黑色素瘤细胞纤溶酶原激活物的纯化和表征。
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瓦伦P波尔克,与N, Ranby M,纽约T, Jornvall H
黑色素瘤细胞纤溶酶原激活物的纯化和表征。
欧元。1983 5月16日,132 (3):681 - 6。
- PubMed ID
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6682760 (在PubMed]
- 文摘
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人类黑色素瘤细胞系的纤溶酶原激活物纯化和免疫吸附作为一个主要步骤。细胞培养在抑肽酶的存在为了避免蛋白质水解。涉及三步纯化抗体吸附在猪组织纤溶酶原激活物在色谱arginine-Sepharose和交联葡聚糖g - 150。所有的溶剂包含渐变- 80(0.01%),除了最后一步,抑肽酶。最终产品的特定活动220000 IU /毫克来衡量谁尿激酶标准。获得的催化剂具有明显的72000先生,由单链分子。四种不同的活化剂的证据是变异发生。第一,知道以前,一串形式可以proteolytically裂解成two-chain形式。其次,一条项链和two-chain分子表现出两种形式与分子量的差异大约3000(可能是由于碳水化合物的差异)。第三,一串准备包含两个变量,每个构成大约50%的材料和不同的长度由三个n端氨基酸。 Finally, a possible positional microheterogeneity was detected. Digestion with plasmin yields the two-chain form with disulfide-bonded polypeptide chains, 'A' and 'B' (from the N-terminal and C-terminal parts, respectively). At the same time, the variability of the original N terminus is removed. The A chain keeps the two Mr variants (now about 40000 and 37000, respectively). The B chain (Mr about 33000) contains the active site of the molecule, as demonstrated by labelling with [3H]diisopropyl phosphofluoridate, and is homologous to the enzymatically active chains of thrombin, plasmin and other serine proteases. In contrast to these enzymes, the plasminogen activator is enzymatically active in the one-chain form. A speculative explanation for this activity may possibly be the presence of an epsilon-amino group of a lysine residue at a position close to the bond cleaved in the two-chain form.