de los Santos T, Wang J, Reich E
Department of Pharmacological Sciences, State University of New York at Stony Brook 11794-8651, USA.
Ciba Found Symp. 1997;212:66-76; discussion 76-83. doi: 10.1002/9780470515457.ch5.
We describe limited chemical proteolysis of microplasminogen/microplasmin (mPlg/mPlm) and their reconstitution from isolated fragments. A V141-->M141 substitution in methionineless human mPlg/mPlm allowed the protein(s) to be cleaved in CNBr/formic acid. The resulting two fragments (141 and 118 residues, respectively), each internally disulfide bonded, were separated by preparative non-reducing gradient SDS-PAGE, and could then be mixed to reconstitute the characteristic mPlg/mPlm, including their activation by urokinase (uPA) and streptokinase (SK), and inhibition by macromolecular inhibitors. The isolated larger, N-terminal fragment, which contains the mPlg activation site in a normal disulfide configuration, was not cleaved by uPA in the absence of its smaller C-terminal companion, showing that the linear amino acid sequence is not by itself sufficient to confer substrate character, even when its conformation is constrained by the disulfide structure.
我们描述了微纤溶酶原/微纤溶酶（mPlg/mPlm）的有限化学蛋白酶解及其从分离片段的重构。在无蛋氨酸的人mPlg/mPlm中进行V141→M141替换，使得该蛋白能够在溴化氰/甲酸中被切割。产生的两个片段（分别为141个和118个残基），各自内部形成二硫键，通过制备性非还原梯度SDS-PAGE分离，然后可以混合以重构具有特征性的mPlg/mPlm，包括它们被尿激酶（uPA）和链激酶（SK）激活以及被大分子抑制剂抑制。分离出的较大的N端片段，其在正常二硫键构型中包含mPlg激活位点，在没有较小的C端伴侣时不会被uPA切割，这表明即使线性氨基酸序列的构象受到二硫键结构的限制，其本身也不足以赋予底物特性。
