Ultrafiltration-based assay for heparanase activity.
Heparanase, a mammalian endoglycosidase that specifically cleaves heparan sulfate (HS), has been found in many tissues. Platelet, liver, and placenta have been abundant sources for the study of the enzyme. Notably, certain malignant cells also have been found to produce large amounts of the enzyme, the levels of which often correlate with their invasive and metastatic properties. To study roles of heparanase in various biological situations, a reliable method measuring the enzyme activity is indispensable. In the past, measurement of heparanase enzyme activity was done either by the detection of the degradation of fluorescent or radiolabeled HS chains by gel filtration procedures or by the use of radiolabeled substrate conjugated to solid matrices for the easy separation of degraded HS chains. A newly developed procedure, presented in this article, measures degradation of radiolabeled HS chains in the aqueous buffer by detecting their degradation products using an ultrafiltration device, the Centricon 30. This procedure has several advantages over previous assay procedures that involved tedious processing such as gel filtration chromatography of each sample or the preparation of substrate HS proteoglycans conjugated to a solid matrix. The simplicity of the new procedure allows a short setup time and a rapid processing of a large number of samples. Furthermore, the enzymatic reaction during the aqueous phase allows kinetic analyses in standard conditions.
Tsuchida
Sachio
S
Biochemistry, Department of Hard Tissue Engineering, Graduate School, Tokyo Medical and Dental University, 1-5-45 Yushima, Bunkyo-ku, Tokyo 113-8549, Japan.
Podyma-Inoue
Katarzyna A
KA
Yanagishita
Masaki
M
D000818:Animals; D005966:Glucuronidase / Q000737:chemistry* / Q000378:metabolism; D006497:Heparan Sulfate / Q000737:chemistry*; D007700:Kinetics; D009362:Neoplasm Metastasis / Q000503:physiopathology; D009369:Neoplasms / Q000378:metabolism / Q000473:pathology; D051381:Rats; D014462:Ultrafiltration; D000099175:Heparanase
D005966:Glucuronidase; D006497:Heparan Sulfate; D000099175:Heparanase