Accurate, quantitative assays for the hydrolysis of soluble type I, II, and III 3H-acetylated collagens by bacterial and tissue collagenases.
Accurate, quantitative assays for the hydrolysis of soluble type I, II, and III 3H-acetylated collagens by bacterial and tissue collagenases.
复制标题
通过细菌和组织胶原酶对可溶性 I、II 和 III 型 3H 乙酰化胶原进行水解的准确定量测定。
DOI：
10.1016/0003-2697(86)90558-0
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发表时间：
1986
影响因子：
2.9
通讯作者：
VanWart,HE
中科院分区：
文献类型：
--
作者：
Mallya,SK;Mookhtiar,KA;VanWart,HE
已经开发了通过细菌和组织胶原酶水解可溶性3 H-乙酰化大鼠肌腱I型、牛软骨II型和人羊膜III型胶原的准确和定量测定。在1-30°C范围内的任何温度下在单个反应管中进行测定，并且通过取出作为时间函数的等分试样、用1，10-菲咯啉淬灭和定量水解片段的浓度来监测反应的进展。后者是通过选择性变性的这些片段，通过孵育条件下，在以前的文件中描述的这个问题。该测定给出了中性粒细胞胶原酶水解所有三种类型胶原的百分比，其与凝胶电泳实验的结果一致。所有三种胶原蛋白的水解的初始速率在10倍酶浓度范围内与嗜中性粒细胞或梭菌胶原酶的浓度成比例。所有三种测定法都可以在0.06至2 mg/ml的胶原蛋白浓度范围内进行，并给出组织和细菌胶原酶的线性双倒数图，其可用于评估动力学参数K和kcator Vmax。中性粒细胞胶原酶水解大鼠I型胶原蛋白的试验显示比使用大鼠I型胶原蛋白的可比试验灵敏至少一个数量级。I胶原纤维或凝胶作为底物。
Accurate and quantitative assays for the hydrolysis of soluble3H-acetylated rat tendon type I, bovine cartilage type II, and human amnion type III collagens by both bacterial and tissue collagenases have been developed. The assays are carried out at any temperature in the 1–30°C range in a single reaction tube and the progress of the reaction is monitored by withdrawing aliquots as a function of time, quenching with 1,10-phenanthroline, and quantitation of the concentration of hydrolysis fragments. The latter is achieved by selective denaturation of these fragments by incubation under conditions described in the previous paper of this issue. The assays give percentages of hydrolysis of all three collagen types by neutrophil collagenase that agree well with the results of gel electrophoresis experiments. The initial rates of hydrolysis of all three collagens are proportional to the concentration of both neutrophil or Clostridial collagenases over a 10-fold range of enzyme concentrations. All three assays can be carried out at collagen concentrations that range from 0.06 to 2 mg/ml and give linear double reciprocal plots for both tissue and bacterial collagenases that can be used to evaluate the kinetic parameters Kmand kcator Vmax. The assay developed for the hydrolysis of rat type I collagen by neutrophil collagenase is shown to be more sensitive by at least one order of magnitude than comparable assays that use rat type. I collagen fibrils or gels as substrate.