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Molecular and Cellular Biochemistry

, Volume 75, Issue 2, pp 123–132 | Cite as

Aminopeptidase P from bovine lung: solubilization, properties, and potential role in bradykinin degradation

  • Arthur T. Orawski
  • Jean P. Susz
  • William H. Simmons
Original Article

Abstract

Aminopeptidase P was solubilized from bovine lung by sodium deoxycholate extraction of salt-washed, delipidated lung acetone powders. Hydrolysis of the standard aminopeptidase P substrate, Gly-Pro-Hyp, as well as cleavage of Arg-Pro-Pro and the Arg1-Pro2 bond of bradykinin, co-eluted from a Mono Q anion exchange column and demonstrated identical inhibitory profiles suggesting that all activities were functions of the same enzyme. The metal chelator, 1,10-phenanthroline, completely inhibited activity suggesting that aminopeptidase P is a metallopeptidase. 2-Mercaptoethanol was both a potent and specific inhibitor of the enzyme (at 4 mM). A variety of other peptidase inhibitors showed either no effect or failed to completely inhibit even at high concentrations. The inhibitory profile and substrate specificity differ considerably from previous reports claiming to study the properties of this enzyme. Evidence is provided that aminopeptidase P may have an important role in the pulmonary degradation of the potent vasoactive peptide, bradykinin.

Keywords

aminopeptidase P bovine lung bradykinin metabolism 

Abbreviations

X-β-NA

aminoacyl-β-naphthylamide

DFP

diisopropylphosphofluoridate

HPLC

high performance liquid chromatography

APP

aminopeptidase P

APM

aminopeptidase M

DAP IV

dipeptidyl-aminopeptidase IV

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References

  1. 1.
    Vane JR: The fate of angiotensin I. Handbook Exp Pharm 37:17–40, 1974Google Scholar
  2. 2.
    Ferreira SH, Bakhle YS: Inactivation of bradykinin and related peptides in lung. In: Metabolic Functions of the Lung. Bakhle YS, Vane JR (eds) Marcel Dekker Inc, New York, 1977, pp 33–53Google Scholar
  3. 3.
    Ferreira SH, Greene LJ, Christiana M, Salgado O, Krieger EM: The fate of circulating biologically active peptides in the lungs. In: Ciba Foundation Symposium 78. Metabolic Activities of the Lung. Excerpta Medica, New York, 1980, pp 129–140Google Scholar
  4. 4.
    Ryan JW, Roblero J, Stewart JM: Inactivation of bradykinin in rat lung. Adv Exp Med Biol 8:263–281, 1970Google Scholar
  5. 5.
    Roblero J, Ryan J, Stewart JM: Assay of kinins by their effects on blood pressure. Res Commun Chem Pathol Pharmacol 6:207–212, 1973Google Scholar
  6. 6.
    Ryan JW, Carlin G, Berryer P, Chung A: Potentiation of bradykinin by inhibitors of angiotensin converting enzyme. Adv Exp Biol Med 156A:613–620, 1983Google Scholar
  7. 7.
    Ryan JW, Ryan US, Chung A, Fisher GH: Metabolism of bradykinin by endothelial cells in culture. Adv Exp Biol Med 156BB:775–781, 1983Google Scholar
  8. 8.
    Orawski AT, Kuppy T, Simmons WH: Membrane-bound peptidases in bovine lung: potential role in bradykinin degradation. Fed Proc Fed Am Soc Exp Biol (Abstr) 42:382, 1983Google Scholar
  9. 9.
    Szechinski J, Behal FJ: Human lung kininase. Fed Proc Fed Am Soc Exp Biol (Abstr) 41:1452, 1982Google Scholar
  10. 10.
    Simmons WH, Burkholder DE, Brecher AS: The hydrolysis of biologically active peptides by bovine lung tissue factor (thromboplastin). Proc Soc Exp Biol Med 152:576–584, 1976Google Scholar
  11. 11.
    Nemerson Y, Pitlick F: Purification and characterization of the protein component of tissue factor. Biochemistry 9:5100–5105, 1970Google Scholar
  12. 12.
    Dehm P, Nordwig A: The cleavage of prolyl peptides by kidney peptidases; partial purification of a “X-prolyl-aminopeptidase” from swine kidney microsomes. Eur J Biochem 17:364–371, 1970Google Scholar
  13. 13.
    Felgenhauer K, Glenner GG: The enzymatic hydrolysis of amino acid β-naphthylamides. II. Partial purification and properties of a particle-bound cobalt-activated rat kidney aminopeptidase. J Histochem Cytochem 14:401–413, 1966Google Scholar
  14. 14.
    Gray WR: End-group analysis using dansyl chloride. Methods in Enzymology 2513:121–138, 1972Google Scholar
  15. 15.
    Sidorowicz W, Szechinski J, Canizaro P, Behal FJ: Cleavage of the Arg1-Pro2 bond of bradykinin by a human lung peptidase: isolation, characterization, and inhibition by several R-lactam antibiotics. Proc Soc Exp Biol Med 175:503–509, 1984Google Scholar
  16. 16.
    Szechinski J, Hsia W-C, Behal FJ: A kininase and a kininconverting enzyme: two distinct alpha aminoacyl peptide hydrolases from bovine lung. Enzyme 29:21–31, 1983Google Scholar
  17. 17.
    Yaron A, Berger A: Aminopeptidase P. Methods in Enzymology 19:521–534, 1970Google Scholar
  18. 18.
    AchutaMurthy PN, Orawski AT, Simmons WH: Partial purification and properties of aminopeptidase P from rat lung (Abstr). Fed Proc Fed Am Soc Exp Biol 44:876, 1985Google Scholar
  19. 19.
    Behal FJ, Sidorowicz W, Canizaro PC: Kinin cleavage by intact human erythrocytes. Fed Proc Fed Am Soc Exp Biol (Abstr) 43:652, 1984Google Scholar
  20. 20.
    McDonald JK, Schwabe C: Intracellular exopeptidases. In: Proteinases in mammalian cells and tissues. Barrett AJ (ed) North Holland Publishing Company, Amsterdam, 1977, pp 311–391Google Scholar
  21. 21.
    Davis NC, Smith EL: Purification and some properties of prolidase of swine kidney. J Biol Chem 224:261–275, 1957Google Scholar
  22. 22.
    Sjostrom H, Noren O, Josefsson L: Purification and specificity of pig intestinal prolidase. Biochim Biophys Acta 327:457–470, 1973Google Scholar
  23. 23.
    Yoshimoto T, Tsuru D: Substrate specificity of aminopeptidase M: evidence that the commercial preparation is contaminated by dipeptidyl aminopeptidase IV and prolidase. J Biochem 94:619–622, 1983Google Scholar
  24. 24.
    Simmons WH, Orawski AT: Degradation of bradykinin by bovine lung microsomes. Fed Proc Fed Am Soc Exp Biol (Abstr) 44:1243, 1985Google Scholar
  25. 25.
    Churchill M, Orawski AT, Achuta-Murthy PN, Simmons WH: Degradation of bradykinin by isolated perfused rat lung. Fed Proc Fed Am Soc Exp Biol (Abstr) 45:678, 1986Google Scholar
  26. 26.
    Stewart JM, Roblero J: Studies on the pulmonary inactivation of bradykinin. IN: Vasoactive polypeptides and inhibitors of proteolytic enzymes. Ludescher O (ed) K.K. Bayer-Yakuhin, Tokyo, 1967, pp 52–55Google Scholar

Copyright information

© Martinus Nijhoff Publishers 1987

Authors and Affiliations

  • Arthur T. Orawski
  • Jean P. Susz
    • 2
  • William H. Simmons
    • 1
  1. 1.Department of Biochemistry and BiophysicsLoyola University of Chicago Stritch School of MedicineMaywoodUSA
  2. 2.Spinal Cord Injury Research LabVeterans Administration Medical CenterWest RoxburyUSA

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