Radiolabeled heparan sulfate immobilized on microplate as substrate for the detection of heparanase activity.
We developed a quantitative assay to monitor the enzymatic activity of heparanase, a protein responsible for the degradation of heparan sulfate (HS) present on cell surface and extracellular matrix. Our assay is based on a new procedure to immobilize radiolabeled HS to a solid support by a single end which is adaptable to a microplate format, thus allowing the rapid analysis of numerous samples. First, HS was radiolabeled by partial de-N-acetylation and re-N-acetylation with [3H] acetic anhydride, second, after reductive amination at the reducing terminus, it was covalently linked to an amino-reactive biotin analog, and third it was immobilized on a streptavidin-coated plate. The degradation of our solid-phase tritiated HS by heparanase was monitored by measuring the soluble radioactivity released in the well. The heparanase-induced release of radioactivity was linear with respect either to time or to the amount of enzyme and was inhibited by heparin or high ionic strength. The linearity of this assay for time and enzyme concentrations could be useful to determine the potency of heparanase inhibitors. Moreover, this assay was shown to be suitable for monitoring HS-degrading activity of either heparanase endogenously expressed by the HCT 116 tumor cell line or recombinant forms of this protein.
Department of Biochemistry, IRBM/Merck Research Laboratories, Via Pontina Km 30,600, 00040, Pomezia, Italy.
Caterina
C
Nardella
Christian
C
Steinkühler
D001710:Biotin; D002240:Carbohydrate Sequence; D002457:Cell Extracts / Q000737:chemistry; D045744:Cell Line, Tumor; D002850:Chromatography, Gel; D005966:Glucuronidase / Q000302:isolation & purification / Q000378:metabolism*; D006497:Heparan Sulfate / Q000737:chemistry* / Q000378:metabolism*; D006801:Humans; D007553:Isotope Labeling; D008969:Molecular Sequence Data; D015394:Molecular Structure; D009994:Osmolar Concentration; D019809:Streptavidin; D013379:Substrate Specificity; D014316:Tritium; D000099175:Heparanase
D001710:Biotin; D002457:Cell Extracts; D005966:Glucuronidase; D006497:Heparan Sulfate; D019809:Streptavidin; D014316:Tritium; D000099175:Heparanase