Sugasawara R, Harper E
Biochemistry. 1984 Oct 23;23(22):5175-81. doi: 10.1021/bi00317a014.
Three collagenases from Clostridium histolyticum, designated C1, C2, and C3, with apparent molecular weights of 96 000, 92 000, and 76 000 were purified. Peptide maps of the enzymes prepared by digestion with Staphylococcus aureus V-8 protease were found to be similar. Cleavage of native C1 with alpha-chymotrypsin or V-8 protease yielded C2 and C3. This suggested that proteolysis of the Mr 96 000 collagenase may have occurred in vivo, producing the other two lower molecular weight enzymes. Previously prepared antiserum directed against a form of the bacterial enzyme similar by molecular weight and charge to collagenase C3 and Fab' fragments generated from this antiserum inhibited the collagenolytic activity. C1, C2, and C3 were immunologically identical by Ouchterlony double diffusion, and C3 was able to compete with C1 for the antiserum binding site. The ability of each enzyme to bind to antiserum raised against the bacterial collagenase supported the hypothesis that these three proteins were closely related. Zinc analyses of C1 and C3 resulted in a value of 1.14 mol of zinc/mol of C1 and 0.82 mol of zinc/mol of C3. C1 did not contain carbohydrate as measured by gas-liquid chromatography or periodic acid-Schiff staining.
从溶组织梭菌中纯化出了三种胶原酶，分别命名为C1、C2和C3，其表观分子量分别为96000、92000和76000。经金黄色葡萄球菌V-8蛋白酶消化制备的这些酶的肽图相似。用α-胰凝乳蛋白酶或V-8蛋白酶切割天然C1可产生C2和C3。这表明，分子量为96000的胶原酶可能在体内发生了蛋白水解，产生了另外两种分子量较低的酶。先前制备的针对一种分子量和电荷与胶原酶C3相似的细菌酶形式的抗血清以及由此抗血清产生的Fab'片段可抑制胶原分解活性。通过Ouchterlony双向扩散法可知，C1、C2和C3在免疫上是相同的，并且C3能够与C1竞争抗血清结合位点。每种酶与针对细菌胶原酶产生的抗血清结合的能力支持了这三种蛋白质密切相关的假说。对C1和C3进行锌分析得出，C1的锌含量为1.14摩尔锌/摩尔C1，C3的锌含量为0.82摩尔锌/摩尔C3。通过气液色谱法或过碘酸-希夫染色法测定，C1不含碳水化合物。
