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Home / Equine Research Bank / Biochem J / Isolation and some molecular parameters of elastase-like nor...
The Biochemical journal1976; 153(2); 389-396; doi: 10.1042/bj1530389

Isolation and some molecular parameters of elastase-like normal proteinases from horse blood leucocytes.

Abstract: Cytoplasmic granules were isolated from horse blood polymorphonuclear leucocytes by the heparin method and extracted with 0.9% NaCl by repeated freezing. Soluble proteins were separated on a column of Sephadex G-75 followed by chromatography on a column of CM-Sephadex with a NaCl gradient. Gel filtration, density-gradient centrifugation, isoelectric focusing and 0.1% sodium dodecyl sulphate/polyacrylamide-gel electrophoresis at pH 7.0 and at pH 4.5 were used to determine molecular parameters of proteinases. Three enzymes hydrolysing both casein and N-benzyloxycarbonyl-L-alanine nitrophenyl ester were found in the granule extract: proteinase 1, mol.wt. 38000, pI5.3; proteinase 2A, mol.wt. 24500, pI8.8; and proteinase 2B, mol.wt. 20500, pI above 10. The latter two elastase-like proteinases were purified to apparent homogeneity.
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Publication Date: 1976-02-01 PubMed ID: 6008PubMed Central: PMC1172584DOI: 10.1042/bj1530389Google Scholar: Lookup
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  • Journal Article

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.

The research is about the isolation and study of elastase-like proteinases, particularly three enzymes that have the capability to hydrolyse both casein and N-benzyloxycarbonyl-L-alanine nitrophenyl ester, from horse blood leucocytes.

Methodology

  • Cytoplasmic granules were isolated from horse blood polymorphonuclear leucocytes using the heparin method.
  • The isolated granules were then extracted with 0.9% NaCl through repeated freezing.
  • The soluble proteins were separated on a column of Sephadex G-75, followed by chromatography on a column of CM-Sephadex with a NaCl gradient.

Determining Molecular Parameters of Proteinases

  • The molecular parameters were determined using gel filtration, density-gradient centrifugation, isoelectric focusing, and 0.1% sodium dodecyl sulphate/polyacrylamide-gel electrophoresis at pH 7.0 and at pH 4.5.

Results

  • Three enzymes were found in the granule extract. These enzymes are capable of hydrolysing both casein and N-benzyloxycarbonyl-L-alanine nitrophenyl ester, marking them as proteinases.
  • The three identified enzymes were: proteinase 1, with a molecular weight of 38000 and an isoelectric point of 5.3; proteinase 2A, with a molecular weight of 24500 and an isoelectric point of 8.8; and proteinase 2B, with a molecular weight of 20500 and an isoelectric point of above 10.
  • Among the identified proteinases, proteinase 2A and proteinase 2B, both elastase-like proteinases, were purified to apparent homogeneity.

Conclusion

  • The research successfully isolated and identified three elastase-like proteinases from horse blood leucocytes. These proteinases have been tested and confirmed to hydrolyse both casein and N-benzyloxycarbonyl-L-alanine nitrophenyl ester.

Cite This Article

APA
Dubin A, Koj A, Chudzik J. (1976). Isolation and some molecular parameters of elastase-like normal proteinases from horse blood leucocytes. Biochem J, 153(2), 389-396. https://doi.org/10.1042/bj1530389

Publication

The Biochemical journal
ISSN: 0264-6021
NlmUniqueID: 2984726R
Country: England
Language: English
Volume: 153
Issue: 2
Pages: 389-396

Researcher Affiliations

Dubin, A
    Koj, A
      Chudzik, J

        MeSH Terms

        • Animals
        • Caseins
        • Cerebroside-Sulfatase / analysis
        • Chromatography, Gel
        • Chromatography, Ion Exchange
        • Electrophoresis, Polyacrylamide Gel
        • Horses / blood
        • Isoelectric Focusing
        • Isoelectric Point
        • Leukocytes / analysis
        • Leukocytes / enzymology
        • Molecular Weight
        • Peptide Hydrolases / blood
        • Peptide Hydrolases / isolation & purification
        • Proteins / analysis
        • Ultracentrifugation

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        Citations

        This article has been cited 7 times.
        1. Pemberton AD, Belham CM, Huntley JF, Plevin R, Miller HR. Sheep mast cell proteinase-1, a serine proteinase with both tryptase- and chymase-like properties, is inhibited by plasma proteinase inhibitors and is mitogenic for bovine pulmonary artery fibroblasts. Biochem J 1997 May 1;323 ( Pt 3)(Pt 3):719-25.
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        2. Pemberton AD, Huntley JF, Miller HR. Sheep mast cell proteinase-1: characterization as a member of a new class of dual-specific ruminant chymases. Biochem J 1997 Feb 1;321 ( Pt 3)(Pt 3):665-70.
          doi: 10.1042/bj3210665pubmed: 9032451google scholar: lookup
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        7. Koj A, Chudzik J, Dubin A. Substrate specificity and modifications of the active centre of elastase-like neutral proteinases from horse blood leucocytes. Biochem J 1976 Feb 1;153(2):397-402.
          doi: 10.1042/bj1530397pubmed: 6009google scholar: lookup
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