van Weely S, van den Berg M, Barranger J A, Sa Miranda M C, Tager J M, Aerts J M
E. C. Slater Institute for Biochemical Research, University of Amsterdam, The Netherlands.
J Clin Invest. 1993 Mar;91(3):1167-75. doi: 10.1172/JCI116276.
The properties of control and 370Asn-->Ser glucocerebrosidase, the frequently encountered mutated form of the enzyme in type 1 Gaucher disease, were studied in vitro as well as in situ. The catalytic properties of purified 370Asn-->Ser glucocerebrosidase were highly dependent on the assay conditions. The enzyme was deficient in activity towards substrate and in reactivity with the irreversible inhibitor conduritol B-epoxide (CBE) when activated by the bile salt taurocholate. In the presence of more physiological activators, the lysosomal activator protein saposin C and phosphatidylserine, the 370Asn-->Ser enzyme was near normal in kinetic properties at pH values approximately 5, but not at higher pH. In intact fibroblasts, the enzymic activity of the 370Asn-->Ser glucocerebrosidase and its reactivity with CBE were found to be clearly deficient. However, in intact lymphoblasts from the same patients, the behavior of the mutant enzyme was near normal. The catalytic efficiency of 370Asn-->Ser glucocerebrosidase in situ was also found to be highly pH dependent. When intact lymphoblasts were cultured in the presence of permeant weak bases, which increase the pH of acidic intracellular compartments, the catalytic efficiency of the mutant enzyme, as assessed by its reactivity with CBE, became markedly impaired. Our findings indicate that the intralysosomal pH in the intact cell can be expected to have a critical influence on the activation state of 370Asn-->Ser glucocerebrosidase and its ability to hydrolyse substrate. This phenomenon may partly underly the marked heterogeneity in clinical manifestation of Gaucher disease among patients with this mutated form of glucocerebrosidase.
我们对1型戈谢病中常见的突变形式——对照型和370位天冬酰胺突变为丝氨酸的葡萄糖脑苷脂酶的性质进行了体外和原位研究。纯化后的370位天冬酰胺突变为丝氨酸的葡萄糖脑苷脂酶的催化性质高度依赖于检测条件。当用胆盐牛磺胆酸盐激活时，该酶对底物的活性以及与不可逆抑制剂环醇氧化物（CBE）的反应性均不足。在存在更多生理性激活剂——溶酶体激活蛋白鞘脂激活蛋白C和磷脂酰丝氨酸的情况下，370位天冬酰胺突变为丝氨酸的酶在pH值约为5时动力学性质接近正常，但在较高pH值时则不然。在完整的成纤维细胞中，发现370位天冬酰胺突变为丝氨酸的葡萄糖脑苷脂酶的酶活性及其与CBE的反应性明显不足。然而，在来自同一患者的完整淋巴细胞中，突变酶的表现接近正常。还发现370位天冬酰胺突变为丝氨酸的葡萄糖脑苷脂酶在原位的催化效率也高度依赖于pH值。当完整的淋巴细胞在可渗透的弱碱存在下培养时，弱碱会增加酸性细胞内区室的pH值，通过其与CBE的反应性评估，突变酶的催化效率会明显受损。我们的研究结果表明，完整细胞内溶酶体的pH值可能对370位天冬酰胺突变为丝氨酸的葡萄糖脑苷脂酶的激活状态及其水解底物的能力产生关键影响。这种现象可能部分解释了携带这种葡萄糖脑苷脂酶突变形式的戈谢病患者临床表现存在明显异质性的原因。
