1987 Fiscal Year Final Research Report Summary
Protein mapping of Na^+-dependent D-glucose transporter
| Project/Area Number |
61570043
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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 |
General physiology
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| Research Institution | Osaka University Medical School |
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Principal Investigator |
FUKUHARA Yoshifumi Department of Medicine, Osaka University Medical School, 医学部, 助手 (90165308)
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| Co-Investigator(Kenkyū-buntansha) |
HORIO Masaru Osaka University Hospita, 医学部附属病院, 医員 (20273633)
MIKAMI Hiroshi Department of Medicine, Osaka University Medical School, 医学部, 助手 (30181861)
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| Project Period (FY) |
1986 – 1987
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| Keywords | D-glucose transport / Brush border membrane / Basolateral membrane / Vesicle / Proximal tubule / aminoglycoside / electron spin resonance / ベジクル |
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| Research Abstract |
Renal brush border membrane (BBM) and basolateral membrane (BLM) vesicle preparations are an ideal experimental ystem for the quantitative studies of coupled transport events and the identificatio of membrane transporter proteins.
In this research project, we developed the preparation methods for BBM and BLM vesicles from outer cortex (OC; mainly contains proximal tubular SL and S2 segments) and outer medulla (OM; mainly contains S3 segement). Each vesicle preparation was enriched good enough to do further experiments, evaluated by marker enzyme activities.
Effects of aminoglycoside on rabbit OC- and OM-BBM vesicles were then investigated. When aminoglycoside was administered in vivo, gentamicin only affected the OC D-glucose transport site, While netilmicin did not. Gentamicin and netilmicin inhibited D-glucose transport concentration-dependently in either OC- or OM-BBM vesicles, when added in vitro. Inhibition constants reveal that gentamicin was more toxic than netilmicin. The effect of gentamicin on the membrane fluidity of BBM vesicles was studied by electron spin resonance using 5- or 16-doxyl stearic acid as a spin label probe. Gentamicin-induced decrease in the membrane fluidity of OC-BBM vesicles was observed only when 5-SAL was used. The above effects of gentamicin were completely diminished after removing gentamicin by washing procedure.
The identification of transporter protein by radioactive ligand on electropheretic gel remains to be further developed.
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