TY - JOUR
T1 - Ultrafiltration-based assay for heparanase activity
AU - Tsuchida, Sachio
AU - Podyma-Inoue, Katarzyna A.
AU - Yanagishita, Masaki
PY - 2004/8/1
Y1 - 2004/8/1
N2 - 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.
AB - 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.
KW - Enzyme activity
KW - Heparan sulfate proteoglycan
KW - Heparanase
KW - Ultrafiltration
UR - http://www.scopus.com/inward/record.url?scp=3042799327&partnerID=8YFLogxK
U2 - 10.1016/S0003-2697(04)00377-X
DO - 10.1016/S0003-2697(04)00377-X
M3 - Article
C2 - 15246007
AN - SCOPUS:3042799327
SN - 0003-2697
VL - 331
SP - 147
EP - 152
JO - Analytical Biochemistry
JF - Analytical Biochemistry
IS - 1
ER -