Natowicz M R, Chi M M, Lowry O H, Sly W S
Proc Natl Acad Sci U S A. 1979 Sep;76(9):4322-6. doi: 10.1073/pnas.76.9.4322.
Human beta-glucuronidase (beta-D-glucuronide glucuronosohydrolase, EC 3.2.1.31), like many other glycoprotein lysosomal hydrolases, is subject to receptor-mediated endocytosis by fibroblasts. Prior work demonstrated charge heterogeneity in beta-glucuronidase and showed that high-uptake forms are more acidic than slowly internalized forms. Considerable indirect evidence implicated mannose 6-phosphate as an essential part of the recognition marker on high-uptake enzyme forms. Here we report the purification of beta-glucuronidase from human spleen and demonstrate enzymatically that mannose 6-phosphate is released on acid hydrolysis of pure enzyme varies directly with its susceptibility to pinocytosis by fibroblasts. Enzyme forms resolved by CM-Sephadex chromatography differed over an 18-fold range in uptake rate and in mannose 6-phosphate content. The most acidic forms had 4.4 mol of mannose 6-phosphate per mol of enzyme. The mannose 6-phosphate was released from the enzyme by treatment with endoglycosidase H with concomitant loss of susceptibility to adsorptive endocytosis. Thus, these studies provide direct evidence that mannose 6-phosphate is present on high-uptake enzyme forms, that it is present in the recognition marker for uptake, and that it is present on oligosaccharide that is released by endoglycosidase H.
人β-葡萄糖醛酸酶（β-D-葡萄糖醛酸葡萄糖醛酸水解酶，EC 3.2.1.31）与许多其他糖蛋白溶酶体水解酶一样，可被成纤维细胞通过受体介导的内吞作用摄取。先前的研究表明β-葡萄糖醛酸酶存在电荷异质性，并显示高摄取形式比缓慢内化形式的酸性更强。大量间接证据表明，6-磷酸甘露糖是高摄取酶形式识别标记的重要组成部分。在此，我们报告了从人脾脏中纯化β-葡萄糖醛酸酶，并通过酶学方法证明，纯酶经酸水解后释放的6-磷酸甘露糖与其被成纤维细胞胞饮作用的敏感性直接相关。经CM-葡聚糖凝胶柱层析分离的酶形式，其摄取速率和6-磷酸甘露糖含量在18倍的范围内有所不同。酸性最强的形式每摩尔酶含有4.4摩尔的6-磷酸甘露糖。用内切糖苷酶H处理该酶可释放出6-磷酸甘露糖，同时其对吸附性内吞作用的敏感性也随之丧失。因此，这些研究提供了直接证据，证明6-磷酸甘露糖存在于高摄取酶形式中，它存在于摄取的识别标记中，并且它存在于内切糖苷酶H释放的寡糖上。
