Protein mapping of Na^+-dependent D-glucose transporter

Na^依赖性D-葡萄糖转运蛋白的蛋白质图谱

• 批准号: 61570043
• 负责人: FUKUHARA Yoshifumi
• 金额: $ 1.28万
• 依托单位: Osaka University Medical School
• 依托单位国家: 日本
• 项目类别: Grant-in-Aid for General Scientific Research (C)
• 财政年份: 1986
• 资助国家: 日本
• 起止时间: 1986 至 1987
• 项目状态: 已结题
• 来源: https://kaken.nii.ac.jp/grant/KAKENHI-PROJECT-61570043/
• 关键词: D-glucose transport; Brush border membrane; Basolateral membrane; Vesicle; Proximal tubule; aminoglycoside; electron spin resonance; ベジクル; 刷子縁膜ベジクル; 側底膜ベジクル; 輸送担体蛋白; アミノグリコシド系抗生物質; ネフロン不均一性

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.

肾刷状缘膜（BBM）和基底外侧膜（BLM）囊泡的制备是研究偶联转运事件和膜转运蛋白鉴定的理想实验工具（OC;主要包括近端管状SL和S2段）和外髓质（OM;主要包括S3段）。每种囊泡制备物都被富集到足够好的程度以进行进一步的实验，通过标记酶活性来评估。然后研究氨基糖苷类对兔OC-和OM-BBM囊泡的影响。氨基糖苷类药物在体内给药时，庆大霉素只影响OC D-葡萄糖转运位点，而奈替米星则不影响。庆大霉素和奈替米星浓度依赖性地抑制D-葡萄糖转运在OC-或OM-BBM囊泡，当在体外添加。抑制常数表明庆大霉素的毒性大于奈替米星。以5-或16-羟基硬脂酸为自旋标记探针，用电子自旋共振技术研究了庆大霉素对BBM囊泡膜流动性的影响。庆大霉素诱导的OC-BBM囊泡的膜流动性的降低仅在使用5-SAL时观察到。用洗涤法去除庆大霉素后，庆大霉素的上述作用完全减弱。

项目成果

Masaru Horio: Biochim.Biophys.Acta.

堀尾胜：Biochim.Biophys.Acta。

Masaru Horio: Biochim. Biophys.Acta. 858. 153-160 (1986)

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Masaru Horio: "Effect of acidosis on glutamine transport by isolated rat renal brush border and basolateral membrane vesicles." Biochimica et Biophysica Acta.

Masaru Horio：“酸中毒对离体大鼠肾刷状缘和基底外侧膜囊泡谷氨酰胺转运的影响。”

E.Drioli;M.Nokagaki eds;Yoshimasa Orita: "Disorder of conpled transport in rcnal brush border membraue vesicles from rabbits with experimeuntal Fanconi syadrome induccd by anbydro-4-epitetracycline as a modcl of membrane disease.In :Membranes and Membrane

E.Drioli;M.Nokagaki eds;Yoshimasa Orita：“实验性范可尼综合症兔子的肾刷状缘膜囊泡中的复合运输障碍，由 anbydro-4-epitetracycline 诱导，作为膜疾病的一种模式。在：膜和膜

Toshiki moriyama: Biochemical Pharmacalogy.

森山俊树：生化药理学。