Kazakova O V, Orekhovich V N
Int J Pept Protein Res. 1975;7(1):23-9. doi: 10.1111/j.1399-3011.1975.tb02411.x.
An insoluble preparation of rat liver cathepsin D was obtained by coupling the enzyme to Enzacryl Polyacetal (EPA-cathepsin) and to CNBr-activated Sepharose 4B. EPA-cathepsin was active toward the synthetic hexapeptides (Gly-Phe-Leu)2 and did not split hemoglobin. The optimum pH of splitting was displaced upward by 1.5 units to pH 5.0. The enzyme exhibited maximum activity at 60 degrees C. No appreciable loss of activity was seen on storage of the enzyme for 4 months or after repeated use of the preparations. Coupling of rat liver cathepsin D to activated Sepharose gave preparations active towards both protein and synthetic substrates. The preparations were totally inactive in acid media and exhibited maximum activity at pH 7.0, that is, under physiological conditions. Optimum temperature was 65 degrees. The specific activity of the preparations (pH 7.0, 65 degrees) was 60-110 percent that of the free enzyme in acid media. Proteolytic activity of the Sepharose-coupled cathepsin D was not inhibited by pepstatin, whereas that of the free enzyme was fully inhibited by this reagent. A sarcoma cathepsin, similar in some of its properties to the rat liver enzyme, was also coupled to CNBr-activated Sepharose 4B. The preparation split protein substrates at pH 7.0 and possessed enhanced thermostability. The enzymes fixed on Sepharose showed increased stability.
通过将大鼠肝脏组织蛋白酶D与恩扎克里尔聚缩醛（EPA - 组织蛋白酶）以及溴化氰活化的琼脂糖4B偶联，获得了一种不溶性制剂。EPA - 组织蛋白酶对合成六肽（甘氨酸 - 苯丙氨酸 - 亮氨酸）₂有活性，且不分解血红蛋白。分解的最适pH值向上偏移1.5个单位至pH 5.0。该酶在60℃时表现出最大活性。该酶储存4个月或制剂重复使用后，活性未见明显损失。将大鼠肝脏组织蛋白酶D与活化的琼脂糖偶联得到的制剂对蛋白质和合成底物均有活性。这些制剂在酸性介质中完全无活性，在pH 7.0即生理条件下表现出最大活性。最适温度为65℃。制剂（pH 7.0，65℃）的比活性是酸性介质中游离酶比活性的60 - 110%。琼脂糖偶联的组织蛋白酶D的蛋白水解活性不受胃蛋白酶抑制剂的抑制，而游离酶的蛋白水解活性则被该试剂完全抑制。一种肉瘤组织蛋白酶，其某些性质与大鼠肝脏酶相似，也与溴化氰活化的琼脂糖4B偶联。该制剂在pH 7.0时能分解蛋白质底物，并且具有增强的热稳定性。固定在琼脂糖上的酶显示出更高的稳定性。
