Korant B, Towatari T, Kelley M, Brzin J, Lenarcic B, Turk V
Central Research and Development Department, DuPont Company, Experimental Station, Wilmington.
Biol Chem Hoppe Seyler. 1988 May;369 Suppl:281-6.
The interactions of two cystatins with a viral cysteine protease were studied using several types of assays. Complex formation between the protease and inhibitor was directly demonstrated using a gel retardation assay. It was also shown that the formation of enzyme inhibitor complexes could occur after first binding either the enzyme or the inhibitor to filter paper, and ultimately decorating the complex with antibody and radio-labelled protein A or by preparing one of the protein ligands with an internal radiolabel. The procedure can be adapted to provide a method for screening expression libraries for protease or inhibitor genes. The inhibition of a cysteine protease by a cystatin was shown not to directly involve binding to the active site thiol of the enzyme, but rather to be the result of a steric block in the active site region which prevents large affinity labels and protein substrates from reaching the active site.
使用多种类型的测定法研究了两种半胱氨酸蛋白酶抑制剂与一种病毒半胱氨酸蛋白酶的相互作用。通过凝胶阻滞测定法直接证明了蛋白酶与抑制剂之间形成了复合物。还表明，酶抑制剂复合物的形成可以在酶或抑制剂首先与滤纸结合之后发生，并最终用抗体和放射性标记的蛋白A对复合物进行标记，或者通过用内部放射性标记制备其中一种蛋白质配体来实现。该方法可适用于提供一种筛选蛋白酶或抑制剂基因表达文库的方法。结果表明，一种半胱氨酸蛋白酶抑制剂对半胱氨酸蛋白酶的抑制作用并非直接涉及与酶活性位点巯基的结合，而是活性位点区域空间位阻的结果，这种空间位阻阻止了大的亲和标记物和蛋白质底物到达活性位点。
