INTRALYSOSOMAL HYDROLYSIS OF GLYCYL-L-PHENYLALANINE 2-NAPHTHYLAMIDE
INTRALYSOSOMAL HYDROLYSIS OF GLYCYL-L-PHENYLALANINE 2-NAPHTHYLAMIDE
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DOI：
10.1042/bj2190965
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发表时间：
1984-01-01
影响因子：
4.1
通讯作者：
WATTIAUX, R
中科院分区：
文献类型：
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作者：
JADOT, M;COLMANT, C;WATTIAUX, R
甘油丙氨酸2-萘酰胺（GLY-L-PHE-2-NNAP）（一种组织蛋白酶C底物）与总线粒体分数在大鼠肝脏中孵育时，会诱导该酶的游离且无法证明的活性增加。在Triton X-100的情况下，ZnSO4（1 mM）大大抑制了用Gly-L-Phe-2-NNAP作为底物测量的组织蛋白酶C的总活性。确定自由活动时，抑制明显不太明显。仍然存在的高活性取决于溶酶体的完整性。当溶酶体接受能够破坏其膜的治疗时，N-乙酰基葡萄糖胺酶的游离活性增加。当从孵育培养基中省略盐酸硫胺胺时，组织蛋白酶C活性会降低。在这些条件下，处于37级。 C，自由活性等于总活性，尽管溶酶体完好无损，如N-乙酰葡萄糖氨基酶的低游离活性所表明。 ZnSO4（1 mm）明显地抑制了总活性，而> 80％的游离活动仍然存在。 Gly-l-phe-2-NNAP可能导致溶酶体在这些细胞器内部的水解导致破坏。在存在ZnSO4的情况下，由于Zn2+对牙入杂体外水解的优先抑制作用，内溶液内水解变得显而易见。在没有氢氯化硫胺的情况下，这是可以测量的，因为由于反应的减速，Gly-L-Phe-2-NNAP溶酶体的破坏延迟了。强调了GLY-L-PHE-2-NNAP和相关二肽基萘胺在溶酶体 - 膜 - 渗透性研究中的有用性。
Glycyl-L-phenylalanine 2-naphthylamide (Gly-L-Phe-2-NNap), a cathepsin C substrate, induces an increase of the free and unsedimentable activities of this enzyme when incubated with a total mitochondrial fraction of rat liver. ZnSO4 (1 mM) considerably inhibits the cathepsin C total activity, measured with Gly-L-Phe-2-NNap as the substrate, in the presence of Triton X-100. The inhibition is markedly less pronounced when the free activity is determined; a high activity remains that depends on the integrity of the lysosomes; it decreases as the free activity of N-acetylglucosaminidase increases when lysosomes are subjected to treatments able to disrupt their membrane. Cathepsin C activity is reduced when thioethylamine hydrochloride is omitted from the incubation medium. Under these conditions at 37.degree. C, the free activity equals the total activity, although the lysosomes are intact, as indicated by the low free activity of N-acetylglucosaminidase. ZnSO4 (1 mM) strikingly inhibits the total activity, whereas > 80% of the free activity remains. Gly-L-Phe-2-NNap possibly caused a disruption of the lysosomes as a result of its hydrolysis inside these organelles. In the presence of ZnSO4, intralysosomal hydrolysis becomes apparent, owing to a preferential inhibition by Zn2+ of extralysosomal hydrolysis; in the absence of thioethylamine hydrochloride, it is measurable because the disruption of lysosomes by Gly-L-Phe-2-NNap is delayed as a result of a slow-down of the reaction. The usefulness of Gly-L-Phe-2-NNap and related dipeptidyl naphthylamides in lysosomal-membrane-permeability studies is emphasized.