Trackman Philip C, Bais Manish V
Boston University Henry M. Goldman School of Dental Medicine, Boston, MA, United States.
Boston University Henry M. Goldman School of Dental Medicine, Boston, MA, United States.
Methods Cell Biol. 2018;143:147-156. doi: 10.1016/bs.mcb.2017.08.009. Epub 2017 Oct 28.
Increasing interest in the multifunctional lysyl oxidase family of proteins is evident from the growth in the number of new publications each year. The enzymes have unique properties of strong affinities to extracellular matrix components, relative insolubility in typical buffers, low catalytic rates, and often low abundance. Here we provide detailed protocols to extract and assay lysyl oxidase enzymes from tissue samples, cell culture cell layers, and media. Buffer conditions and procedures are optimized based on the characteristics mentioned above, while avoiding the use of radioactive substrates. Peroxidase/Amplex Red-based coupled reactions have proven to be the most useful in this context under specified conditions, and permit calculation of specific enzyme activities in absolute amounts of nanomoles of product/unit time/mg protein.
每年新发表文章数量的增长表明，人们对多功能赖氨酰氧化酶蛋白家族的兴趣与日俱增。这些酶具有独特的性质，对细胞外基质成分具有很强的亲和力，在典型缓冲液中相对不溶，催化速率低，且丰度往往较低。在此，我们提供了从组织样本、细胞培养细胞层和培养基中提取和检测赖氨酰氧化酶的详细方案。基于上述特性对缓冲液条件和操作步骤进行了优化，同时避免使用放射性底物。在特定条件下，基于过氧化物酶/氨基苯磺酸红的偶联反应已被证明是最有用的，并且可以计算出以每单位时间/毫克蛋白质产生的纳摩尔数表示的比酶活性。
