5'-NUCLEOTIDASES OF CHICKEN GIZZARD AND HUMAN PANCREATIC ADENOCARCINOMA CELLS ARE ANCHORED TO THE PLASMA-MEMBRANE VIA A PHOSPHATIDYLINOSITOL GLYCAN
5'-NUCLEOTIDASES OF CHICKEN GIZZARD AND HUMAN PANCREATIC ADENOCARCINOMA CELLS ARE ANCHORED TO THE PLASMA-MEMBRANE VIA A PHOSPHATIDYLINOSITOL GLYCAN
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DOI：
10.1042/bj2620033
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发表时间：
1989-08-15
影响因子：
4.1
通讯作者：
MANNHERZ, HG
中科院分区：
文献类型：
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作者：
STOCHAJ, U;FLOCKE, K;MANNHERZ, HG
我们分析了质膜 5''-核苷酸酶的膜锚定，这是一种可以介导与细胞外基质成分结合的胞外酶。我们证明，通过磷脂酶 C 和 D 处理，从鸡砂囊和人胰腺腺癌细胞系中获得的纯化酶均完全转化为亲水形式，裂解糖基磷脂酰肌醇 (GPI)。这些数据表明存在用于从两个来源获得的5''-核苷酸酶的膜锚定的糖脂接头。在相同条件下孵育质膜表明，大约一半的 AMPase 活性对 GPI 水解磷脂酶具有抗性。对纯化的鸡肫 5'' 核苷酸酶的酶学特性的研究表明，在去除磷脂酰肌醇接头后仅发生了微小的变化。然而，膜锚的裂解导致对伴刀豆球蛋白 A 抑制的敏感性增加。组织分级分离后，可以获得膜结合蛋白或可溶性蛋白形式的鸡砂囊 5''-核苷酸酶；后者被怀疑是通过内源性磷脂酶从质膜释放的。与纯化的鸡肫 5''-核苷酸酶发生免疫交叉反应的较高分子量蛋白质被检测为可溶性和膜结合形式。
We have analysed the membrane anchorage of plasma-membrane 5''-nucleotidase, an ectoenzyme which can mediate binding to components of the extracellular matrix. We demonstrated that the purified enzyme obtained from chicken gizzard and human pancreatic adenocarcinoma cell line were both completely transformed into a hydrophilic form by treatment with phospholipases C and D, cleaving glylcosylphopshatidylinositol (GPI). These data indicate the presence of a glycolipid linker employed for membrane anchoring of the 5''-nucleotidase obtained from both sources. Incubation of plasma membranes under identical conditions revealed that about half of the AMPase activity was resistant to GPI-hydrolysing phopsholipases. Investigation of the enzymic properties of purified chicken gizzard 5''nucleotidase revealed only minor changes after removal of the phosphatidylinositol linker. However, cleavage of the membrane anchor resulted in an increased sensitivity towards inhibition by concanavalin A. After tissue fractionation, chicken gizzard 5''-nucleotidase could be obtained as either a membrane-bound or a soluble protein; the latter is suspected to be released from the plasma membrane by endogenous phospholipases. Higher-molecular-mass proteins immuno-cross-reactive with the purified chicken gizzard 5''-nucleotidase were detected as both soluble and membrane-bound forms.