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

(1)利用聚戊烯醇产率较低的酵母突变株#进行补充试验，筛选出三个无性系：#-1、#-2、#-4。在这些酵母中检测到较短的C50-60聚戊烯醇和较长的二元醇。(2)化学合成了三种生物素化的聚戊烯醇(法尼醇、C55-聚戊烯醛和C80-聚戊烯醛)，并分析了它们对聚戊烯醇体外还原反应的影响。与Triton X-100、CHAPS、辛基葡萄糖苷、脱氧胆酸和吐温80相比，C-80单独具有抑制作用。用十二烷基硫酸钠凝胶电泳法检测到一个与C80-化合物有关的蛋白质(50000，Da)。(3)分析了两种蕨类植物(日本的Warabi和Zenmai)都含有C80，C85，C90多糖醇。还分析了一种海藻(日本为Wakame)中含有C90、C95、C100多戊烯醇和C80、C85、C90多烯醇。(4)用大鼠胸腺细胞和人HL-60细胞将全反式香叶基香叶醇(GGOH)转化为2，3-二氢香叶基香叶酸(2，3-Digga)。在0.1%胎牛血清存在下，2，3-Digga具有诱导HL-60细胞凋亡的作用。在caspase-3抑制剂的共同作用下，细胞的凋亡受到抑制，并出现脂滴。另一方面，相同的化合物在10%胎牛血清存在的情况下表现出诱导脂滴的能力。在酰辅酶A合成酶抑制剂的共同作用下，脂滴的形成受到抑制，并观察到细胞凋亡。GGA在0.1%FBS中也可诱导HL-60细胞的凋亡，但在10%FBS中可诱导HL-60细胞分化为中性粒细胞。(5)利用突变的细菌十一烯基二磷酸合成酶的研究，揭示了Arg残基在P-环手Asp29、Arg33、Arg80残基结构中对催化活性的重要性。此外，Tyr71、Tyr148、Ty210残基对异戊烯基二磷酸和法尼基二磷酸的结合也很重要。

(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.