Wilkening G, Linke T, Sandhoff K
Kekulé Institut für Organische Chemie und Biochemie, Universität Bonn, D-53121 Bonn, Germany.
J Biol Chem. 1998 Nov 13;273(46):30271-8. doi: 10.1074/jbc.273.46.30271.
According to a recent hypothesis (Sandhoff, K., and Kolter, T. (1996) Trends Cell Biol. 6, 98-103), glycolipids, which originate from the plasma membrane, are exposed to lysosomal degradation on the surface of intralysosomal vesicles. Taking the interaction of membrane-bound lipid substrates and lysosomal hydrolases as an experimental model, we studied the degradation of glucosylceramides with different acyl chain lengths by purified glucocerebrosidase in a detergent-free liposomal assay system. Our investigation focused on the stimulating effect induced by lysosomal components such as sphingolipid activator protein C (SAP-C or saposin C), anionic lysosomal lipids, bis(monoacylglycero)phosphate, and dolichol phosphate, as well as degradation products of lysosomal lipids, e.g. dolichols and free fatty acids. The size of the substrate-containing liposomal vesicles was varied in the study. Enzymatic hydrolysis of glucosylceramide carried by liposomes made of phosphatidylcholine and cholesterol was rather slow and only weakly accelerated by the addition of SAP-C. However, the incorporation of anionic lipids such as bis(monoacylglycero)phosphate, dolichol phosphate, and phosphatidylinositol into the substrate carrying liposomes stimulated glucosylceramide hydrolysis up to 30-fold. Dolichol was less effective. SAP-C activated glucosylceramide hydrolysis under a variety of experimental conditions and was especially effective for the increase of enzyme activity when anionic lipids were inserted into the liposomes. Glucosylceramides with short acyl chains were found to be degraded much faster than the natural substrates. Dilution experiments indicated that the added enzyme molecules associate at least partially with the membranes and act there. Surface plasmon resonance experiments demonstrated binding of SAP-C at concentrations up to 1 microM to liposomes. At higher concentrations (2.5 microM SAP-C), liposomal lipids were released from the liposome coated chip. A model for lysosomal glucosylceramide hydrolysis is discussed.
根据最近的一种假说（桑德霍夫，K.，和科尔特，T.（1996年）《细胞生物学趋势》6，98 - 103），源自质膜的糖脂在溶酶体内小泡表面会受到溶酶体降解作用。以膜结合脂质底物与溶酶体水解酶的相互作用作为实验模型，我们在无去污剂脂质体测定系统中，研究了纯化的葡萄糖脑苷脂酶对不同酰基链长度的葡萄糖神经酰胺的降解作用。我们的研究集中于溶酶体成分如鞘脂激活蛋白C（SAP - C或鞘磷脂C）、阴离子溶酶体脂质双（单酰甘油）磷酸酯、磷酸多萜醇以及溶酶体脂质的降解产物如多萜醇和游离脂肪酸所诱导的刺激作用。在该研究中，含底物脂质体小泡的大小有所变化。由磷脂酰胆碱和胆固醇制成的脂质体所携带的葡萄糖神经酰胺的酶促水解相当缓慢，添加SAP - C后仅略有加速。然而，将双（单酰甘油）磷酸酯、磷酸多萜醇和磷脂酰肌醇等阴离子脂质掺入携带底物的脂质体中，可使葡萄糖神经酰胺水解加速达30倍。多萜醇的效果较差。在各种实验条件下，SAP - C均能激活葡萄糖神经酰胺水解，当阴离子脂质插入脂质体时，其对酶活性增加的作用尤为显著。发现短酰基链的葡萄糖神经酰胺比天然底物降解得快得多。稀释实验表明，添加的酶分子至少部分与膜结合并在膜上起作用。表面等离子体共振实验证明，浓度高达1微摩尔的SAP - C可与脂质体结合。在更高浓度（2.5微摩尔SAP - C）时，脂质体脂质从脂质体包被芯片上释放出来。文中讨论了溶酶体葡萄糖神经酰胺水解的模型。
