Gonzales M L, Basu A, de Haas G H, Dijkman R, van Oort M G, Okolo A A, Glew R H
Department of Microbiology, Biochemistry, and Molecular Biology, School of Medicine, University of Pittsburgh, Pennsylvania 15261.
Arch Biochem Biophys. 1988 Apr;262(1):345-53. doi: 10.1016/0003-9861(88)90197-x.
The study of the acidic lipid requirement of human spleen glucocerebrosidase was extended to include two new series of acidic lipids, namely, monoacylglycol sulfates and diacylglycerol sulfates. Lysosomal glucocerebrosidase was extracted with sodium cholate and 1-butanol to render its beta-glucosidase activity dependent upon exogenous lipids. Maximum reactivation of control glucocerebrosidase was obtained with nonanoylglycol sulfate (NGS) and diheptanoylglycerol sulfate (DHGS). However, the effects of these lipids were markedly dependent on the nature of buffer used in the assay medium; specifically, 0.2 M sodium citrate-phosphate (pH 5.5) was much more effective than 0.2 M sodium acetate (pH 5.5) in permitting these lipids to reactivate glucocerebrosidase. In contrast, the marked activation of glucocerebrosidase by phosphatidylserine and galactocerebroside 3-sulfate (sulfatide) that was achievable in the sodium acetate buffer was totally inhibited by citrate or phosphate ions. The effects of NGS and DHGS on the kinetic parameters of control glucocerebrosidase were to lower the Km for the substrate, 4-methylumbelliferyl-beta-D-glucoside from 5.5 mM to approximately 2 mM (in sodium citrate-phosphate buffer) and markedly increase the Vmax. Furthermore, with DHGS, significant activation was achieved at concentrations below the lipid's critical micellar concentration. None of the monoacylglycol- or diacylglycerol sulfates were capable of stimulating mutant glucocerebrosidases from either type 1 (Ashkenazi-Jewish) or type 2 Gaucher's disease patients. Like control glucocerebrosidase, the type 1 glucocerebrosidase was unresponsive to phosphatidylserine and sulfatide when the beta-glucosidase assay was conducted in 0.2 M sodium citrate-phosphate buffer. Based on the differential action of these lipid activators in the two buffers and their effects on the mutant enzymes, we propose that, with regard to the lipid requirement of glucocerebrosidase, there are two classes of acidic lipids--one comprised of phosphatidylserine and sulfatide and the other comprised of the likes of NGS, DHGS, or sodium taurodeoxycholate. It appears that control glucocerebrosidase and the mutant enzyme of the patient with type 1 Gaucher's disease is reconstitutable with the first class of lipids whereas the glucocerebrosidase of the type 2 patient is not. The observations in this report are interpreted in terms of a model which postulates that normal glucocerebrosidase possesses at least two distinct lipid binding domains.
对人脾葡萄糖脑苷脂酶酸性脂质需求的研究扩展至包括两个新的酸性脂质系列，即单酰基甘油硫酸盐和二酰基甘油硫酸盐。用胆酸钠和1-丁醇提取溶酶体葡萄糖脑苷脂酶，使其β-葡萄糖苷酶活性依赖于外源性脂质。对照葡萄糖脑苷脂酶用壬酰基甘油硫酸盐（NGS）和二庚酰基甘油硫酸盐（DHGS）可实现最大程度的再激活。然而，这些脂质的作用明显取决于测定介质中所用缓冲液的性质；具体而言，在使这些脂质再激活葡萄糖脑苷脂酶方面，0.2M柠檬酸钠 - 磷酸盐（pH 5.5）比0.2M醋酸钠（pH 5.5）有效得多。相比之下，在醋酸钠缓冲液中可实现的磷脂酰丝氨酸和半乳糖脑苷脂3 - 硫酸盐（硫脂）对葡萄糖脑苷脂酶的显著激活被柠檬酸盐或磷酸盐离子完全抑制。NGS和DHGS对对照葡萄糖脑苷脂酶动力学参数的影响是将底物4 - 甲基伞形酮基 - β - D - 葡萄糖苷的Km从5.5mM降至约2mM（在柠檬酸钠 - 磷酸盐缓冲液中），并显著增加Vmax。此外，对于DHGS，在低于脂质临界胶束浓度的浓度下可实现显著激活。单酰基甘油或二酰基甘油硫酸盐均不能刺激来自1型（阿什肯纳兹 - 犹太人）或2型戈谢病患者的突变葡萄糖脑苷脂酶。与对照葡萄糖脑苷脂酶一样，当在0.2M柠檬酸钠 - 磷酸盐缓冲液中进行β - 葡萄糖苷酶测定时，1型葡萄糖脑苷脂酶对磷脂酰丝氨酸和硫脂无反应。基于这些脂质激活剂在两种缓冲液中的不同作用及其对突变酶的影响，我们提出，就葡萄糖脑苷脂酶的脂质需求而言，存在两类酸性脂质 - 一类由磷脂酰丝氨酸和硫脂组成，另一类由NGS、DHGS或牛磺脱氧胆酸钠等组成。似乎对照葡萄糖脑苷脂酶和1型戈谢病患者的突变酶可用第一类脂质重构，而2型患者的葡萄糖脑苷脂酶则不能。本报告中的观察结果根据一个模型进行解释，该模型假定正常葡萄糖脑苷脂酶至少具有两个不同的脂质结合结构域。
