PURIFICATION AND SEPARATION OF INDIVIDUAL COLLAGENASES OF CLOSTRIDIUM-HISTOLYTICUM USING RED-DYE LIGAND CHROMATOGRAPHY
PURIFICATION AND SEPARATION OF INDIVIDUAL COLLAGENASES OF CLOSTRIDIUM-HISTOLYTICUM USING RED-DYE LIGAND CHROMATOGRAPHY
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DOI：
10.1021/bi00308a035
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发表时间：
1984-01-01
期刊：
影响因子：
2.9
通讯作者：
VANWART, HE
中科院分区：
文献类型：
--
作者：
BOND, MD;VANWART, HE
对溶组织菌培养滤液中存在的6种胶原酶进行了纯化，使其达到均匀性。羟基磷灰石、Sephacryl S-200和l-精氨酸- affi - gel 202层析可以去除棕色色素和绝大多数对酪蛋白、苯甲酰- l-精氨酸乙酯和弹性蛋白有活性的污染蛋白酶。活性红120染料配体色谱法将具有非常相似的物理化学性质的胶原酶细分为4个部分。最后的纯化是通过deae -纤维素和SP-Sephadex层析实现的。所有6种胶原蛋白，代号。alpha。度量。,可以利用。，。delta，。和.zeta。根据它们的纯化顺序，它们对胶原蛋白具有高度活性，而缺乏其他蛋白水解活性。在十二烷基硫酸钠-聚丙烯酰胺凝胶上，每一种都有一个单带。α的两个不同亚种。并可以利用。所分离的酶分子量和活性相同，但等电点不同。其他胶原酶有一些不太明显的微观异质性。根据它们对天然胶原蛋白和合成肽2-呋喃丙烯酰-l -亮氨酸甘酰基-l -脯氨酸-l -丙氨酸（FALGPA）的活性，将6种胶原酶分为2类。I类胶原酶。度量。和.gamma.)具有高的胶原酶活性和中等的FALGPA活性，II类胶原酶（.delta., .epsilon.）。和.zeta.)具有中等胶原酶和高FALGPA活性。我们还研究了这6种胶原酶与其他文献中分离出的胶原酶之间的关系。
Six collagenases present in the culture filtrate of C. histolyticum were purified to homogeneity. Chromatography over hydroxylapatite, Sephacryl S-200 and L-arginine-Affi-Gel 202 removes the brown pigment and the great majority of the contaminating proteinases active against casein, benzoyl-L-arginine ethyl ester and elastin. Reactive Red 120 dye ligand chromatography subdivides the collagenases, which have very similar physicochemical properties, among 4 fractions. The final purification is achieved by chromatography over DEAE-cellulose and SP-Sephadex. All 6 collagenases, designated .alpha., .beta., .gamma., .delta., .epsilon. and .zeta. by the order of their purification, are highly active against collagen and devoid of other proteolytic activities. Each exhibits a single band on sodium dodecyl sulfate-polyacrylamide gels. Two distinct subspecies of the .alpha. and .gamma. enzymes were isolated, which have the same MW and activity but different isoelectric points. There is some less pronounced microheterogeneity for the other collagenases. On the basis of their activities toward native collagen and the synthetic peptide 2-furanacryloyl-L-leucylglycyl-L-prolyl-L-alanine (FALGPA), the 6 collagenases are divided into 2 classes. Class I collagenases (.alpha., .beta. and .gamma.) have high collagenase activity and moderate FALGPA activity, class II collagenases (.delta., .epsilon. and .zeta.) have moderate collagenase and high FALGPA activities. The relationship between these 6 collagenases and others reported to have been isolated in the literature was also examined.