Studies on Biogenesis of the Lysosomal Membranes.

溶酶体膜生物发生的研究。

基本信息

批准号：
62480459
负责人：
KATO Keitaro
金额：
$ 4.35万
依托单位：
Kyushu University
依托单位国家：
日本
项目类别：
Grant-in-Aid for General Scientific Research (B)
财政年份：
1987
资助国家：
日本
起止时间：
1987 至 1988
项目状态：
已结题

项目摘要

To clarify the mechanisms of lysosome formation, it is essential to understand the biogenesis and the pathway of the intracellular transport of lysosomal membrane glycoproteins.In this project I attempted to study the biosynthesis of acid phosphatases(APase), which are located both in lysosomal matrix and membranes, and dipeptidyl peptidase IV(DPP-IV) which are located in the plasma membrane of bile canaliculi(Pl), lysosomal membranes(Ly) and contents(Lc).50% of activity of the whole APases activity in rat liver lysosomes localizes in the lysosomal contents(C-APase) and the other is tightly associated with the lysosomal membranes(M-APase). Although C-APase(Mr. 48K) and M-APase(Mr. 67K) have a different molecular weight, these two enzyme have the same amino terminal sequences and show the same reactivity against antibodies prepared from rabbits immunized with the purified C-APase. In the in vivo experiment, after 30 min labeling with [^<35>S]methionine APase having a molecular weight of … More 67 k was seen both in the golgi and the lysosomal membrane fraction. Appearance of C-APase in the lysosomal contents took a longer chase period. From these results, we could conclude that APase is synthesized on the rough er as a membrane integrated protein and transported into the lysosomes in a manner independent of the mannose-6-phosphate receptor system. After reaching the lysosomes, M-APase is solubilized into the lysosomal matrix by a limited proteolytic cleavage.Concerning the biosynthesis of DPP-IV, after labeling rats with [^<35>S]me-thionine the plasma membranes, lysosomal membranes and contents were isolated from the rat liver homogenates. Thereafter, Pl-,Ly- and Lc-DPP-IV were separately purified and subjected to the measurement of the each radioactivities. It was revealed from the kinetic analysis that these three enzymes took 2h to reach their final destination. Since half life of Pl-DPP-IV and lysosomal ones are different, shuttling of the enzyme between the bile canaliculi membrane and lysosomal membranes seems not to occur. Less

为了阐明溶酶体形成的机制，有必要了解溶酶体膜糖蛋白的生物发生和细胞内转运途径。在这个项目中，我试图研究酸性磷酸酶（APase）的生物合成，它们都位于溶酶体基质中和膜，以及位于胆小管（Pl）质膜中的二肽基肽酶IV（DPP-IV），溶酶体膜(Ly)和内容物(Lc)。大鼠肝脏溶酶体中全部APase活性的50%位于溶酶体内容物(C-APase)，另一部分与溶酶体膜(M-APase)紧密相关。尽管C-APase(Mr. 48K)和M-APase(Mr. 67K)具有不同的分子量，但这两种酶具有相同的氨基末端序列并显示出与用纯化的 C-APase 免疫的兔子制备的抗体相同的反应性。在体内实验中，用[^ 35 S]蛋氨酸 APase 标记 30 分钟后，发现两者的分子量均为 67 k。在高尔基体和溶酶体膜部分中，溶酶体内容物中 C-APase 的出现需要较长的追踪时间，从这些结果我们可以得出结论，APase 是在粗糙的表面上合成的。 M-APase 作为膜整合蛋白，以不依赖于 6-磷酸甘露糖受体系统的方式转运到溶酶体中。到达溶酶体后，M-APase 通过有限的蛋白水解裂解溶解到溶酶体基质中。关于 DPP 的生物合成。 -IV，用[^ 35 S]甲硫氨酸标记大鼠后的质膜、溶酶体膜和内容物然后，从大鼠肝脏匀浆中分离出Pl-、Ly-和Lc-DPP-IV，并进行各自放射性活性的测量。从动力学分析表明，这三种酶需要2小时才能达到它们的活性。由于 Pl-DPP-IV 和溶酶体的半衰期不同，因此酶在胆小管膜和溶酶体膜之间的穿梭似乎不会发生。