A SOLID-PHASE SUBSTRATE OF HEPARANASE - ITS APPLICATION TO ASSAY OF HUMAN-MELANOMA FOR HEPARAN-SULFATE DEGRADATIVE ACTIVITY
A SOLID-PHASE SUBSTRATE OF HEPARANASE - ITS APPLICATION TO ASSAY OF HUMAN-MELANOMA FOR HEPARAN-SULFATE DEGRADATIVE ACTIVITY
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DOI：
10.1016/0003-2697(86)90209-5
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发表时间：
1986-08-15
影响因子：
2.9
通讯作者：
NICOLSON, GL
中科院分区：
文献类型：
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作者：
NAKAJIMA, M;IRIMURA, T;NICOLSON, GL
我们在黑色素瘤细胞中发现了一种与肿瘤转移相关的硫酸乙酰肝素 (HS) 降解内切糖苷酶，它是一种独特的内切 β-葡萄糖醛酸酶（乙酰肝素酶），能够在链内位点特异性切割 HS（M. Nakajima、T. Irimura、N. DiFerrante 和 G.L. Nicolson，1984，J. Biol. Chem. 259， 2283-2290）。为了对乙酰肝素酶进行快速和微量定量测定，我们通过使用一个共价键将放射性标记的 HS 交联到琼脂糖凝胶珠上，开发了一种固相 HS 底物。来自牛肺的HS被部分N-脱硫并用[14C]乙酸酐标记。游离的 HS 氨基被完全乙酰化，还原性末端糖被还原活化。还原末端带有氨基的 HS 衍生物与氨基反应性琼脂糖珠偶联。在 D-糖二酸 1,4-内酯（一种有效的外切-β-葡萄糖醛酸酶抑制剂）存在下，将固相 HS 底物与 B16 黑色素瘤细胞提取物一起孵育，导致 [14C]HS 片段的时间和剂量依赖性释放。使用新开发的固相 HS 底物测试了人黑色素瘤细胞系的 HS 降解内切糖苷酶。测试的人类恶性黑色素瘤细胞具有高水平的 HS 降解活性，与高度转移的小鼠 B16-F10 黑色素瘤细胞相当。
We found a tumor metastasis-associated heparan sulfate (HS)-degrading endoglycosidase in melanoma cells that is a unique endo-.beta.-glucuronidase (heparanse) capable of specifically cleaving HS at intrachain sites (M. Nakajima, T. Irimura, N. DiFerrante, And G.L. Nicolson, 1984, J. Biol. Chem. 259, 2283-2290). To perform rapid and microscale quantitative assays of heparanase we developed a solid-phase HS substrate by crosslinking radiolabeled HS onto agarose gel beads using one covalent linkage. The HS from bovine lung was partially N-desulfated and labeled with [14C]acetic anhydride. Free HS amino groups were completely acetylated, and reducing terminal saccharides were reductively animated. The HS derivatives with amino groups at their reducing termini were coupled to amino-reactive agarose beads. Incubation of the solid-phase HS substrates with B16 melanoma cell extracts in the presence of D-saccharic acid 1,4-lactone (a potent exo-.beta.-glucuronidase inhibitor) resulted in the time- and dose-dependent release of [14C]HS fragments. Human melanoma cell lines were tested for HS-degrading endoglycosidase using the newly developed solid-phase HS substrates. The human malignant melanoma cells tested had high levels of HS-degrading activity that were comparable to those of highly metastatic murine B16-F10 melanoma cells.