1993 Fiscal Year Final Research Report Summary
MECHANISM FOR THE MEMBRANE TRANSPORT OF GLYCOLATE AND ISOLATION OF ITS TRANSPORTER FROM THE PLASMA MEMBRANE IN MICROALGAE
| Project/Area Number |
04640622
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| Research Category |
Grant-in-Aid for General Scientific Research (C)
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| Allocation Type | Single-year Grants |
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| Research Field |
植物生理学
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| Research Institution | NIIGATA UNIVERSITY |
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Principal Investigator |
SHIRAIWA Yoshihiro NIIGATA UNIV., FAC.OF SCI.ASSOCIATE PROF., 理学部, 助教授 (40126420)
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| Project Period (FY) |
1992 – 1993
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| Keywords | Isolated plasma membrane / Plasma membarne ATPase / Proton-ATPase / Glycolate transport / Glycolate transporter / Active transport / Microalgae / Chlorella |
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| Research Abstract |
Uptake of glycolate by Chlorella vulgaris 211-11h cells adapted to air-level CO_2 concentration was an energy-dependent-active process which is mediated by carrier-protein. The glycolate taken up was metabolized via the glycolate pathway and the rate of uptake was limited by that of the metabolism of glycolate. Therefore, isolation and purification of the plasma membranes was performed to study on the transport mechanism of glycolate through the plasma membrane. Before this work, there was little informations on methods for the isolation of plasma membrane from cells with a rigid cell wall like Chlorella cells.
1. We improved methos for isolating plasma membranes from Chlorella cells using an aqueous two-phase partitioning method and a surcrose-density gradient centrifugation.
2. Plasma membrane ATPase is a Mg-dependent H^+-ATPase of which Km and Vmax values are 1.64 mM and 4.3 mumol Pi/mg protein/min.
3. The activity of glycolate uptake by intact cell of the Chlorella was specifically inhibited by a membrane-impermeable modifier of proteins, diisothiocyanostilbene (DIDS) without affecting the transport of CO^2 and photosynthesis.
4. Using a H^+-labeled DIDS was used for labeling some proteins which may involve a transporter protein of glycolate. Membrane proteins solubilized with detergents were separated by giycerol-gradient centrifugation and the radiolabelled fraction isolated was separated by polyacrylamide gel electrophoresis. Some radioactive bands which may be candidates of the glycolate transporter protein, were detected using a radio-fluorography method in low-CO_2 grown cells. However, it was difficult to identify a polypeptide of glycolate transporter.
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