Human skin collagenase in recessive dystrophic epidermolysis bullosa. Purification of a mutant enzyme from fibroblast cultures.
Human skin collagenase in recessive dystrophic epidermolysis bullosa. Purification of a mutant enzyme from fibroblast cultures.
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隐性营养不良性大疱性表皮松解症中的人皮肤胶原酶。
DOI：
10.1172/jci110577
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发表时间：
1982
期刊：
影响因子：
--
通讯作者：
Bauer,EA
中科院分区：
文献类型：
--
作者：
Stricklin,GP;Welgus,HG;Bauer,EA
隐性营养不良性大疱性表皮松解症是一种遗传性皮肤病，其特征是皮肤成纤维细胞在培养过程中胶原酶合成增加。由于部分纯化的隐性营养不良性大疱性表皮松解症胶原酶的初步研究表明，该蛋白本身是异常的，因此我们努力将该酶纯化为均一酶，以便与正常人皮肤成纤维细胞胶原酶进行详细的生化和免疫学比较。从患者身上获得的隐性营养不良性大疱性表皮松解症皮肤成纤维细胞具有增加的酶合成，在大规模的组织培养中生长，并收集作为胶原酶来源的无血清和有血清的培养液。隐性营养不良性大疱性表皮松解症胶原酶经盐析、离子交换和凝胶过滤层析纯化为均一组分。与正常酶相比，隐性营养不良性大疱性表皮松解症胶原酶与羧甲基纤维素结合的钙离子浓度至少是正常酶的10倍。此外，这种酶对层析操作明显更不稳定，特别是当使用无血清介质时。然而，从含血清的培养液中快速纯化得到了一种酶活相当于正常人体皮肤胶原酶的制剂。与正常酶一样，隐性营养不良性大疱性表皮松解症胶原酶被分泌为两个密切相关的酶原∼60,000和∼55,000道尔顿，可被胰酶激活，分别形成∼50,000和∼45,000道尔顿的酶活性物种。氨基酸分析表明，正常和隐性营养不良性大疱性表皮松解症胶原酶之间略有差异。溴化氰消化显示了来自每个来源的酶所独有的多肽。隐性营养不良性大疱性表皮松解症原酶在60°C时明显比正常情况下更不耐热，这一发现与突变酶对已知的人类皮肤胶原酶激活剂和稳定剂--钙离子的亲和力降低了近四倍有关。除了对这种金属辅因子的亲和力改变外，对结构改变的隐性营养不良性大疱性表皮松解症胶原酶的动力学分析表明，它对人I-V型胶原的反应速度和底物专一性与正常酶相同。同样，两种来源的酶表现出相同的激活能量和氚同位素效应。抗血清分别被提高到正常和可能突变的前胶原酶，尽管在双向扩散分析中它们表现出相同的反应，但在酶抑制和定量沉淀研究中存在免疫学差异。这些研究表明，隐性营养不良性大疱性表皮松解症的特征是酶活性正常但结构异常的胶原酶合成增加。
Recessive dystrophic epidermolysis bullosa, a genodermatosis characterized by dermolytic blister formation in response to minor trauma, is characterized by an incresaed collagenase synthesis by skin fibroblasts in culture. Since preliminary studies of partially purified recessive dystrophic epidermolysis bullosa collagenase suggested that the protein itself was aberrant, efforts were made to purify this enzyme to homogeneity, so that detailed biochemical and immunologic comparisons could be made with normal human skin fibroblast collagenase. Recessive dystrophic epidermolysis bullosa skin fibroblasts obtained from a patient documented to have increased synthesis of the enzyme were grown in large scale tissue culture and both serum-free and serum-containing medium collected as a source of collagenase. The recessive dystrophic epidermolysis bullosa collagenase was purified to electrophoretic homogeneity using a combination of salt precipitation, ion-exchange, and gel-filtration chromatography. In contrast to the normal enzyme, the recessive dystrophic epidermolysis bullosa collagenase bound to carboxymethyl-cellulose at Ca2+concentrations at least 10 times higher than those used with the normal enzyme. Additionally, this enzyme was significantly more labile to chromatographic manipulations, particularly when serum-free medium was used. However, rapid purification from serum-containing medium yielded a preparation enzymatically equivalent to normal human skin collagenase. Like the normal enzyme, the recessive dystrophic epidermolysis bullosa collagenase was secreted as a set of two closely related zymogens of ∼60,000 and ∼55,000 daltons that could be activated by trypsin to form enzymically active species of ∼50,000 and ∼45,000 daltons, respectively. Amino acid analysis suggested slight variations between the normal and recessive dystrophic epidermolysis bullosa collagenases. Cyanogen bromide digests demonstrated peptides unique to the enzyme from each source. The recessive dystrophic epidermolysis bullosa proenzyme was significantly more thermolabile at 60°C than the normal, a finding that correlated with an approximate fourfold decrease in the affinity of the mutant enzyme for Ca2+, a known activator and stabilizer of human skin collagenase. Aside from the altered affinity for this metal cofactor, kinetic analysis of the structurally altered recessive dystrophic epidermolysis bullosa collagenase revealed that its reaction rates and substrate specificity for human collagen types I-V were identical to those for the normal enzyme. Likewise, enzymes from both sources displayed identical energies of activation and deuterium isotope effects. Antisera were raised to the normal and putatively mutant procollagenases respectively, and, although they displayed a reaction of identity in double diffusion analysis, immunologic differences were present in enzyme inhibition and quantitative precipitation studies. These studies indicate that recessive dystrophic epidermolysis bullosa is characterized by the increased synthesis of an enzymically normal, but structurally aberrant, collagenase.Images
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大疱性表皮松解症发病机制的研究。
影响因子：
6.5
作者：
R. Pearson
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R. Pearson
人皮肤成纤维细胞胶原酶的胶原底物特异性。
DOI：
--
发表时间：
1981
期刊：
The Journal of biological chemistry
影响因子：
--
作者：
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通讯作者：
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影响因子：
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作者：
W. E. Groves;W. E. Groves;Francis C. Davis;Francis C. Davis;B. H. Sells;B. H. Sells
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影响因子：
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作者：
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通讯作者：
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DOI：
--
发表时间：
1977
影响因子：
11.1
作者：
E. Bauer
通讯作者：
E. Bauer