Yonekawa O, Voskuilen M, Nieuwenhuizen W
Gaubius Laboratory IVVO-TNO, Leiden, The Netherlands.
Biochem J. 1992 Apr 1;283 ( Pt 1)(Pt 1):187-91. doi: 10.1042/bj2830187.
In previous publications [e.g. Voskuilen, Vermond, Veeneman, Van Boom, Klasen, Zegers & Nieuwenhuizen (1987) J. Biol. Chem. 262, 5944-5946] we have shown that fibrin(ogen) chain fragment A alpha-(148-160) contains a site that contributes to the acceleration of Glu-plasminogen activation by tissue-type plasminogen activator (t-PA). In contrast with fibrin, this peptide, however, does not enhance the rate of mini-plasminogen activation. Therefore, possibly more stimulatory sites than A alpha-(148-160) are present in fibrin. In the present investigation we have localized a possible second type of stimulatory site in the fibrin(ogen) molecule. A whole CNBr digest of fibrinogen was applied to a Bio-Gel P-2 column run in water, pH 4. Two peaks with stimulatory activity were observed, one at the void volume and one between the void volume and the total volume. The former contained the previously described stimulating fragment FCB-2 [which comprises A alpha-(148-160)]; the latter had not been observed before and was characterized further. The stimulating material in the low-M(r) fraction of the Bio-Gel P-2 column was precipitated at pH 8.3 in a virtually pure form. It has a high tryptophan content, and an M(r) of 6500 as assessed by SDS/PAGE. On reduction, a main band of M(r) 2500 is seen, plus a weakly staining band of M(r) 4000. These properties plus the amino acid sequence data identify the fragment as FCB-5. FCB-5 consists of two chains, i.e. gamma-(311-336) and gamma-(337-379), linked by a single disulphide bond between Cys-gamma-326 and Cys-gamma-339. Both these chains and the disulphide bond appear to be essential for rate enhancement. FCB-5 enhances the activation rates of Glu-, mini- and micro-plasminogen, with all five kringles, only kringle V and without kringles respectively. FCB-5 binds t-PA, but none of the plasminogen forms binds to FCB-5. This indicates that the rate enhancements induced by FCB-5 are due to an effect on t-PA.
在之前的出版物中[例如Voskuilen、Vermond、Veeneman、Van Boom、Klasen、Zegers和Nieuwenhuizen（1987年），《生物化学杂志》262卷，5944 - 5946页]，我们已经表明纤维蛋白（原）链片段Aα-(148 - 160)含有一个有助于组织型纤溶酶原激活剂（t-PA）加速Glu-纤溶酶原激活的位点。然而，与纤维蛋白不同的是，该肽并不能提高微型纤溶酶原的激活速率。因此，纤维蛋白中可能存在比Aα-(148 - 160)更多的刺激位点。在本研究中，我们在纤维蛋白（原）分子中定位了一个可能的第二种刺激位点类型。将纤维蛋白原的完整溴化氰消化产物应用于在pH 4的水中运行的Bio-Gel P-2柱。观察到两个具有刺激活性的峰，一个在空体积处，一个在空体积和总体积之间。前者包含先前描述的刺激片段FCB-2[其包含Aα-(148 - 160)]；后者以前未被观察到，并对其进行了进一步表征。Bio-Gel P-2柱低分子量部分的刺激物质在pH 8.3时以几乎纯的形式沉淀。它具有高色氨酸含量，通过SDS/PAGE评估其分子量为6500。还原后，可见一条分子量为2500的主带，加上一条分子量为4000的弱染色带。这些特性以及氨基酸序列数据将该片段鉴定为FCB-5。FCB-5由两条链组成，即γ-(311 - 336)和γ-(337 - 379)，通过Cys-γ-326和Cys-γ-339之间的单个二硫键连接。这两条链和二硫键似乎对速率增强都是必不可少的。FCB-5提高了Glu-、微型和微型纤溶酶原的激活速率，它们分别具有所有五个kringle结构域、仅kringle V结构域和无kringle结构域。FCB-5结合t-PA，但纤溶酶原的任何形式都不与FCB-5结合。这表明FCB-5诱导的速率增强是由于对t-PA的作用。
