Characterization of human glucosylsphingosine glucosyl hydrolase and comparison with glucosylceramidase.
Characterization of human glucosylsphingosine glucosyl hydrolase and comparison with glucosylceramidase.
复制标题
人葡萄糖基鞘氨醇葡萄糖基水解酶的表征以及与葡萄糖神经酰胺酶的比较。
DOI：
10.1111/j.1432-1033.1985.tb08655.x
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发表时间：
1985
期刊：
影响因子：
--
通讯作者：
Suzuki,K
中科院分区：
文献类型：
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作者：
Vaccaro,AM;Muscillo,M;Suzuki,K
本文对体外培养的人成纤维细胞和胎盘中的神经鞘氨醇葡萄糖水解酶的性质进行了详细的研究，并与葡萄糖神经酰胺酶进行了比较。这两种活性在高雪病患者的组织中是缺乏的，表现出微小但一致的差异。神经氨酸酶的最适pH为4.8，葡萄糖神经酰胺的最适pH为5.3。在油酸存在的情况下，牛磺胆酸盐能激活10倍以上的葡萄糖神经酰胺酶，而仅能激活约30%的心苷水解酶。Triton X-100对葡萄糖神经酰胺酶有刺激作用，但对神经氨酸的水解有强烈的抑制作用。磷脂可使葡萄糖神经酰胺酶活力提高数倍，对神经氨酸的酶解有一定的抑制作用。神经氨酸水解酶活性的热稳定性略高于葡萄糖神经酰胺酶活性。两种底物的Km值相似，心苷的Km值为1.70×10−5M，葡萄糖神经酰胺的Km值为2.7×10−5M。然而，葡萄糖神经酰胺的V值是精神苷水解物的100倍。精神苷是一种有效的非竞争性抑制物(Ki=1.8×10−5M)，而葡萄糖神经酰胺是一种弱的精神苷水解酶抑制物。在葡萄糖神经酰胺的溶解度范围内，心苷的水解率不能被抑制超过50%。此外，葡萄糖神经酰胺抑制作用的Dixon图呈反常斜率。在成纤维细胞、胎盘线粒体-溶酶体组分和部分纯化的胎盘制剂中，这两种活性的比例相似。这些发现最好的解释是假设，虽然这两种底物被单一的酶水解，但它们共享一个重叠但不相同的催化部位，同时结合到各自底物特有的疏水部位。
Properties of glucosylsphingosine (gluco‐psychosine) glucosyl hydrolase were studied in detail in cultured human fibroblasts and placenta and were compared with those of glucosylceramidase. The two activities, that are deficient in tissues of Gaucher patients, showed minor but consistent differences. The pH optima were 4.8 for psychosine hydrolysis and 5.3 for glucosylceramide hydrolysis. In the presence of oleic acid, taurocholate activated glucosylceramidase more than 10‐fold, while it activated psychosine hydrolysis only by about 30%. Triton X‐100 was stimulatory for glucosylceramidase but was strongly inhibitory for psychosine hydrolysis. Phospholipids, that increase many times glucosylceramidase activity, were moderately inhibitory to enzymatic hydrolysis of psychosine. The psychosine hydrolase activity was slightly more heat‐stable than the glucosylceramidase activity. TheKmvalues for the two substrates were similar; 1.7 × 10−5M for psychosine and 2.7 × 10−5M for glucosylceramide. TheVfor glucosylceramide was, however, 100‐times that for psychosine hydrolysis. Psychosine acted as a potent non‐competitive inhibitor (Ki= 1.8 × 10−5M), while glucosylceramide was a weak inhibitor against psychosine hydrolysis. Within the limit of glucosylceramide solubility, psychosine hydrolysis could not be inhibited by more than 50%. Furthermore, the Dixon plot of glucosylceramide inhibition showed an anomalous slope. The ratio of the two activities was similar in fibroblasts, in the placenta mitochondria‐lysosomal fraction and in a partially purified placental preparation. These findings are best explained by the hypothesis that, although the two substrates are hydrolyzed by a single enzyme, they share an overlapping but not identical catalytic site while binding to hydrophobic sites unique for the respective substrates.