Wallén P, Pohl G, Bergsdorf N, Rånby M, Ny T, Jörnvall H
Eur J Biochem. 1983 May 16;132(3):681-6. doi: 10.1111/j.1432-1033.1983.tb07418.x.
The plasminogen activator from a human melanoma cell line was purified with immunoadsorption as a major step. The cells were cultured in the presence of aprotinin in order to avoid proteolysis. A three-step purification involved adsorption on antibodies to porcine tissue plasminogen activator before chromatographies on arginine-Sepharose and Sephadex G-150. All solvents contained Tween-80 (0.01%) and, except for the last step, aprotinin. The final product had a specific activity of about 220000 IU/mg measured against the WHO urokinase standard. The activator obtained has an apparent Mr of 72000 and consists of single-chain molecules. Evidence was obtained that four different types of activator variants occur. First and known previously, the one-chain form can be proteolytically cleaved into a two-chain form. Secondly, both the one-chain and two-chain molecules exhibit two forms with molecular weight differences of about 3000 (possibly due to carbohydrate differences). Thirdly, the one-chain preparations contain two variants, each constituting about 50% of the material and differing in length by three N-terminal amino acids. 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.
以免疫吸附为主要步骤，对来自人黑色素瘤细胞系的纤溶酶原激活剂进行了纯化。为避免蛋白水解，细胞在抑肽酶存在的条件下培养。三步纯化过程包括在精氨酸 - 琼脂糖和葡聚糖凝胶G - 150柱层析之前，先吸附于抗猪组织纤溶酶原激活剂抗体上。所有溶剂均含有吐温 - 80（0.01%），除最后一步外，还含有抑肽酶。以世界卫生组织尿激酶标准品为对照，最终产物的比活性约为220000 IU/mg。所获得的激活剂表观分子量为72000，由单链分子组成。已获得证据表明存在四种不同类型的激活剂变体。首先，也是之前已知的，单链形式可被蛋白水解切割成双链形式。其次，单链和双链分子均呈现两种分子量相差约3000的形式（可能是由于碳水化合物差异）。第三，单链制剂包含两种变体，每种变体约占材料的50%，且N端氨基酸长度相差三个。最后，检测到可能存在的位置微异质性。用纤溶酶消化产生具有二硫键连接的多肽链“A”和“B”（分别来自N端和C端部分）的双链形式。同时，原始N端的变异性被消除。A链保留两种分子量变体（现在分别约为40000和37000）。B链（分子量约为33000）含有分子的活性位点，这通过用[³H]二异丙基磷酰氟标记得以证明，并且与凝血酶、纤溶酶和其他丝氨酸蛋白酶的酶活性链同源。与这些酶不同，纤溶酶原激活剂在单链形式下具有酶活性。对此活性的一种推测性解释可能是在双链形式中靠近切割键的位置存在赖氨酸残基的ε - 氨基。
