Nitric Oxide：from a mysterious labile factor to the molecule of the Nobel Prize Recent progress in nitric oxide ressarch

NO is now known to be an important messenger molecule in biology.It regulates a variety of functions within cells and tissues including vasodilation,neurotransmission and immunological process.This review will focus on the nitric oxide synthase gene family and recent progress on molecular genetic analysis of NOS1,NOS2 and NOS3 genes.     

羧酸盐对鸭肝脂肪酸合成酶的抑制作用

本文研究了一系列羧酸盐对鸭肝脂肪酸合成酶的抑制规律,抑制动力学以及对几个含羧基的巯基试剂修饰该酶的保护动力学,并计算了相应的在酶上结合的平衡常数。提出羧酸盐结合于该酶缩合中心区域一个可结合羧基负离子的部位,该部位也是丙二酰基的结合部位,它可使携带了丙二酰基的活臂向缩合中心定向运动,并为缩合活性所必需。     

头状轮生链霉菌谷氨酰胺合成酶的研究 Ⅱ.酶的性质和调节

头状轮生链霉菌(Streptoverticillium caespitosus)谷氨酰胺合成酶(Glutamine synthetase,GS)生物活性的最适pH为7。测活系统中必须加入Mn~(2+)或Mg~(2+),二者分别作激活离子时得到的酶的参数不同。Mn~(2+)对GS有稳定作用。一些金属离子如Ca~(2+)等强烈地抑制生物活性。GS的最适温度为50℃,对ATP、谷氨酸和NH_4Cl的Km值分别为1.75、2.78和5mmol/L。NH_4Cl的浓度小于10mmol/L时对酶有正协同效应,大于10mmol/L时对酶有负协同效应。GS可被氨阻遏,比活能被硝酸盐促进。一些代谢物对GS有累积反馈抑制作用。发酵过程中加入氨后无“氨休克”现象,高氨条件下的GS不受蛇毒磷酸二酯酶(SVPDE)的作用,所以此酶无腺苷酰化—去腺苷酰化调节。     

大肠杆菌亮氨酰－tRNA合成酶LeuRS67R的纯化及其动力学研究

本实验室已得到的亮氨酰－ｔＲＮＡ合成酶（ＬｅｕＲＳ）基因,与文献相比,６７位氨基酸残基由Ｈｉｓ变为Ａｒｇ,此酶被定名为ＬｅｕＲＳ６７Ｒ。我们从该基因与ｐＵＣ１９重组质粒的大肠杆菌ＴＧ１转化子ＴＧ１－９１中得到ＬｅｕＲＳ的高表达,粗抽液中ＬｅｕＲＳ的表达量在转化子中比在宿主菌ＴＧ１中高２０倍以上。用三步拉层析得到电泳一条带的酶,其比活为１７８９单位／毫克。测定其动力学常数,氨酰化活力对Ｌｅｕ、ＡＴＰ的Ｋｍ值分别为０．０２７ｍｍｏｌ／Ｌ、０．４７ｍｍｏｌ／Ｌ,Ｋｃａｔ值分别为３．５～５．１ｓ－１。ＡＴＰ－ＰＰｉ交换活力对Ｌｅｕ、ＡＴＰ的Ｋｍ值分别为０．０３ｍｍｏｌ／Ｌ、１．０ｍｍｏｌ／Ｌ,Ｌｃａｔ值分别为１４０～１５５ｓ－１。此结果与从野生型大肠杆菌Ｋ－１２中提纯的ＬｅｕＲＳ的动力学常数差别很小,６７位氨基酸残基在与活性中心无直接关系的域可能是大肠杆菌的种间差异。     

大肠杆菌精氨酰－tRNA合成酶的Lys306为酶活力所必需

将大肠杆菌精氨酰ｔＲＮＡ合成酶（ＡｒｇＲＳ）上Ｌｙｓ３０６用基因点突变的方法分别变为Ａｌａ和Ａｒｇ的密码子;得到变种基因ａｒｇｓ３０６ＫＡ和ａｒｇｓ３０６ＫＲ。变种基因重组在ｐＵＣ１８上,转化到大肠杆菌ＴＧ１中,转化子中ＡｒｇＲＳ及其变种ＡｒｇＲＳ３０６ＫＡ和ＡｒｇＲＳ３０６ＫＲ所表达的蛋白量至少为ＴＧ１表达ＡｒｇＲＳ蛋白量的１００倍。细胞粗抽提液中ＡｒｇＲＳ的比活ＴＧ１、转化子ｐＵＣ１８－ａｒｇｓ、ｐＵＣ１８－ａｒｇｓ３０６ＫＡ和ｐＵＣ１８－ａｒｇｓ３０６ＫＲ分别为１．６５、２１０、１．８和３８单位／毫克。结果表明ＡｒｓＲＳ的Ｌｙｓ３０６为Ａｌａ取代使活力完全丧失;若被Ａｒｇ取代,则活力丧失８０％以上。Ｌｙｓ３０６为ＡｒｇＲＳ活力所必需。     

Physical mapping of three fruit ripening genes：Endopolygalacturonase,ACC oxidase and ACC synthase from apple（Malus x domestica） in an apple rootstock A106（Malus sieboldii

The apple rootstock,A106(Malus sieboldii),had 17 bivalents in pollen mother cells at meiotic metaphase 1,and 17 chromosomes in a haploid pollen cell.Karyotypes were prepared from root-tip cells with 2n=34 chromosomes,Seven out of 82 karyotypes(8.5%) showed one pari of satellites at the end of the short arm of chromosome 3.C-bands were shown on 6 pairs of chromosomes 2,4,6,8,14,and 16 near the telomeric regions of short arms.Probes for three ripening-related genes from Malus x domestica:endopolygalacturonase(EPG,0.6kb),ACC oxidase(1.2kb),and ACC synthase(2kb)were hybridized in situ to metaphase chromosomes of A106.Hybridization sites for the EPG gene were observed on the long arm of chromosome 14 in 15 out of 16 replicate spreads and proximal to the centromere of chromosomes 6 and 11.For the ACC oxidase gene,hylridization sites were observed in the telomeric region of the short arm of chromosomes 5 and 11 in 87% and 81% of 16 spreads respectively,proxiaml to the centromere of chromosome 1 in 81% of the spreads,and on the long arm of chromosome 13 in 50% of the spreads. Physical mapping of three fruit ripening genes in an apple rootstock A106.Twenty five spreads were studied for the ACC synthase gene and hybridization sites were observed in the telomeric region of the short arm of chromosome 12 in 96% of the spreads.chromosomes 9 and 10 in 76% of the spreads,and chromosome 17 in 56% of the spreads.     

鸭肝脂肪酸合成酶的胍变性与失活

报道了鸭肝脂肪酸俣成酶在胍变性过程中构变性过程中构象变化和活性变化的关系,首次验证了邹承鲁提出的酶活性部位构象的理论适用于多功能复合酶,同时该酶变性及复性均可测定出多个阶段,且证明有无活性稳态酶存在,在低浓度盐酸胍溶液中该酶的全反应活性和其中两个还原部位单独活性被同步可逆抑制,随着胍浓度增高,出现不可逆失活且程度和速度均迅速提高,在０．５４ｍｏｌ／Ｌ胍中该酶全反应活性在１．５分种内已有一半不可逆失     

肝癌组织中GD3合成酶的纯化

ＧＤ３合成酶是膜嵌合蛋白,定位于高尔基体,经过制作微粒体,Ｔｒｉｔｏｎ增溶、ＡＨ－Ｓｅｐｈａｒｏｓｅ及ＧＭ３－Ｇｌａｓｓｂｅａｄｓｅ及ＧＭ３－Ｇｌａｓｓｂｅａｄｓ亲和层析等步骤,二乙基亚硝胺诱发大鼠肝癌组织中的ＳＴ２被纯化了３１５９７倍,得率为０．３５％。ＳＤＳ－聚丙烯酰胺凝胶电泳后银染色呈１条蛋白着色带,分子量为５５ｋｄ。