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2. 发明授权

US5300630A Oncofetal fibronectin epitope 失效
标题翻译： Oncofetal纤连蛋白表位
公开(公告)号：US5300630A
公开(公告)日：1994-04-05
申请号：US955488
申请日：1992-10-02
申请人： Hidemitsu Matsuura , Sen-itiroh Hakomori , Koiti Titani
发明人： Hidemitsu Matsuura , Sen-itiroh Hakomori , Koiti Titani
IPC分类号： C12N9/10 , C12Q1/48 , G01N33/573 , G01N33/574 , A61K37/02 , C07K7/06
CPC分类号： C12Q1/48 , C12N9/1051 , G01N33/573 , G01N33/57492 , G01N2333/4742 , G01N2333/91091
摘要： An onco-developmentally regulated .alpha.-N-acetylgalactosaminyltransferase isolated as a component of a particulate membrane fraction separated from cell and tissue homogenates and having the following characteristics:(a) Activity in Various Cells and Tissues--present in human fetal lung fibroblasts, hepatoma tissues and placenta, but absent from normal adult liver, lung, kidney and spleen tissues;(b) Substrate Specificity--acts on polypeptides comprising a sequence val-thr-his-pro-gly-tyr by catalyzing u-N-acetylgalactosaminylation at the thr residue of the sequence;(c) Requirements for Metal Ion--requires metal ion for activity in 25 mM tris buffer, pH 7.6;(d) Optimal pH--optimal pH is about 7.6 assayed in hepatoma cell homogenate in tris buffer and at a pH of about 6 to about 7 assayed in hepatoma cell homogenate enzyme activity is higher in 2(N-morpholino)ethanesulfonic acid than in cacodylate buffer; and(e) Km--apparent Km for UDP - GalNAc is about 48 .mu.M.A method of detecting tumor cells comprising the steps of:(1) homogenizing tissue suspected of having tumor cells;(2) incubating the homogenate with labeled .alpha.-N-acetylgalactosamine (.alpha.-GalNAc) and a polypeptide substrate comprising a sequence of val-thr-his-pro-gly-tyr under conditions sufficient to catalyze .alpha.-N-acetylgalactosaminylation of the thr residue to thereby form labeled polypeptide substrate comprising val-thr(m-GalNAc)-his-pro-gly-tyr; and(3) determining the presence of said labeled polypeptide substrate.A diagnostic test kit for detecting tumor cells in a sample suspected of containing tumor cells comprising as separate components:(a) labeled m-N-acetylgalactosamine; and(b) polypeptide substrate comprising a sequence of val-thr-his-pro-gly-tyr.
摘要翻译：作为分离自细胞和组织匀浆的颗粒膜级分的组分分离发育调节的α-N-乙酰半乳糖胺基转移酶，具有以下特征：（a）各种细胞和组织中的活性 - 存在于人胎儿肺成纤维细胞，肝癌组织和胎盘，但不存在正常成年肝，肺，肾，脾组织; （b）底物特异性 - 通过在序列的残基处催化u-N-乙酰半乳糖胺化而对包含序列val-thr-his-gly-tyr的多肽进行作用; （c）对金属离子的要求 - 要求金属离子在25mM tris缓冲液（pH 7.6）中活性; （d）在Tris缓冲液中，在肝癌细胞匀浆中的最佳pH-最佳pH约7.6，在约6〜约7的pH下测定肝癌细胞匀浆酶活性在2（N-吗啉代）乙磺酸中比在二甲胂酸中高缓冲; 和（e）UDP-GalNAc的Km-表观Km为约48μM。一种检测肿瘤细胞的方法，包括以下步骤：（1）使怀疑具有肿瘤细胞的组织匀浆; （2）在足以催化α-N-乙酰半乳糖胺化的条件下，将匀浆与标记的α-N-乙酰半乳糖胺（α-GalNAc）和包含val-thr-his-pro-gly-tyr序列的多肽底物一起温育残留物，由此形成包含val-thr（m-GalNAc）-his-pro-gly-tyr的标记多肽底物; 和（3）确定所述标记的多肽底物的存在。一种用于检测怀疑含有肿瘤细胞的样品中的肿瘤细胞的诊断测试试剂盒，其包含单独的组分：（a）标记的m-N-乙酰半乳糖胺; 和（b）包含val-thr-his-pro-gly-tyr序列的多肽底物。

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