Nakamura S, Iwanaga S, Suzuki T
J Biochem. 1975 Dec;78(6):1247-66. doi: 10.1093/oxfordjournals.jbchem.a131023.
A blood coagulation factor, Factor XIII, was highly purified from bovine fresh plasma by a method similar to those used for human plasma Factor XIII. The isolated Factor XIII consisted of two subunit polypeptides, a and b chains, with molecular weights of 79,000 +/- 2,000 and 75,000 +/- 2,000, respectively. In the conversion of Factor XIII to the active enzyme, Factor XIIIa, by bovine thrombin [EC 3.4.21.5], a peptide was liberated. This peptide, designated tentatively as "activation peptide," was isolated by gel-filtration on a Sephadex G-75 column. It contained a total of 37 amino acid residues with a masked N-terminal residue and C-terminal arginine. The whole amino acid sequence of "Activation peptide" was established by the dansyl-Edman method and standard enzymatic techniques, and the masked N-terminal residue was identified as N-acetylserine by using a rat liver acylamino acid-releasing enzyme. This enzyme specifically cleaved the N-acetylserylglutamyl peptide bond serine and the remaining peptide, which was now reactive to 1-dimethylamino-naphthalene-5-sulfonyl chloride. A comparison of the sequences of human and bovine "Activation peptide" revealed five amino acids replacements, Ser-3 to Thr; Gly-5 to Arg; Ile-14 to Val; Thr-18 to Asn, and Pro-26 to Leu. Another difference was the deletion of Leu-34 in the human peptide. Adsorption chromatography on a hydroxylapatite column in the presence of 0.1% sodium dodecyl sulfate was developed as a preparative procedure for the resolution of the two subunit polypeptides, a or a' chain and b chain, constituting the protein molecule of Factor XIII or Factor XIIIa. End group analyses on the isolated pure chains revealed that the structural change of Factor XIII during activation with thrombin occurs only in the N-terminal portion of the a chain, not in the N-terminal end of the b chain or in the C-terminal ends of the a and b chains. From these results, it was concluded that the activation of bovine plasma Factor XIII by thrombin must be accompanied by a limited proteolysis of the arginyl-glycyl bond located in the N-terminal region of the a chain, liberating the "Activation peptide." The possibility of activating Factor XII with other porteinases was examined using Factor Xa [EC 3.4.21.6], Factor XIIa, kallikreins [EC 3.4.21.8], urokinase [EC 3.4.99.26], trypsin [EC 3.4.21.4], ficin [EC 3.4.22.3], papain [EC 3.4.22.2], and bromelain [EC 3.4.22.4]. Among these enzymes, only bromelain and trypsin showed clear activating effects.
采用与从人血浆中纯化 XIII 因子相似的方法，从牛新鲜血浆中高度纯化了一种凝血因子 XIII。分离得到的 XIII 因子由两条亚基多肽，即 a 链和 b 链组成，其分子量分别为 79,000±2,000 和 75,000±2,000。在用牛凝血酶[EC 3.4.21.5]将 XIII 因子转化为活性酶 XIIIa 的过程中，释放出一种肽。该肽暂定为“激活肽”，通过在 Sephadex G - 75 柱上进行凝胶过滤分离得到。它总共含有 37 个氨基酸残基，N 端残基被封闭，C 端为精氨酸。“激活肽”的完整氨基酸序列通过丹磺酰 - 埃德曼法和标准酶学技术确定，使用大鼠肝脏酰基氨基酸释放酶鉴定出被封闭的 N 端残基为 N - 乙酰丝氨酸。该酶特异性切割 N - 乙酰丝氨酰 - 谷氨酰肽键中的丝氨酸，剩余的肽现在对 1 - 二甲基氨基萘 - 5 - 磺酰氯有反应。对人和牛“激活肽”的序列进行比较，发现有五个氨基酸替换，即 Ser - 3 替换为 Thr；Gly - 5 替换为 Arg；Ile - 14 替换为 Val；Thr - 18 替换为 Asn，以及 Pro - 26 替换为 Leu。另一个差异是人类肽中 Leu - 34 的缺失。在 0.1%十二烷基硫酸钠存在下，在羟基磷灰石柱上进行吸附色谱法，作为分离构成 XIII 因子或 XIIIa 蛋白分子的两条亚基多肽，即 a 链或 a'链和 b 链的制备方法。对分离得到的纯链进行末端基团分析表明，凝血酶激活 XIII 因子的过程中，结构变化仅发生在 a 链的 N 端部分，而不是 b 链的 N 端或 a 链和 b 链的 C 端。从这些结果可以得出结论，凝血酶激活牛血浆 XIII 因子时，必然伴随着 a 链 N 端区域的精氨酰 - 甘氨酰键的有限蛋白水解，释放出“激活肽”。使用 Xa 因子[EC 3.4.21.6]、XIIa 因子、激肽释放酶[EC 3.4.21.8]、尿激酶[EC 3.4.99.26]、胰蛋白酶[EC 3.4.21.4]、无花果蛋白酶[EC 3.4.22.3]、木瓜蛋白酶[EC 3.4.22.2]和菠萝蛋白酶[EC 3.4.22.4]研究了用其他蛋白酶激活 XII 因子的可能性。在这些酶中，只有菠萝蛋白酶和胰蛋白酶显示出明显的激活作用。
