| Budget Amount *help |
¥6,800,000 (Direct Cost: ¥6,800,000)
Fiscal Year 1988: ¥1,000,000 (Direct Cost: ¥1,000,000)
Fiscal Year 1987: ¥5,800,000 (Direct Cost: ¥5,800,000)
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| Research Abstract |
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
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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
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