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. 1983 Jan 1;209(1):91–97. doi: 10.1042/bj2090091

Isolation and characterization of a specific enterokinase inhibitor from kidney bean (Phaseolus vulgaris).

R T Jacob, P G Bhat, T N Pattabiraman
PMCID: PMC1154059  PMID: 6405734

Abstract

A specific enterokinase inhibitor from kidney bean (Phaseolus vulgaris) was purified to homogeneity. It showed a single protein band on sodium dodecyl sulphate/polyacryl-amide-gel electrophoresis in the presence of mercaptoethanol, and the Mr was 31000. Aspartic acid was identified as the N-terminus of the inhibitor. The Mr by gel chromatography on Sephadex G-200 was found to be 60000, indicating the dimeric nature of the inhibitor. The inhibitor was found to be a glycoprotein. The monosaccharide moieties were glucose, mannose, glucuronic acid and glucosamine in the proportions 3.15%, 5.0%, 0.85% and 1.3% respectively. The inhibitor was most active on pig enterokinase, followed by bovine and human enterokinases. Maximal inhibitory activity was elicited by preincubation of the inhibitor with the enzyme for 15 min. Digestion with pepsin resulted in loss of inhibitory activity. The inhibitor was stable to exposure to a wide range of pH values (2-10), and exposure to pH above 10 resulted in loss of inhibitory activity. Modification of arginine residues by cyclohexane 1,2-dione and ninhydrin led to complete loss of enterokinase-inhibitory activity.

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Selected References

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

  1. Abe O., Ohata J., Utsumi Y., Kuromizu K. Proteinase inhibitor from Vicia angustifolia L. var. segetalis Koch. Isolation, characterization, and chemical modification. J Biochem. 1978 Jun;83(6):1737–1748. doi: 10.1093/oxfordjournals.jbchem.a132088. [DOI] [PubMed] [Google Scholar]
  2. BITTER T., MUIR H. M. A modified uronic acid carbazole reaction. Anal Biochem. 1962 Oct;4:330–334. doi: 10.1016/0003-2697(62)90095-7. [DOI] [PubMed] [Google Scholar]
  3. CRESTFIELD A. M., MOORE S., STEIN W. H. The preparation and enzymatic hydrolysis of reduced and S-carboxymethylated proteins. J Biol Chem. 1963 Feb;238:622–627. [PubMed] [Google Scholar]
  4. Chaplin M. F. The use of ninhydrin as a reagent for the reversible modification of arginine residues in proteins. Biochem J. 1976 May 1;155(2):457–459. doi: 10.1042/bj1550457. [DOI] [PMC free article] [PubMed] [Google Scholar]
  5. ELLMAN G. L. Tissue sulfhydryl groups. Arch Biochem Biophys. 1959 May;82(1):70–77. doi: 10.1016/0003-9861(59)90090-6. [DOI] [PubMed] [Google Scholar]
  6. ERLANGER B. F., KOKOWSKY N., COHEN W. The preparation and properties of two new chromogenic substrates of trypsin. Arch Biochem Biophys. 1961 Nov;95:271–278. doi: 10.1016/0003-9861(61)90145-x. [DOI] [PubMed] [Google Scholar]
  7. Haynes R., Osuga D. T., Feeney R. E. Modification of amino groups in inhibitors of proteolytic enzymes. Biochemistry. 1967 Feb;6(2):541–547. doi: 10.1021/bi00854a023. [DOI] [PubMed] [Google Scholar]
  8. Hochstrasser K., Illchmann K., Werle E. Uber pflanzliche Proteaseninhibitoren. VI. Reindarstellung der polyvalenten Proteaseninhibitoren aus Arachis hypogaea. Hoppe Seylers Z Physiol Chem. 1969 Aug;350(8):929–932. [PubMed] [Google Scholar]
  9. Jacoby G. A. The induction and repression of amino acid oxidation in Pseudomonas fluorescens. Biochem J. 1964 Jul;92(1):1–8. doi: 10.1042/bj0920001. [DOI] [PMC free article] [PubMed] [Google Scholar]
  10. LEVVY G. A., MCALLAN A. The N-acetylation and estimation of hexosamines. Biochem J. 1959 Sep;73:127–132. doi: 10.1042/bj0730127. [DOI] [PMC free article] [PubMed] [Google Scholar]
  11. Lau A., Ako H., Werner-Washburne M. Survey of plants for enterokinase inhibitors. Biochem Biophys Res Commun. 1980 Feb 27;92(4):1243–1249. doi: 10.1016/0006-291x(80)90420-9. [DOI] [PubMed] [Google Scholar]
  12. Liepnieks J. J., Light A. The preparation and properties of bovine enterokinase. J Biol Chem. 1979 Mar 10;254(5):1677–1683. [PubMed] [Google Scholar]
  13. Spiro R. G. Studies on fetuin, a glycoprotein of fetal serum. I. Isolation, chemical composition, and physiochemical properties. J Biol Chem. 1960 Oct;235(10):2860–2869. [PubMed] [Google Scholar]
  14. TREVELYAN W. E., PROCTER D. P., HARRISON J. S. Detection of sugars on paper chromatograms. Nature. 1950 Sep 9;166(4219):444–445. doi: 10.1038/166444b0. [DOI] [PubMed] [Google Scholar]
  15. Tur-Sinal A., Birk Y., Gertler A., Rigbi M. A basic trypsin- and chymotrypsin-inhibitor from groundnuts (Arachis hypogaea). Biochim Biophys Acta. 1972 May 18;263(3):666–672. doi: 10.1016/0005-2795(72)90048-7. [DOI] [PubMed] [Google Scholar]
  16. Weber K., Pringle J. R., Osborn M. Measurement of molecular weights by electrophoresis on SDS-acrylamide gel. Methods Enzymol. 1972;26:3–27. doi: 10.1016/s0076-6879(72)26003-7. [DOI] [PubMed] [Google Scholar]
  17. Wilimowska-Pelc A., Mejbaum-Katzenellenbogen W. A simple method for isolating trypsin from trichloroacetic acid extracts of bovine pancreas. Anal Biochem. 1978 Oct 15;90(2):816–820. doi: 10.1016/0003-2697(78)90173-2. [DOI] [PubMed] [Google Scholar]

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