利用超分子体系模拟生物体G蛋白耦联受体型信号转导过程

以生物体G蛋白耦联受体型信号转导过程为模拟原型,利用自组装方法构建了具有分子间信号转导能力的超分子体系。带正电荷的肽脂质分子与人工受体共同形成脂质体,乳酸脱氢酶(LDH)(效应器)通过静电作用吸附到脂质体上,乳酸脱氢酶的竞争性抑制剂(Cu2+)抑制LDH活性(相对酶活性为11.6%),进而共同组成了一个酶活性处于关闭状态的超分子系统。向超分子体系中加入信号分子——磷酸吡哆醛(PLP)后,PLP通过静电作用吸附到带正电荷的脂质体表面,并与人工受体形成希夫碱;希夫碱与LDH竞争铜离子,解除铜离子对LDH活性的抑制,恢复LDH的催化活性,使相对酶活性达到83.0%。实验结果表明,超分子体系完成了从信号分子到效应器(酶)活性变化的信号转导过程,可以利用超分子体系构建调控酶催化活性的超分子器件。     

不同去垢剂对琥珀酸脱氢酶溶膜效果的比较研究

比较了3种溶膜剂Triton X-100、Tween-20和脱氧胆酸钠对琥珀酸脱氢酶（SDH）的溶膜效果。其中Triron X-100的效果为最好。并且，在1％的Triton X-100溶液中SDH能够较长时间保持活力。     

五种医用酶的硫酸按分级分离研究

采用硫酸铵分级沉淀法从一种兔肌匀浆上清中分步分离出醛缩酶、乳酸脱氢酶、丙酮酸激酶、肌酸激酶和磷酸甘油醛脱氢酶等五种医用酶。该法步骤简单、分离周期短、酶活回收率高、具有较大的实用价值。     

两种策略实现1,3-丙二醇关键酶基因的共表达

甘油脱水酶(GDHt)和1,3-丙二醇氧化还原酶(PDOR)是甘油歧化为1,3-丙二醇(1,3-PD)的两个关键酶。采用多顺反子重组和质粒共存两种策略,对来自克雷伯肺炎杆菌(Klebsiela pneumoniae)的两个关键酶进行共表达。构建表达载体pET-28a-dhaB1B2B3-dhaT将两酶基因dhaB1B2B3和dhaT用SD序列相隔,在E.coli BL21(DE3)高水平共表达了GDHt 3个亚基和PDOR,表达蛋白分别约占菌体总蛋白的18%、9%、7%和9%。质粒pET-28a-dhaB1B2B3和pET-22b-dhaT共转化E.coli BL21(DE3)得到稳定的双质粒系统,48h后84%的细胞能同时含有两种质粒,GDHt 3亚基和PDOR分别约占菌体总蛋白的16%、8%、6%和14%。两种酶在两种表达方法下均显示高于原始菌株的酶活力。     

琥珀酸脱氢酶的纯化研究

以玉米黄化苗为生物材料，将其捣碎后，通过差速离心获得线粒体，使用超声波将其破碎，用2％Ttiton X-100溶膜，超速离心，硫酸铵沉淀，DEAE－C32层析纯化琥珀酸脱氢酶，纯化倍数为12倍，电泳图谱显示一条带。     

A novel salicylaldehyde dehydrogenase-NahV involved in catabolism of naphthalene from Pseudomonas putida ND6

A novel salicylaldehyde dehydrogenase involved in catabolism of naphthalene from Pseudomonas putida ND6, NahV, has been identified. NahV exhibited lower identity in amino acid sequence with the classic salicylaldehyde dehydrogenase, NahF, from P. putida ND6. This is the first report of an isofunctional enzyme of bacterial salicylaldehyde dehydrogenase. Comparison of Km and Vmax values of NahV and NahF demonstrated that NahF has a more efficient catalytic reaction than NahV, while NahV has much higher affinity for salicylaldehyde and NAD . Both enzymes exhibited broad substrate speci- ficities and catalyzed the oxidation of salicylaldehyde, 5-chlorosalicylaldehyde, formaldehyde, m-nitrobenzaldehyde, o-nitrobenzaldehyde, o-methoxybenxaldehyde, glutaraldehyde, caprylic aldehyde, and glyoxal. However, the relative rates at which the substituted analogs are transformed differ considerably. NahV activity could be enhanced by Fe2 , Cu2 and Zn2 ; whereas NahF activity could only be stimulated by Fe2 . NahF is more stable than NahV at elevated temperatures. Dot-blot hybridization analyses showed that nahF-like genes occurred in all naphthalene-degradation bacteria isolated in this study, whereas nahV-like genes were present in only some naphthalene-degrading bacteria.     

Protein synthesis during germination of heterothallic yeast ascospores

Protein synthesis during ascospore germination of the heterothallicSaccharomyces cerevisiae strain AP-3 was investigated. Protein synthesis in the germinating ascospores appeared to begin approximately 20 min following glucose initiation. Since RNA synthesis did not start until approximately 70 min after the onset of germination, strain AP-3 ascospores must contain RNA which is ready for immediate translation. Both trehalase and glyceraldehyde-3-phosphate dehydrogenase activities were found to be affected by the onset of germination. Trehalase activity was found to increase severalfold following 60 min of spore germination but remained relatively constant over the subsequent 120 min examined. Dehydrogenase activity was not detectable in AP-3 ascospores but was measurable in germinating ascospores.     

Distinct but parallel evolutionary patterns between alcohol and aldehyde dehydrogenases: addition of fish/human betaine aldehyde dehydrogenase divergence

Alcohol dehydrogenases (ADHs) of the MDR type (medium-chain dehydrogenases/reductases) have diverged into two evolutionary groups in eukaryotes: a set of 'constant' enzymes (class III) typical of basal enzymes, and a set of 'variable' enzymes (remaining classes) suggesting 'evolving' forms. The variable set has larger overall variability, different segment variability, and variability also in functional segments. Using a major aldehyde dehydrogenase (ALDH) from cod liver and fish ALDHs deduced from the draft genome sequence of Fugu rubripes (Japanese puffer fish), we found that ALDHs form more complex patterns than the ADHs. Nevertheless, ALDHs also group into 'constant' and 'variable' sets, have separate segment variabilities, and distinct functions. Betaine ALDH (class 9 ALDH) is 'constant,' has three segments of variability, all non-functional, and a limited fish/human divergence, reminiscent of the ADH class III pattern. Enzymatic properties of fish betaine ALDH were also determined. Although all ALDH patterns are still not known, overall patterns are related to those of ADH, and group separations may be distinguished. The results can be interpreted functionally, support ALDH isozyme distinctions, and assign properties to the multiplicities of the ADH and ALDH enzymes.     

大鼠离体心脏缺灌和再灌早期乳酸脱氢酶和肌酸激酶的释放规律

用Ｌａｎｇｅｎｄｏｅｆｆ法灌流雄性Ｗｉｓｔａｒ大鼠心脏，平衡１０ｍｉｎ，缺灌１０ｍｉｎ，于再灌３ｍｉｎ内每隔１５ｓｅｃ收集一次冠脉流份，连续检测冠脉流份中ＣＫ、ＬＤＨ以及ＧＯＴ的活性（Ｕ／Ｌ），比较它们释放的动态特征。结果表明，再灌３ｍｉｎ内，３种酶都呈双相释放。第１峰出现在再灌１５ｓｅｃ，酶活性为对照值的５一９倍，它主要反映缺灌损伤。再灌９０一１２０ｓｅｃ又出现第Ⅱ峰，主要代表再灌损伤。无论是再灌３ｍｉｎ内酶释放总量，还是第Ⅰ，第Ⅱ峰的峰值，依酶活性高低排列的次序都是ＣＫ＞ＬＤＨ＞ＧＯＴ。此外，在部分动物身上进行了反复两次缺灌再灌实验，结果发现，第二次缺灌后ＣＫ释放量明显高于第一次缺灌值。ＬＤＨ的Ⅰ峰和Ⅱ峰出现时间与第一次无差别，只是３分钟内酶释放总量高于第一次。说明在本实验条件下，未见预缺灌处理对随后再次缺灌有保护作用。缺灌期心率逐渐减慢，收缩力逐渐减弱，但多数标本仍有微弱的收缩。这使缺灌期冠脉流份的酶活测定成为可能。实验发现，早在缺灌１ｍｉｎ时心肌细胞释放的酶，其活性可达对照值的２倍（ｐ＜０．０５），至缺灌１０ｍｉｎ时ＣＫ和ＬＤＨ的值可达对照值的１０倍左右。这一现象进一步证实，再灌１５ｓｅｃ内大量酶的     

Genetic control of malate dehydrogenase inNicotiana suaveolens andN. glutinosa

Summary The study of malate dehydrogenase patterns in leaves ofNicotiana suaveolens, N. glutinosa and their interspecific hybrid has been carried out, in order to propose a model of its genetic control. Two possible explanations for the genetic control of malate dehydrogenase have been postulated forN. suaveolens, and at least two loci appear to be implicated in the control of this system inN. glutinosa.Acknowledgments. The authors thank Dr M. Pérez de la Vega for his valuable advice.