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Home / Equine Research Bank / Biochem J / Substrate specificity and modifications of the active centre...
The Biochemical journal1976; 153(2); 397-402; doi: 10.1042/bj1530397

Substrate specificity and modifications of the active centre of elastase-like neutral proteinases from horse blood leucocytes.

Abstract: Two proteinases (2A and 2B) purified from the granular fraction of horse blood leucocytes degrade casein (Km values 12.8 and 6mg/ml respectively) with maximum activity at pH 7.4 and in the presence of 2m-urea. Urea-denatured haemoglobin, fibrinogen, albumin and resorcin/fuchsin-stained elastin are digested at a slower rate. The enzymes hydrolyse synthetic substrates of elastase, N-benzyloxycarbonyl-L-alanine 4-nitrophenyl ester (Km 0.114 and 0.178 mM) and N-acetyl-tri-L-alanine methyl ester (Km 5.55 and 0.98 mM), but they do not hydrolyse synthetic substrates of trypsin, chymotrypsin and thrombin. The examined proteinases are completely inhibited by 2 mM-di-isopropyl phosphorfluoridate and show a sensitivity to butyl and octyl isocyanates similar to that of pancreatic elastase. The pH-dependence of their photoinactivation in the presence of Rose Bengal indicates the presence of histidine in the active centre. Proteinase 2A rather insensitive to iodination by IC1 as is pancreatic elastase, whereas proteinase 2B is totally inactivated after incorporation of five iodine atoms per enzyme molecule.
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Publication Date: 1976-02-01 PubMed ID: 6009PubMed Central: PMC1172585DOI: 10.1042/bj1530397Google Scholar: Lookup
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Summary

This research summary has been generated with artificial intelligence and may contain errors and omissions. Refer to the original study to confirm details provided. Submit correction.

This research investigates the specificity and modification of enzyme centres in two proteinases derived from horse blood leucocytes, and their degradation rates and inhibition potential.

Overview and Objective of the Research

The study focuses on two proteinases (labelled 2A and 2B) that are extracted from the granular fraction of horse blood leucocytes. The primary aim was to examine how these proteinase enzymes degrade certain substances and how they respond under different conditions and in the presence of inhibitors.

Substrate Specificity and Degradation

  • Both of the proteinase enzymes degraded casein, a protein found in milk, with maximum activity seen at pH 7.4 and in the presence of 2m-urea.
  • They both also digested other enzymes such as urea-denatured haemoglobin, fibrinogen, albumin, and resorcin/fuchsin-stained elastin, but at a slower rate.
  • The proteinases were seen to hydrolyse or break down synthetic substrates of elastase, another enzyme.
  • However, the proteinases did not show any activity against synthetic substrates of other enzymes including trypsin, chymotrypsin, and thrombin.

Inhibitors and Sensitivity

  • The two proteinases were completely inhibited or stopped by a compound called di-isopropyl phosphorfluoridate.
  • Both proteinase 2A and 2B showed a similar sensitivity to butyl and octyl isocyanates as that of pancreatic elastase.

Active Centre Modifications

  • The study indicates the presence of histidine in the active centre of the proteinase enzymes. Histidine is an essential amino acid that is important to purify, form, and modify proteins.
  • Proteinase 2A showed resistance or insensitivity to iodination by IC1, similar to that of pancreatic elastase.
  • However, proteinase 2B was completely inactivated after the incorporation of five iodine atoms per enzyme molecule.

In conclusion, this study helps in understanding the behaviour, activity, and modification potential of specific proteinase enzymes derived from horse blood leucocytes, which could be influential in further biochemical and pharmaceutical studies.

Cite This Article

APA
Koj A, Chudzik J, Dubin A. (1976). Substrate specificity and modifications of the active centre of elastase-like neutral proteinases from horse blood leucocytes. Biochem J, 153(2), 397-402. https://doi.org/10.1042/bj1530397

Publication

The Biochemical journal
ISSN: 0264-6021
NlmUniqueID: 2984726R
Country: England
Language: English
Volume: 153
Issue: 2
Pages: 397-402

Researcher Affiliations

Koj, A
    Chudzik, J
      Dubin, A

        MeSH Terms

        • Animals
        • Binding Sites
        • Caseins
        • Chymotrypsin
        • Cyanates
        • Fibrinogen
        • Hemoglobins
        • Horses / blood
        • Hydrogen-Ion Concentration
        • Iodine
        • Isoflurophate
        • Kinetics
        • Leukocytes / enzymology
        • Pancreatic Elastase
        • Peptide Hydrolases / metabolism
        • Protease Inhibitors
        • Rose Bengal
        • Serum Albumin
        • Trypsin

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        Citations

        This article has been cited 5 times.
        1. Koivunen AL, Maisi P, Konttinen YT, Sandholm M. Gelatinolytic activity in tracheal aspirates of horses with chronic obstructive pulmonary disease. Acta Vet Scand 1997;38(1):17-27.
          doi: 10.1186/BF03548504pubmed: 9129343google scholar: lookup
        2. Kordula T, Dubin A, Schooltink H, Koj A, Heinrich PC, Rose-John S. Molecular cloning and expression of an intracellular serpin: an elastase inhibitor from horse leucocytes. Biochem J 1993 Jul 1;293 ( Pt 1)(Pt 1):187-93.
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        3. Dubin A, Potempa J, Travis J. Structural and functional characterization of elastases from horse neutrophils. Biochem J 1994 Jun 1;300 ( Pt 2)(Pt 2):401-6.
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          doi: 10.1007/BF00916242pubmed: 7049937google scholar: lookup
        5. Dubin A, Koj A, Chudzik J. Isolation and some molecular parameters of elastase-like normal proteinases from horse blood leucocytes. Biochem J 1976 Feb 1;153(2):389-96.
          doi: 10.1042/bj1530389pubmed: 6008google scholar: lookup
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