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Related Experiment Videos

Human heparanase. Purification, characterization, cloning, and expression.

M Toyoshima1, M Nakajima

  • 1Discovery Research, Takarazuka Research Institute, Novartis Pharma K. K., 10-66 Miyuki-cho, Takarazuka 665-8666, Japan. motowa.nakajima@pharma.novartis.com

The Journal of Biological Chemistry
|August 14, 1999
PubMed
Summary
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Researchers purified human heparanase, an enzyme that degrades heparan sulfate, and cloned its gene. This enzyme plays a role in inflammation and cancer metastasis.

Area of Science:

  • Biochemistry
  • Molecular Biology
  • Cell Biology

Background:

  • Heparan sulfate proteoglycans are crucial extracellular matrix components involved in cell signaling.
  • Heparanase, an enzyme cleaving heparan sulfate, is implicated in inflammation and cancer progression.

Purpose of the Study:

  • To purify and characterize human heparanase.
  • To clone the cDNA encoding human heparanase.
  • To investigate the functional expression of cloned heparanase.

Main Methods:

  • Purification of human heparanase using sequential column chromatography.
  • Enzyme activity assay via gel permeation chromatography of labeled heparan sulfate.
  • SDS-PAGE for molecular mass determination.
  • cDNA cloning using N-terminal and internal peptide sequences.

Related Experiment Videos

  • Stable transfection of NIH3T3 and COS-7 cells for expression studies.

    • Main Results:

    • Human heparanase was purified to homogeneity with a molecular mass of 50 kDa.
    • A cDNA encoding human heparanase was successfully cloned.
    • Transfected cells exhibited high heparanase activity.
    • No homologous proteins were identified in homology searches.

    Conclusions:

    • The study successfully purified and cloned human heparanase.
    • The cloned heparanase is functionally active when expressed in mammalian cells.
    • The lack of homology suggests a unique protein structure or function.