A SELECTIVE COLORIMETRIC ASSAY FOR CATHEPSIN-L USING Z-PHE-ARG-4-METHOXY-BETA-NAPHTHYLAMIDE
A SELECTIVE COLORIMETRIC ASSAY FOR CATHEPSIN-L USING Z-PHE-ARG-4-METHOXY-BETA-NAPHTHYLAMIDE
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DOI：
10.1016/0300-9084(93)90042-q
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发表时间：
1993-01-01
期刊：
影响因子：
3.9
通讯作者：
SINGH, H
中科院分区：
文献类型：
--
作者：
KAMBOJ, RC;PAL, S;SINGH, H
在细胞内蛋白酶中，在弱酸性pH下作用的硫醇蛋白酶，如组织蛋白酶B (EC 3.4.22.1)、组织蛋白酶H (EC 3.4.22.16)和组织蛋白酶L (EC 3.4.22.15)更有可能在溶酶体蛋白质分解代谢中发挥重要作用。其中，组织蛋白酶 L 发挥着重要作用，主要是因为它对细胞和基质蛋白具有高降解活性。然而，由于缺乏专门测量这种蛋白酶活性的特定底物，对粗匀浆和亚细胞组分中组织蛋白酶 L 的研究一直受到阻碍。唯一可被组织蛋白酶 L 水解的合成底物 α-N-苄氧基羰基-L-Phe-L-Arg-4-甲氧基-β-萘酰胺 (Z-Phe-Arg-NNapOMe) 也能同样被组织蛋白酶 B 水解。该底物被操纵作为组织蛋白酶 L 的选择性底物。在 pH 5.0 的 4 M 尿素存在下，组织蛋白酶 B （唯一一种也水解 Z-Phe-Arg-NNapOMe 的组织蛋白酶）被灭活，因此，在这些条件下，使用该底物定量的酶活性仅归因于组织蛋白酶 L。使用这种新开发的组织蛋白酶 L 特异性比色测定方法，建立了该蛋白酶在山羊脑组织中的亚细胞和区域分布。在溶酶体部分中恢复了约 80% 的组织蛋白酶 L 活性，从而确定了其溶酶体性质。在大脑的各个部分中，大脑的活动最高，其次是小脑、垂体、脑桥、丘脑、延髓和下丘脑。
Among the intracellular proteinases, the thiol proteinases such as cathepsin B (EC 3.4.22. 1), cathepsin H (EC 3.4.22.16) and cathepsin L (EC 3.4.22.15) which act at slightly acidic pHs are more likely to play an important role in lysosomal protein catabolism. Out of these, cathepsin L plays a major role primarily because it has high degradative activity on cellular and matrix proteins. However, the studies on cathepsin L in crude homogenates and subcellular fractions have always been hampered by the lack of a specific substrate to exclusively measure the activity of this proteinase. The only synthetic substrate alpha-N-benzyloxycarbonyl-L-Phe-L-Arg-4-methoxy-beta-naphthylamide (Z-Phe-Arg-NNapOMe) which is hydrolysed by cathepsin L is hydrolysed equally well by cathepsin B. This substrate was manipulated to act as a selective substrate for cathepsin L. In presence of 4 M urea at pH 5.0, cathepsin B (the only other cathepsin which also hydrolyses Z-Phe-Arg-NNapOMe) was inactivated and, therefore, under these conditions, the enzyme activity quantitated by using this substrate is only due to cathepsin L. Using this newly-developed colorimetric assay method specific for cathepsin L, the subcellular and regional distribution of this proteinase were established in goat brain tissue. About 80% cathepsin L activity was recovered in the lysosomal fraction thus establishing its lysosomal nature. Among the various brain parts, highest activity was found in cerebrum followed by cerebellum, pituitary body, pons-varolli, thalamus, medulla-oblongata and hypothalamus.