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. 1978 Dec 1;175(3):1051–1067. doi: 10.1042/bj1751051

Purification and characterization of two human erythrocyte arylamidases preferentially hydrolysing N-terminal arginine or lysine residues.

K K Mäkinen, P L Mäkinen
PMCID: PMC1186168  PMID: 743227

Abstract

Two arylamidases (I and II) were purified from human erythrocytes by a procedure that comprised removal of haemoglobin from disrupted cells with CM-Sephadex D-50, followed by treatment of the haemoglobin-free preparation subsequently with DEAE-cellulose, gel-permeation chromatography on Sephadex G-200, gradient solubilization on Celite, isoelectric focusing in a pH gradient from 4 to 6, gel-permeation chromatography on Sephadex G-100 (superfine), and finally affinity chromatography on Sepharose 4B covalently coupled to L-arginine. In preparative-scale purifications, enzymes I and II were separated at the second gel-permeation chromatography. Enzyme II was obtained as a homogeneous protein, as shown by several criteria. Enzyme I hydrolysed, with decreasing rates, the L-amino acid 2-naphtylamides of lysine, arginine, alanine, methionine, phenylalanine and leucine, and the reactions were slightly inhibited by 0.2 M-NaCl. Enzyme II hydrolysed most rapidly the corresponding derivatives of arginine, leucine, valine, methionine, proline and alanine, in that order, and the hydrolyses were strongly dependent on Cl-. The hydrolysis of these substrates proceeded rapidly at physiological Cl- concentration (0.15 M). The molecular weights (by gel filtration) of enzymes I and II were 85 000 and 52 500 respectively. The pH optimum was approx. 7.2 for both enzymes. The isoelectric point of enzyme II was approx. 4.8. Enzyme I was activated by Co2+, which did not affect enzyme II to any noticeable extent. The kinetics of reactions catalysed by enzyme I were characterized by strong substrate inhibition, but enzyme II was not inhibited by high substrate concentrations. The Cl- activated enzyme II also showed endopeptidase activity in hydrolysing bradykinin.

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Selected References

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  1. Bury A. F., Coolbear T., Savery C. R. Separation and properties of two arylamidases from rat cardiac-muscle extracts. Biochem J. 1977 Jun 1;163(3):565–570. doi: 10.1042/bj1630565. [DOI] [PMC free article] [PubMed] [Google Scholar]
  2. DAVIS B. J. DISC ELECTROPHORESIS. II. METHOD AND APPLICATION TO HUMAN SERUM PROTEINS. Ann N Y Acad Sci. 1964 Dec 28;121:404–427. doi: 10.1111/j.1749-6632.1964.tb14213.x. [DOI] [PubMed] [Google Scholar]
  3. Eisenthal R., Cornish-Bowden A. The direct linear plot. A new graphical procedure for estimating enzyme kinetic parameters. Biochem J. 1974 Jun;139(3):715–720. doi: 10.1042/bj1390715. [DOI] [PMC free article] [PubMed] [Google Scholar]
  4. Garner C. W., Behal F. J. Hydrophobic binding sites of human liver alanine aminopeptidase. Arch Biochem Biophys. 1977 Aug;182(2):667–673. doi: 10.1016/0003-9861(77)90547-1. [DOI] [PubMed] [Google Scholar]
  5. Greiff D., Brooker D., Mackey S. Cryobiology of platelets. I. Aminopeptidase activity as a measure of intactness of platelets in vitro. Cryobiology. 1969 Nov-Dec;6(3):194–199. doi: 10.1016/s0011-2240(69)80349-4. [DOI] [PubMed] [Google Scholar]
  6. Hopsu V. K., Mäkinen K. K., Glenner G. G. Purification of a mammalian peptidase selective for N-terminal arginine and lysine residues: aminopeptidase B. Arch Biochem Biophys. 1966 Jan;114(3):557–566. doi: 10.1016/0003-9861(66)90380-8. [DOI] [PubMed] [Google Scholar]
  7. Hopsu V. K., Mäkinn K. K., Glenner G. G. Characterization of aminopeptidase B: substrate specificity and affector studies. Arch Biochem Biophys. 1966 Jun;114(3):567–575. doi: 10.1016/0003-9861(66)90381-x. [DOI] [PubMed] [Google Scholar]
  8. King T. P. Separation of proteins by ammonium sulfate gradient solubilization. Biochemistry. 1972 Feb 1;11(3):367–371. doi: 10.1021/bi00753a010. [DOI] [PubMed] [Google Scholar]
  9. Klimek R. Clinical studies on the balance between isooxytocinases in the blood of pregnant women. Clin Chim Acta. 1968 May;20(2):233–238. doi: 10.1016/0009-8981(68)90155-1. [DOI] [PubMed] [Google Scholar]
  10. Klimek R., Malolepszy E. Blood levels of cystine aminopeptidase (oxytocinase) in patients with toxic and simple goiter. Clin Chim Acta. 1968 Dec;22(4):491–495. doi: 10.1016/0009-8981(68)90097-1. [DOI] [PubMed] [Google Scholar]
  11. Knuuttila M., Virtanen K., Söderling E., Mäkinen K. K. A chloride-activated aminopeptidase in rat inflammatory exudate: properties and evidence of the origin of the enzyme. Biochem Biophys Res Commun. 1978 Mar 30;81(2):374–381. doi: 10.1016/0006-291x(78)91543-7. [DOI] [PubMed] [Google Scholar]
  12. Kurtz A. B., Wachsmuth E. D. Identification of plasma angiotensinase as aminopeptidase. Nature. 1969 Jan 4;221(5175):92–93. doi: 10.1038/221092a0. [DOI] [PubMed] [Google Scholar]
  13. LOWRY O. H., ROSEBROUGH N. J., FARR A. L., RANDALL R. J. Protein measurement with the Folin phenol reagent. J Biol Chem. 1951 Nov;193(1):265–275. [PubMed] [Google Scholar]
  14. Mäkinen K. K., Brummer R., Scheinin A. Arylaminopeptidase activity in dental pulp. Acta Odontol Scand. 1970 Jun;28(3):377–387. doi: 10.3109/00016357009032041. [DOI] [PubMed] [Google Scholar]
  15. Mäkinen K. K. Evidence for the aggregation of aminopeptidase B during storage and breakdown of the aggregate by substrate and serum albumin. Biochim Biophys Acta. 1972 Jul 21;271(2):413–418. doi: 10.1016/0005-2795(72)90216-4. [DOI] [PubMed] [Google Scholar]
  16. Mäkinen K. K., Hopsu-Havu V. K. The presence of enzymes resembling aminopeptidase B in several rat organs. Ann Med Exp Biol Fenn. 1967;45(2):230–234. [PubMed] [Google Scholar]
  17. Mäkinen K. K., Hyyppä T. A biochemical study of the origin of arginine aminopeptidases in human gingival fluid. Arch Oral Biol. 1975 Aug;20(8):509–514. doi: 10.1016/0003-9969(75)90213-7. [DOI] [PubMed] [Google Scholar]
  18. Mäkinen K. K., Luostarinen V., Varrela J., Rekola M., Luoma S. Arginine aminopeptidase reactions to laser in vivo and in vitro. Biochem Med. 1975 Jun;13(2):192–195. doi: 10.1016/0006-2944(75)90155-6. [DOI] [PubMed] [Google Scholar]
  19. Mäkinen K. K., Oksala E. Evidence on the involvement in inflammation of an enzyme resembling aminopeptidase B. Clin Chim Acta. 1973 Dec 27;49(3):301–309. doi: 10.1016/0009-8981(73)90226-x. [DOI] [PubMed] [Google Scholar]
  20. Mäkinen K. K., Paunio K. U. A histochemical method for the demonstration of aminopeptidase B activity. J Histochem Cytochem. 1972 Mar;20(3):192–194. doi: 10.1177/20.3.192. [DOI] [PubMed] [Google Scholar]
  21. Mäkinen K. K., Virtanen K. K. Aminopeptidase B in human serum. Clin Chim Acta. 1976 Mar 1;67(2):213–218. doi: 10.1016/0009-8981(76)90262-x. [DOI] [PubMed] [Google Scholar]
  22. Mäkinen K. K., Virtanen K. K. Aminopeptidases of mechanically strained and normal rat gingiva, with special reference to aminopeptidase B. Acta Odontol Scand. 1974;32(2):115–124. doi: 10.3109/00016357409002540. [DOI] [PubMed] [Google Scholar]
  23. Mäkinen P. L., Mäkinen K. K. Fractionation and properties of aminopeptidase B during purification and storage. Int J Pept Protein Res. 1972;4(4):241–255. doi: 10.1111/j.1399-3011.1972.tb03425.x. [DOI] [PubMed] [Google Scholar]
  24. Nagatsu I., Nagatsu T., Yamamoto T., Glenner G. G., Mehl J. W. Purification of aminopeptidase A in human serum and degradation of angiotensin II by the purified enzyme. Biochim Biophys Acta. 1970 Feb 11;198(2):255–270. doi: 10.1016/0005-2744(70)90058-6. [DOI] [PubMed] [Google Scholar]
  25. Neef L., Peters J. E., Haschen R. J. Alanin- und Leuzinaminopeptidase in isolierten menschlichen Blutzellen. Z Gesamte Inn Med. 1973 Oct 1;28(19):573–576. [PubMed] [Google Scholar]
  26. SEARCY R. L., GOUGH G. S., KOROTZER J. L., BERGQUIST L. M. Evaluation of a new technique for estimation of urea nitrogen in serum. Am J Med Technol. 1961 Sep-Oct;27:255–262. [PubMed] [Google Scholar]
  27. Virtanen K. K., Mäkinen K. K., Oksala E. Activity of arginine aminopeptidases and phosphatases in inflamed palatal mucosa in denture stomatitis: a histochemical and biochemical study. J Dent Res. 1977 Jun;56(6):674–684. doi: 10.1177/00220345770560061801. [DOI] [PubMed] [Google Scholar]
  28. Yman L. Studies on human serum aminopeptidases. Some properties of oxytocinase, human serum aminopeptidase A and leucine aminopeptidase and their purification from retroplacental serum. Acta Pharm Suec. 1970 Apr;7(2):75–86. [PubMed] [Google Scholar]

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