权益分类	功能权益	普通用户	{{item.name}}会员
{{category.name}}	{{benefitItem.name}}

Molecular analysis and functional change of cis-isoprene-chain elongation enzyme

顺式异戊二烯链延长酶的分子分析及功能变化

基本信息

批准号：
13680667
负责人：
SAGAMI Hiroshi
金额：
$ 2.24万
依托单位：
Tohoku University
依托单位国家：
日本
项目类别：
Grant-in-Aid for Scientific Research (C)
财政年份：
2001
资助国家：
日本
起止时间：
2001 至 2003
项目状态：
已结题

项目摘要

(1)In complimentary experiments using a yeast mutant #64 with low conversion of polyprenol to dolichol, the following three clones, #64-1、 #64-2、#64-4 were screened. In these yeasts shorter C50-60 polyprenols were detected together with longer dolichols.(2)Three biotinylated prenals (farnesal, C55-polyprenal, and C80-polyprenal) were chemically synthesized and their effects on in vitro polyprenol reduction reaction were analyzed. The C-80compound alone showed inhibitory effect and the effect was specific compared with those of Triton X-100, Chaps, octylglucoside, deoxycholic acid, and Tween 80. By SDS elctrophoresis, a protein (50,000, Da) associated with the C80-compound was detected.(3)Two ferns (warabi and zenmai in Japanese) were analyzed to both contain C80, C85, C90 dolichols. A seaweed (Wakame in Japanese) was also analyzed to contain C90,C95,C100 polyprenols and C80, C85, C90 dolichols.(4)All-trans-geranylgeraniol (GGOH) was converted to 2,3-dihydrogeranylgeranoic acid (2,3-diGGA) using rat thymic cells and human HL-60 cells. The 2,3-diGGA showed the ability to induce apoptosis of HL-60 cells in the presence of 0.1% FBS. In the copresence of a caspase-3 inhibior, the apoptosis was repressed and lipid droplets were observed. On the other hand, the same compound showed the ability to induce lipid droplets in the presence of 10% FBS. In the copresence of an acyl-CoA synthethase inhibitor, the lipid droplets formation was repressed and apoptosis was observed. GGA also inducded the apoptosis in 0.1% FBS, but the HL-60 cells were differentiated to neutrophiles in 10% FBS.(5)The study using mutated bacterial undecaprenyl diphosphatesynthase, showd the importance of Arg residue in the structure of P-loop hand Asp29, Arg33, Arg80 residies for catalytic activiy. Also, Tyr71,Tyr148, Ty210 residues were important for the binding of isopentenyl diphosphate and farnesyl diphosphat.

(1)利用低聚戊二醇转化为醇的酵母突变体#64进行补充实验，筛选了以下三个无性系，#64-1,#64-2,#64-4。在这些酵母中检测到较短的C50-60聚戊烯醇和较长的醇缩醇。(2)化学合成了三种生物素化的戊烯醛（法尼醛、c55 -聚戊烯醛和c80 -聚戊烯醛），并分析了它们对体外聚戊烯醇还原反应的影响。与Triton X-100、Chaps、辛基葡萄糖苷、脱氧胆酸和Tween 80相比，c -80单独具有抑制作用，且具有特异性。SDS电泳检测到与c80化合物相关的蛋白（50,000,Da）。(3)分析了两种蕨类植物（日本的warabi和zenmai）均含有C80、C85和C90颗粒。对一种海藻（日本名Wakame）进行了C90、C95、C100聚戊烯醇和C80、C85、C90多酚的分析。(4)利用大鼠胸腺细胞和人HL-60细胞将全反式香叶醇（GGOH）转化为2,3-二氢香叶酸（2,3- digga）。2,3- digga在0.1% FBS的作用下诱导HL-60细胞凋亡。在caspase-3抑制剂的存在下，细胞凋亡被抑制，脂滴被观察到。另一方面，同样的化合物在10% FBS的存在下显示出诱导脂滴的能力。在酰基辅酶a合成酶抑制剂的存在下，脂滴的形成受到抑制，细胞凋亡。GGA在0.1%胎牛血清中也能诱导HL-60细胞凋亡，而在10%胎牛血清中HL-60细胞向中性粒细胞分化。(5)利用突变的细菌脱戊烯基二磷酸合成酶进行研究，发现p环手Asp29、Arg33、Arg80残基结构中Arg残基对催化活性的重要性。此外，Tyr71、Tyr148、Ty210残基对于二磷酸异戊烯基和法尼酯基的结合也很重要。