摘要
ARF GAP是重要的细胞内物质转运调节分子 .最近 ,在人胎肝 c DNA文库中发现一种新基因 ,其编码的氨基酸序列与大鼠的 ARF1 GAP有 32 %同源性 ,故将其命名为“ARFGAP1”.对ARFGAP1进行功能研究 ,利用分子克隆技术构建绿色荧光蛋白 (GFP) - ARFGAP1融合基因表达质粒 (p EGFP- C1 - ARFGAP1 ) ,经脂质体转染将其导入 COS- 7细胞瞬时表达 ,利用绿色荧光确定ARFGAP1的亚细胞定位 .结果显示 ,ARFGAP1位于细胞质部分 ,表达量高时 ,在核周高尔基体区聚集呈团块状或颗粒状 .构建真核表达质粒 pc DNA3.1 /myc- His- ARFGAP1 ,在 COS- 7细胞中表达 ,并用 ARFGAP1和分泌型碱性磷酸酶 (SEAP)真核表达质粒共同转染 COS- 7细胞 ,发现ARFGAP1在细胞中过表达能部分抑制 SEAP的分泌 .结果证明 ,ARFGAP1对细胞的物质转运和分泌功能有调节作用 .
ADP\|ribosylation factors(ARFs)are critical components in vesicular trafficking pathways,which are active in their GTP\|bound form and controlled by GTPase\|activating proteins(GAPs).ARF GAPs are important regulators of intracellular transport.Recently,a novel human gene was cloned from a cDNA library of second trimester human fetal liver.The amino acid sequence encoded by the novel gene has 32% similarity to rat ARF1 GAP and is thus termed as ARFGAP1.The functional characterization of the new gene was studied.The recombinant expression plasmid pEGFP\|Cl\|ARFGAP1 tagged with green fluorescence protein(GFP)was constructed by routine molecular cloning technique.pEGFP\|Cl\|ARFGAP1 was transfected into COS\|7 cells by lipofectin reagent.The transient expression of GFP\|ARFGAP1 fusion protein in COS\|7 was confirmed by Western blotting analysis.Subcellular localization of ARFGAP1 in COS\|7 was determined by fluorescence microscope.The result showed that ARFGAP1 was localized in vesicular structures that are concentrated mainly in the perinuclear region.but were also found throughout the cytosol.Moreover,the recombinant expression plasmid pcDNA 3 1/myc\|His\|ARFGAP1 was constructed and transfected into COS\|7 cells.The expression of ARFGAP1 was verified by Western blotting analysis.It was found that the overexpression of ARFGAP1 in cultured cells reduced the constitutive secretion of secreted form of alkaline phosphatase(SEAP) partly and reproducibly by co\|transfecting COS\|7 cells with pcDNA3.1/myc\|His\|ARFGAP1 and pGEM\|SEAP.It is proposed that ARFGAP1 be involoved in regulation of intracellular transport and cellular secretion.
出处
《中国生物化学与分子生物学报》
CAS
CSCD
北大核心
2001年第2期237-243,共7页
Chinese Journal of Biochemistry and Molecular Biology
基金
国家自然科学基金!项目 (No.39730 310和 30 0 70 386 )&&
关键词
细胞内物质转运
细胞分泌功能
ARFGAP1
分泌型碱性磷酸酶
ADP\|ribosylation factor(ARF), GTPase activating protein(GAP),subcellular localization,secreted alkaline phosphatase
