de Vries A C, Schram A W, van den Berg M, Tager J M, Batenburg J J, van Golde L M
Laboratory of Veterinary Biochemistry, Utrecht University, The Netherlands.
Biochim Biophys Acta. 1987 Dec 14;922(3):259-69. doi: 10.1016/0005-2760(87)90048-8.
We have recently shown that lamellar body fractions purified from human lung contain a distinct acid alpha-glucosidase distinguishable from lysosomal acid alpha-glucosidase in that it does not cross-react with antibodies raised against the lysosomal enzyme and does not bind to concanavalin A (De Vries, A.C.J., Schram, A.W., Tager, J.M., Batenburg, J.J. and Van Golde, L.M.G. (1985) Biochim. Biophys. Acta 837, 230-238). In order to study the relationship between the non-concanavalin A-binding alpha-glucosidase and lamellar bodies more closely a method was developed for the further purification of the organelles. A purified lamellar body preparation isolated from human lung homogenate by discontinuous sucrose density centrifugation was subjected to gel filtration with Sepharose 4B followed by Percoll density gradient centrifugation, which yielded a lamellar body preparation with a phospholipid phosphorus/protein ratio of 12.57 +/- 0.38 (mumol/mg) (n = 3) as compared to a ratio of 3.34 +/- 0.16 (mumol/mg) (n = 3) in the sucrose density gradient preparation. Concomitantly there was a 3.3 +/- 0.1 (n = 3)-fold enrichment in the content of total acid alpha-glucosidase and a 3.2 +/- 0.1 (n = 3) -fold enrichment of non-concanavalin A-binding acid alpha-glucosidase. The new purification method removes adhering proteins without changing the phospholipid composition. During the successive purification steps the concanavalin A-sensitive and -insensitive alpha-glucosidases remained fully lamellar body fraction associated. Differences between a lysosome-enriched fraction and a lamellar body preparation at varying stages of purification with respect to the ratio between soluble acid hydrolases and the membrane-associated lysosomal enzyme glucocerebrosidase indicate that the purified lamellar bodies were not contaminated with lysosomes. The absence of lysosomes in the purified lamellar body fraction was confirmed by experiments with the weak base glycyl-L-phenylalanine-beta-naphthylamide, which is an artificial substrate for the lysosomal enzyme cathepsin C and brings about lysis of lysosomes. Morphological examination by electron microscopy endorses the absence of contaminating vesicles and organelles and showed a structural integrity of the lamellar bodies in the final preparation. The improved isolation procedure strongly suggests that the concanavalin A-insensitive acid alpha-glucosidase is endogenous to lamellar bodies and supports our earlier idea that it can be used as a lamellar body-specific marker enzyme. In addition, the experiments show that lamellar bodies free of lysosomes contain a spectrum of lysosomal-type enzymes.
我们最近发现，从人肺中纯化得到的板层体组分含有一种独特的酸性α-葡萄糖苷酶，它与溶酶体酸性α-葡萄糖苷酶不同，因为它不与针对溶酶体酶产生的抗体发生交叉反应，也不与伴刀豆球蛋白A结合（德弗里斯，A.C.J.，施拉姆，A.W.，塔杰，J.M.，巴滕堡，J.J.和范戈尔，L.M.G.（1985年）《生物化学与生物物理学报》837卷，230 - 238页）。为了更深入地研究不与伴刀豆球蛋白A结合的α-葡萄糖苷酶与板层体之间的关系，我们开发了一种进一步纯化这些细胞器的方法。通过不连续蔗糖密度离心从人肺匀浆中分离得到的纯化板层体制剂，先经琼脂糖4B凝胶过滤，然后进行Percoll密度梯度离心，得到的板层体制剂中磷脂磷/蛋白比为12.57±0.38（μmol/mg）（n = 3），而蔗糖密度梯度制剂中的该比值为3.34±0.16（μmol/mg）（n = 3）。同时，总酸性α-葡萄糖苷酶含量富集了3.3±0.1（n = 3）倍，不与伴刀豆球蛋白A结合的酸性α-葡萄糖苷酶富集了3.2±0.1（n = 3）倍。新的纯化方法去除了附着的蛋白质，而不改变磷脂组成。在连续的纯化步骤中，对伴刀豆球蛋白A敏感和不敏感的α-葡萄糖苷酶仍与板层体组分完全相关。在不同纯化阶段，富含溶酶体的组分与板层体制剂在可溶性酸性水解酶与膜相关溶酶体酶葡萄糖脑苷脂酶的比例方面存在差异，这表明纯化的板层体未被溶酶体污染。用弱碱甘氨酰-L-苯丙氨酸-β-萘酰胺进行的实验证实了纯化的板层体组分中不存在溶酶体，甘氨酰-L-苯丙氨酸-β-萘酰胺是溶酶体酶组织蛋白酶C的人工底物，可导致溶酶体裂解。电子显微镜形态学检查证实不存在污染的囊泡和细胞器，并显示最终制剂中板层体的结构完整性。改进的分离程序有力地表明，不与伴刀豆球蛋白A结合的酸性α-葡萄糖苷酶是板层体的内源性成分，并支持了我们早期的观点，即它可作为板层体特异性标记酶。此外，实验表明不含溶酶体的板层体含有一系列溶酶体类型的酶。
