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. 1977 Oct;74(10):4311–4314. doi: 10.1073/pnas.74.10.4311

Specific lysine labeling by 18OH- during alkaline cleavage of the alpha-1-antitrypsin-trypsin complex.

A B Cohen, L D Gruenke, J C Craig, D Geczy
PMCID: PMC431930  PMID: 303770

Abstract

alpha-1-Antitrypsin is a serum protein that inhibits many proteolytic enzymes. Recently, it was suggested that the alpha-1-antitrypsin-trypsin complex is an acyl ester analogous to the acyl intermediate that forms between trypsin and its substrates. In previous work we showed that the alpha-1-antitrypsin-trypsin complex can be split at high pH, releasing a component of alpha-1-antitrypsin. This component had a new carboxyl-terminal lysine, and it had lost a peptide of about 4000 daltons. In order to determine whether the alpha-1-antitrypsin is bound to trypsin through the new carboxy-terminal lysine, as would be expected if the above hypothesis is correct, we split the complex in the presence of 18OH-. When the new carboxy-terminal lysine was cleaved with carboxypeptidase B, singly labeled, doubly labeled, and unlabeled lysine were recovered. These data support the hypothesis that the alpha-1-antitrypsin-trypsin complex is an acyl ester or a tetrahedral precursor that is transformed into the acyl ester form at high pH. If other enzymes are bound by a similar mechanism, the methods used may be useful in determining which amino acids on alpha-1-antitrypsin bind covalently to each enzyme.

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Selected References

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

  1. Birktoft J. J., Blow D. M. Structure of crystalline -chymotrypsin. V. The atomic structure of tosyl- -chymotrypsin at 2 A resolution. J Mol Biol. 1972 Jul 21;68(2):187–240. doi: 10.1016/0022-2836(72)90210-0. [DOI] [PubMed] [Google Scholar]
  2. Blow D. M., Janin J., Sweet R. M. Mode of action of soybean trypsin inhibitor (Kunitz) as a model for specific protein-protein interactions. Nature. 1974 May 3;249(452):54–57. doi: 10.1038/249054a0. [DOI] [PubMed] [Google Scholar]
  3. Cohen A. B. Mechanism of action of alpha-1-antitrypsin. J Biol Chem. 1973 Oct 25;248(20):7055–7059. [PubMed] [Google Scholar]
  4. Henderson R. Structure of crystalline alpha-chymotrypsin. IV. The structure of indoleacryloyl-alpha-chyotrypsin and its relevance to the hydrolytic mechanism of the enzyme. J Mol Biol. 1970 Dec 14;54(2):341–354. doi: 10.1016/0022-2836(70)90434-1. [DOI] [PubMed] [Google Scholar]
  5. Henderson R., Wright C. S., Hess G. P., Blow D. M. -Chymotrypsin: what can we learn about catalysis from x-ray diffraction? Cold Spring Harb Symp Quant Biol. 1972;36:63–70. doi: 10.1101/sqb.1972.036.01.011. [DOI] [PubMed] [Google Scholar]
  6. Hokin L. E., Dahl J. L., Deupree J. D., Dioxon J. F., Hackney J. F., Perdue J. F. Studies on the characterization of the sodium-potassium transport adenosine triphosphatase. X. Purification of the enzyme from the rectal gland of Squalus acanthias. J Biol Chem. 1973 Apr 10;248(7):2593–2605. [PubMed] [Google Scholar]
  7. Horman I., Hesford F. J. Amino acid mixture analysis by mass spectrometry in the form of their dimethylaminomethylene methyl esters. Biomed Mass Spectrom. 1974 Apr;1(2):115–119. doi: 10.1002/bms.1200010206. [DOI] [PubMed] [Google Scholar]
  8. Horng W. J., Gan J. C. Purification and characterization of human plasma (alpha 1)-antitrypsin. Tex Rep Biol Med. 1974 Summer;32(2):489–504. [PubMed] [Google Scholar]
  9. Johnson D. A., Travis J. Human alpha-1-proteinase inhibitor mechanism of action: evidence for activation by limited proteolysis. Biochem Biophys Res Commun. 1976 Sep 7;72(1):33–39. doi: 10.1016/0006-291x(76)90956-6. [DOI] [PubMed] [Google Scholar]
  10. Moroi M., Yamasaki M. Mechanism of interaction of bovine trypsin with human alpha1-antitrypsin. Biochim Biophys Acta. 1974 Jul 7;359(1):130–141. doi: 10.1016/0005-2795(74)90138-x. [DOI] [PubMed] [Google Scholar]
  11. Shapiro A. L., Viñuela E., Maizel J. V., Jr Molecular weight estimation of polypeptide chains by electrophoresis in SDS-polyacrylamide gels. Biochem Biophys Res Commun. 1967 Sep 7;28(5):815–820. doi: 10.1016/0006-291x(67)90391-9. [DOI] [PubMed] [Google Scholar]
  12. Stroud R. M. A family of protein-cutting proteins. Sci Am. 1974 Jul;231(1):74–88. doi: 10.1038/scientificamerican0774-74. [DOI] [PubMed] [Google Scholar]
  13. Sweet R. M., Wright H. T., Janin J., Chothia C. H., Blow D. M. Crystal structure of the complex of porcine trypsin with soybean trypsin inhibitor (Kunitz) at 2.6-A resolution. Biochemistry. 1974 Sep 24;13(20):4212–4228. doi: 10.1021/bi00717a024. [DOI] [PubMed] [Google Scholar]
  14. Woods K. R., Wang K. T. Separation of dansyl-amino acids by polyamide layer chromatography. Biochim Biophys Acta. 1967 Feb 21;133(2):369–370. doi: 10.1016/0005-2795(67)90078-5. [DOI] [PubMed] [Google Scholar]

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