provided here courtesy of The Biochemical Society

RESOURCES