Acid β-glucosidase:: Intrinsic fluorescence and conformational changes induced by phospholipids and saposin C
Acid β-glucosidase:: Intrinsic fluorescence and conformational changes induced by phospholipids and saposin C
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DOI：
10.1021/bi980785
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发表时间：
1998-08-18
期刊：
影响因子：
2.9
通讯作者：
Grabowski, GA
中科院分区：
文献类型：
--
作者：
Qi, XY;Grabowski, GA
酸性β-葡萄糖苷酶是一种裂解葡萄糖神经酰胺和芳基-β-葡萄糖苷O-β-D-葡萄糖苷键的溶酶体膜蛋白。纯酶的完全活性重建需要磷脂和皂苷C，一种80aa的激活蛋白。酶或激活剂的缺乏会导致高谢病。通过固有的荧光光谱漂移、荧光猝灭和圆二色谱(CD)显示，酸性β-葡萄糖苷酶的构象变化伴随着所选择的磷脂，特别是磷脂/皂苷C复合体的活性重建。带负电荷的磷脂(NCP)与不饱和脂肪酸酰链(UFAC)的界面导致色氨酸荧光光谱中协调的蓝移，CD导致β链结构的丢失。该酶需要脂质体膜中邻近(10-11埃)的不饱和脂肪酸酰链来激活、荧光蓝移和CD光谱的变化。当UFAC和带负电荷的头基存在于同一磷脂上时，活性增强最大。含UFAC的NCPs对水溶液(I-、Cs+、丙烯酰胺：TEMPO)的荧光猝灭有保护作用。只有在NCP/PC脂质体中，碳7、10或16上带有DOXY自旋标记脂肪酸酰链的磷脂酰胆碱才能猝灭酶的荧光。皂苷C(不含色氨酸)仅在含有不饱和脂肪酸的NCP脂质体存在时才能诱导酶的额外活性和荧光光谱的变化。圆二色谱分析表明，皂苷C与酸性β-葡萄糖苷酶只有在NCPs与不饱和脂肪酸存在时才发生相互作用。这些研究表明，酸性β-葡萄糖苷酶需要由含有UFAC的NCPs组成的界面才能穿透到膜的外层小叶。此外，这种相互作用导致皂苷C结合的基本构象变化，并进一步增强了酸性β-葡萄糖苷酶的催化活性。
Acid beta-glucosidase is a lysosomal membrane protein that cleaves the O-beta-D-glucosidic linkage of glucosylceramide and aryl-beta-glucosides. Full activity reconstitution of the pure enzyme requires phospholipids and saposin C, an 80 aa activator protein. The deficiency of the enzyme or activator leads to Gaucher disease. A conformational change of acid beta-glucosidase is shown to accompany activity reconstitution by selected phospholipids or, particularly, phospholipid/saposin C complexes by intrinsic fluorescence spectral shifts, fluorescence quenching, and circular dichroism (CD). Negatively charged phospholipid (NCP) interfaces with unsaturated fatty acid acyl chains (UFAC) induced concordant blueshifts in tryptophanyl fluorescence spectra and a loss of beta-strand structure by CD. The enzyme required an unsaturated fatty acid acyl chain in proximity (10-11 Angstrom) within liposomal membranes for activation, fluorescence blue-shifts, and changes in CD spectra. Activity enhancements were greatest when UFAC and the negatively charged headgroup were present on the same phospholipid. NCPs with UFAC protected the enzyme from fluorescence quenching by aqueous agents (I-, Cs+, acrylamide: TEMPO). Phosphatidylcholine with doxyl spin-labeled fatty acid acyl chains at carbons 7, 10, or 16 quenched enzyme fluorescence only when in NCP/PC liposomes. Saposin C (Trp-free) induced additional activity and fluorescence spectral changes in the enzyme only in the presence of NCP liposomes containing UFA. CD spectral changes indicated saposin C and acid beta-glucosidase interaction only in the presence of NCPs with UFA. These studies show that acid beta-glucosidase requires interfaces composed of NCPs, containing UFAC, for penetration into the outer leaflet of membranes. Furthermore, this interaction induces essential conformational changes for Saposin C binding and further enhancement of acid beta-glucosidase catalytic activity.