Tadpole collagenase. Preparation and purification.
Tadpole collagenase. Preparation and purification.
复制标题
蝌蚪胶原酶。
DOI：
10.1021/bi00874a007
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发表时间：
1966
期刊：
影响因子：
2.9
通讯作者：
J. Gross
中科院分区：
文献类型：
--
作者：
Y. Nagai;C. Lapière;J. Gross
Y. Nagai, f . C. M. Lapiere， J.和J. Gross摘要：报道了从蝌蚪尾鳍和背部皮肤组织培养培养基中制备胶原溶解酶的方法。受甲状腺素刺激的蝌蚪组织的酶产量与未受甲状腺素刺激的蝌蚪组织的酶产量相同，而成熟青蛙的皮肤产生的酶活性很少。通过顺序硫酸铵沉淀，凝胶过滤，淀粉块电泳和deae -纤维素层析，酶浓缩高达300倍。酪蛋白溶解活性明显降低，但在最终产物中仍处于低水平。纯化后的酶在pH值为7.6的溶液中攻击天然小牛皮肤胶原蛋白，在20时将粘度降低40-50%，在27时将粘度降低75%，并阻止胶原蛋白的重构。在组织中，胶原蛋白的快速生理吸收一直无法产生一种酶，这种酶在中性pH和生理温度下会攻击天然胶原蛋白（Mandl, 1961; Woessner, 1965； Lapiere和Gross， 1963）。然而，在蝌蚪尾巴、鳃、肠道、哺乳动物子宫、骨骼和皮肤伤口等培养组织的培养基中检测到胶原溶解活性（Gross and Lapiere, 1962; Gross et al., 1963; Walker et al., 1964; Grillo and Gross, 1964）。这种酶显然是从活细胞中合成的，并在培养室中积累到可检测的水平。冻融组织或暴露于嘌呤霉素的组织均不产生酶活性（Gross and Lapiere, 1962; Eisen and Gross, 1965）。从培养基中提取并纯化了蝌蚪胶原酶（Nagai et al., 1963），并对其攻击胶原蛋白的方式进行了部分表征。阴性分子在单个位点和三个多肽链上被攻击，在分子的“B”端切割出四分之一的长度。
Y. Nagai, f C. M. Lapiere, J and J. Gross abstract: The preparation of a collagenolytic enzyme from the medium of tissue cultures of tadpole tail fin and back skin is described. The yield of enzyme from the tissues of thyroxine-stimulated tadpoles was the same as that from nontreated tadpoles and very little activity was produced by the skin of mature frogs. The enzyme was concentrated as much as 300-fold by sequential ammonium sulfate precipitation, gel filtration, starch block electrophoresis, and DEAE-cellulose chromatography. Caseinolytic activity was considerably reduced but was still present at low levels in the final product. The purified enzyme attacks native calf skincollagen in solution at pH 7.6 reducing the viscosity 40-50% at 20 and 75% at 27, and prevents reconstitution pJL^ xtracts of tissues in which rapid physiologic resorp-tion of collagen occurs have consistently failed to yield an enzyme which, at neutral pH and physiologic tem-perature, will attack native collagen (Mandl, 1961; Woessner, 1965; Lapiere and Gross, 1963). However collagenolytic activity has been detected in the medium of cultured tissues such as tadpole tail, gills, and gut, mammalian uterus, bone, and skin wounds (Gross and Lapiere, 1962; Gross et al., 1963; Walker et al., 1964; Grillo and Gross, 1964). This enzyme apparently is synthesized de nooo from living cells and accumulates in the culture chamber to detectable levels during the period of incubation. No enzyme activity was produced by frozen thawed tissues or by those exposed to puromycin (Gross and Lapiere, 1962; Eisen and Gross, 1965). The tadpole collagenase hasbeen harvested from the culture medium and purified (Nagai et al., 1963) and its mode of attack on collagen partially character-ized. Thenative molecule is attacked at a single locus and across the three polypeptide chains cleaving off one-quarter length of the molecule at the “B” end.