Sambamurti K, Shioi J, Anderson J P, Pappolla M A, Robakis N K
Department of Psychiatry, Mount Sinai School of Medicine, New York, New York.
J Neurosci Res. 1992 Oct;33(2):319-29. doi: 10.1002/jnr.490330216.
The Alzheimer's amyloid precursor (APP) is cleaved by an unidentified enzyme (APP secretase) to produce soluble APP. Fractionation of PC12 cell homogenates in a detergent-free buffer showed the presence of the Kunitz protease inhibitor (KPI)-containing soluble APP (nexin II) in the particulate fraction. Digitonin or sodium carbonate treatment of this fraction solubilized nexin II suggesting that it is contained in the lumen of vesicles. Nexin II production was not affected by lysosomotropic agents, suggesting that APP secretase is not a lysosomal enzyme. Labelling of cell surface proteins by iodination failed to detect full-length APP on the surface of PC12 cells, suggesting that most of this protein is located intracellularly. Furthermore, pulse-chase experiments showed that nexin II is detected in cell extracts before it appears in the culture medium. Cellular nexin II was detected at zero time of chase after only 5 min of pulse labelling with 35S-sulfate, indicated that APP secretase cleavage takes place immediately after APP is sulfated. Temperature block, pulse-chase, and 35S-sulfate-labelling experiments suggested that APP is cleaved by APP secretase intracellularly in the trans-Golgi network (TGN) or in a post-Golgi compartment.
阿尔茨海默病淀粉样前体蛋白（APP）被一种未明确的酶（APP 分泌酶）切割，产生可溶性 APP。在无去污剂缓冲液中对 PC12 细胞匀浆进行分级分离，结果显示在颗粒组分中存在含库尼茨蛋白酶抑制剂（KPI）的可溶性 APP（连接蛋白 II）。用洋地黄皂苷或碳酸钠处理该组分可使连接蛋白 II 溶解，这表明它存在于囊泡腔内。连接蛋白 II 的产生不受溶酶体促渗剂的影响，这表明 APP 分泌酶不是溶酶体酶。通过碘化标记细胞表面蛋白未能在 PC12 细胞表面检测到全长 APP，这表明该蛋白大部分位于细胞内。此外，脉冲追踪实验表明，在连接蛋白 II 出现在培养基中之前，就能在细胞提取物中检测到它。在用 35S - 硫酸盐脉冲标记仅 5 分钟后的追踪零时就检测到了细胞内的连接蛋白 II，这表明 APP 分泌酶的切割在 APP 硫酸化后立即发生。温度阻断、脉冲追踪和 35S - 硫酸盐标记实验表明，APP 在反式高尔基体网络（TGN）或高尔基体后区室中被 APP 分泌酶在细胞内切割。
