树鼩组织乳酸脱氢酶同工酶的研究

对云南树鼩(Tupaia belangeri chinensis)心肌、肺、颌下腺、脾脏、肾脏、肾上腺、骨骼肌和大脑8种组织乳酸脱氢酶(LDH)同工酶进行琼脂糖凝胶电泳分析。结果表明,8种组织均呈现为LDH-M型和LDH-H型两种亚基组合而成的5种不同分子形式同工酶。采用分光光度比色定量法测得5种同工酶组分的相对酶活性。对其H亚基和M亚基百分率以及H/M亚基比率进行了统计分析。并就不同酶谱特征与基因表达状况进行了分析讨论。     

树qu组织乳酸脱氢酶同工酶的研究

对云南树qu（Tupaia beiangeri）心肌、肺、颌下腺、肾脏、肾上腺、骨骼肌和大脑8种组织乳酸脱氢酶（LDH）同工酶进行琼脂糖凝胶电泳分析。结果表明，8种组织均呈现为LDH-M型和LDH-H型两种亚基组合而成的5种不同分子形式同工酶。采用分光光度比色定量法测得5种同工酶组分的相对酶活性。对其H亚基和M亚基百分率以及H/M亚基比率进行了统计分析。并就不同酶谱特征与基因表达状况进行了分析讨论。     

正常树驹乳酸脱氢酶同工酶等的血清学调查

近年来,研究者利用云南树鼩(Tupaia glis yunnalis)做了不少试验,如甲型肝炎、乙型肝炎的实验动物模型。这些实验的一个特点是对树鼩在自然生活状态下的各种生化指标做得比较少。这次,我们对80只树鼩血清乳酸脱氢酶同工酶(LDH)、乙型肝炎表面抗原(HBsAg)、抗甲肝病毒IgG抗体(HAV-IgG)及谷丙转氨酶(SGPT)进行了检测。兹报告     

树鼩血压和心率的无创测定

目的建立健康树鼩的心率、血压正常值参考范围,并探讨不同来源、不同性别、不同年龄树鼩心率、血压的差异。方法随机挑选实验树鼩180只,按来源分为野生成年组、F1代自繁成年组和青幼年组三个组,每组雌雄各半,共60只。采用智能无创血压计（鼠仪）逐只测定HR（心率）、SBP（收缩压）、DBP（舒张压）和MBP（平均动脉压）。结果野生成年树鼩、自繁成年树鼩和青幼年树鼩心率分别为394.33±37.74 BPM、351.61±72.76 BPM和378.19±69.04 BPM,野生和自繁成年树鼩组差异有显著性（P〈0.05）。自繁成年树鼩收缩压、舒张压和平均动脉压均明显低于青幼年树鼩,差异有极显著性（P〈0.01）。野生成年树鼩和自繁成年树鼩相比,收缩压、舒张压和平均动脉压差异均无显著性（P〉0.05）。结论大鼠无创血压计适合于树鼩的血压、心率的测量。通过测定,获得了野生成年树鼩、F1代自繁成年树鼩和青幼年树鼩的心率和血压参考值范围,丰富了树鼩基础生理数据,可为相关研究提供科学参考。     

臭鼩血清蛋白和六种组织乳酸脱氢酶同工酶的研究

本实验对臭鼩的血清蛋白及心肌、骨骼肌、肾脏、脾脏、肝脏,睾丸6种组织器官的乳酸脱氢酶(LDH)同工酶进行了聚丙烯酰胺凝胶盘状电泳的分析研究。臭鼩血清蛋白存在15—17条带,各组织的LDH同工酶均由5条带构成,其中心肌LDH-1、LDH-2和肾脏LDH-1各出现1条亚带。     

东方田鼠乳酸脱氢酶同工酶的研究

目的研究乳酸脱氢同工酶在3种东方田鼠不同器官的分布.方法以大鼠和小鼠作对照,用聚丙烯酰胺凝胶电泳对3个不同地区的东方田鼠(Microtusfortis)血清、红细胞、肝脏、肾脏、肺中乳酸脱氢酶(LDH)同工酶进行分析.结果东方田鼠血浆中只有LDH5,血清中湖南洞庭湖地区、宁夏青铜峡地区的东方田鼠也只有LDH5,而东北地区的东方田鼠则含有LDH1和LDH5,3个地区的东方田鼠肾脏和肺中都有5种LDH同工酶,肝脏中除东北地区的东方田鼠含5种LDH同工酶外,其余两种东方田鼠均以LDH5为主(LDH5占95%以上).结论东北地区东方田鼠乳酸脱氢酶同工酶谱与其他两地的东方田鼠不相同.     

高原鼠兔和高原鼢鼠乳酸脱氢酶同工酶的初步研究

比较异种生物同工酶(Isoenzyme)能从分子水平阐明物种的多样性。近年来,不少学者对许多动物的同工酶进行广泛而深入的研究(Masters 1975,Markert,1975)。其中对乳酸脱氢酶(以下简称LDH)的研究最为详尽(Everse等,1973,Moss,1982)。乳酸脱氢酶同工酶是由H(或B),M(或A)2种亚基组成的一组四聚体分子。H,M亚基分别受不同基因位点控制,并按不同比例组成5种不同的同工酶。     

根际低氧胁迫对网纹甜瓜生长、根呼吸代谢及抗氧化酶活性的影响

采用气雾法栽培系统,研究了根际低氧（10% O₂和5% O₂）胁迫对网纹甜瓜果实发育期间植株生长、根呼吸代谢及抗氧化酶活性的影响.结果表明：与对照相比,低氧胁迫下,网纹甜瓜株高、根长降低,植株鲜、干物质量显著下降;根呼吸速率极显著低于对照（21% O₂）,且5% O₂处理下降幅度大于10% O₂处理;乳酸脱氢酶（LDH）、乙醇脱氢酶（ADH）和丙酮酸脱羧酶（PDC）活性较对照显著升高,而苹果酸脱氢酶（MDH）活性显著降低;根系中超氧化物歧化酶（SOD）、过氧化物酶（POD）、过氧化氢酶（CAT）活性和丙二醛（MDA）含量显著高于对照,其中10% O₂处理抗氧化酶活性升高幅度显著大于5% O₂处理,而MDA含量5% O₂处理高于10% O₂处理.说明网纹甜瓜果实发育期间根际氧浓度降到10%及其以下时,根系有氧呼吸明显受阻,无氧呼吸代谢被促进,同时根系抗氧化酶发生应激反应,但随低氧胁迫时间的延长,根细胞质膜过氧化程度加剧,根系受到伤害,植株生长受到抑制,最终导致果实产量和品质下降.     

水迷宫实验中不同性别树鼩学习记忆能力的比较

目的研究雌雄树鼩空间学习和记忆能力的差异。方法随机选择自繁F1代树鼩20只（雄11只,雌9只）,在相同条件下进行8 d的水迷宫实验,包括前7 d的定位航行实验和第8天的空间探索实验。结果定位航行实验中雌雄逃避潜伏期、游泳总路程差异无显著性（P〉0.05）,但不同时间水平差异有显著性（P〈0.05）;平均游泳速度雌雄差异无显著性（P〉0.05）。空间探索实验中目标象限游泳时间和总时间之比、目标象限游泳路程和总路程之比雌雄差异无显著性（P〉0.05）;穿越目标象限次数和搜索策略雌雄差异有显著性（P〈0.05）。结论水迷宫实验中树鼩在空间学习能力上雌雄无差异,但在空间探索实验中雄性的表现优于雌性。     

江豚(Neomeris phocaenoides)某些组织中的LDH同工酶

1957年T.Wieland运用高压电泳方法测定乳酸脱氢酶(LDH)活性时,发现同一动物个体的不同组织的心、肝、肾、脑和骨胳肌等提取液分别显示出1-6条区带。其后,将这些酶促污性相同而分子形式不同的蛋白质称为同工酶(isoenzyme)。     

Interactions of glyceraldehyde-3-phosphate dehydrogenase with G- and F-actin predicted by Brownian dynamics

Brownian dynamics (BD) was used to simulate the binding of glyceraldehyde-3-phosphate dehydrogenase (GAPDH) to G- and F-actin. High-resolution three-dimensional models (X-ray and homology built) of the proteins were used in the simulations. The electrostatic potential about each protein was predicted by solving the linearized Poisson-Boltzmann equation for use in BD simulations. The BD simulations resulted in complexes of GAPDH with G- or F-actin involving positively charged surface patches on GAPDH (Lyses 24, 69, 110 and 114) and negatively charged residues of the N- and C-termini (Asps 1, 25 and 363 and Glus 2, 4, 224 and 364) of actin. The actin residues all belong to subdomain 1. Although the positively charged surface patches of GAPDH are not close enough to each other to enhance their electrostatic potential, occasionally two subunits of the GAPDH tetramer may simultaneously interact with two neighboring monomers of F-actin. These results are different from those of fructose-1,6-bisphosphate aldolase, where quaternary structure directly influenced binding by two subunits combining their electrostatic potentials (see previous study, Ouporov et al., 1999, Biophys. J. 76: 17-27). Instead, GAPDH uses its quaternary structure to span the distance between two different actin subunits so that it can interact with two different actin subunits simultaneously.     

Electron microscopic analysis of the peripheral and membrane parts of mitochondrial NADH dehydrogenase (complex I).

Two related forms of the respiratory chain NADH dehydrogenase (NADH:ubiquinone reductase or complex I) are synthesized in the mitochondria of Neurospora crassa. Normally growing cells make a large form that consists of 25 subunits encoded by nuclear DNA and six to seven subunits encoded by mitochondrial DNA. Cells grown in the presence of chloramphenicol, however, make a smaller form comprising only 13 subunits, all encoded by nuclear DNA. When the large enzyme is dissected by chaotropic agents (such as NaBr), all those subunits of the large form that are missing in the small form can be isolated as a distinct, so-called hydrophobic fragment. The small enzyme and the hydrophobic fragment make up, with regard to their redox groups, subunit composition and function, two complementary parts of the large-form NADH dehydrogenase. Averaging of electron microscope images of single particles of the large enzyme was carried out, revealing an unusual L-shaped structure with two domains or "arms" arranged at right angles. The hydrophobic fragment obtained by the NaBr treatment corresponds in size and appearance to one of these arms. A three-dimensional reconstruction from images of negatively stained membrane crystals of the large-form NADH dehydrogenase shows a peripheral domain, protruding from the membrane, with weak unresolved density within the membrane. This peripheral domain was removed by washing the crystals in situ with 2 M-NaBr, exposing a large membrane-buried domain, which was reconstructed in three dimensions. A three-dimensional reconstruction of the small enzyme from negatively stained membrane crystals, also described here, shows only a peripheral domain. These results suggest that the membrane protruding arm of the large form corresponds to the small enzyme, whereas the arm lying within the membrane can be identified as the hydrophobic fragment. The two parts of NADH dehydrogenase that can be defined by the separate genetic origin of (most of) their subunits, their independent assembly, and their distinct contributions to the electron pathway can thus be assigned to the two arms of the L-shaped complex I.     

Purification and comparative study of the kinases specific for branched chain alpha-ketoacid dehydrogenase and pyruvate dehydrogenase.

Rat heart branched chain alpha-ketoacid dehydrogenase kinase (BCKDH kinase) and pyruvate dehydrogenase kinase (PDH kinase) were purified from their respective complexes to apparent homogeneity. BCKDH kinase consisted of one subunit with molecular weight 44,000-45,000 Da, whereas PDH kinase consisted of two subunits with molecular weight 48,000 Da (alpha) and 45,000 Da (beta) as previously shown for the bovine kidney enzyme (Stepp et al., 1983, J. Biol. Chem. 258, 9454-9458). Proteolysis maps of BCKDH kinase and the two subunits of PDH kinase were different, suggesting that all subunits are different entities. The alpha subunit of the rat heart PDH kinase could be cleaved selectively by chymotrypsin with concomitant loss of kinase activity, as previously shown for the bovine kidney enzyme, suggesting that the catalytic activity of PDH kinase resides in the alpha subunit. The beta subunit appeared to be a different entity unique to the PDH kinase. Both kinases exhibited marked substrate specificity toward their respective complexes and would not inactivate heterologous complexes. The kinases possessed slightly different substrate specificity toward histones. BCKDH kinase preferentially phosphorylated histones in the order f1 greater than f2B much greater than f2A much greater than f3. The relative order for PDH kinase was the same, but f2A and f3 were considerably better substrates than they were for BCKDH kinase. These observations suggest that the kinases have different requirements for the structure of the protein at their phosphorylation sites.     

Purification and properties of NAD+-dependent glyceraldehyde-3-phosphate dehydrogenase from spinach leaves.

An NAD+-dependent glyceraldehyde-3-phosphate dehydrogenase (D-glyceraldehyde-3-phosphate:NAD+ oxidoreductase (phosphorylating), EC. 1.2.1.12) has been purified from spinach leaves as a homogeneous protein of 150,000 daltons. Kinetic constants of 2.5 . 10(-4) M and 4 . 10(-4) M have been calculated for NAD+ and glyceraldehyde-3-phosphate, respectively. The amino acid composition is characterized by a cysteine content higher than that found in analogous enzymes. On sodium dodecyl sulphate gel electrophoresis, the native enzyme dissociates into two subunits of 37,000 and 14,000 daltons. The two subunits have been isolated in equimolar amounts by gel filtration; end-group analysis shows that alanine is the N-terminal residue of the large subunit, while serine is found at the N-terminus of the small subunit. Comparison of amino acid analysies and peptide maps shows that the two subunits have a different amino acid sequence. These results indicate that the NAD+-dependent glyceraldehyde-3-phosphate, dehydrogenase, isolated from spinach leaves has an atypical oligomeric structure, the protomer being formed by two different subunits.     

Lactate dehydrogenase isozymes in the genus Channa: Species-specific patterns

The lactate dehydrogenase isozyme system for five Channa species (snakehead fish) have been examined by means of polyacrylamide gel electrophoresis. The pattern and distribution of the LDH isozymes in all these species are found to be species-specific with a reversed relative mobility of the A and B subunits as compared to most of the other vertebrates. In addition to these two subunits, an eye-specific LDH E isozyme has also been detected in these species.     

2-Oxoacid dehydrogenase complexes of Escherichia coli: cellular amounts and patterns of synthesis.

The oxidative decarboxylations of pyruvate and 2-oxoglutarate in Escherichia coli are carried out by two large, multienzyme complexes: pyruvate dehydrogenase and 2-oxoglutarate dehydrogenase. The enzyme complexes each contain three subunits: two are unique to the individual complexes, the third is shared between them. Resolution of the polypeptide subunits on two-dimensional gels allowed quantitative analysis of their cellular levels and patterns of synthesis in growing cells. Cells growing in glucose-salts medium were found to contain roughly 85 to 136 pyruvate dehydrogenase complexes and 73 2-oxoglutarate complexes. Lipoamide dehydrogenase, the subunit shared by the two complexes, was found to be in significant excess of its stoichiometric demand in the two enzyme complexes under most growth conditions. The subunits unique to each of the complexes were coordinately regulated over a wide variety of growth conditions and a broad range of expression. The two complexes responded to different, but partially overlapping, regulatory signals. Most importantly, the shared subunit was actively regulated to accommodate its demand in both enzymes. These results are discussed with regard to possible mechanisms of regulation of the enzyme complexes in general and of the shared subunit specifically.     

Cloning, expression, and nucleotide sequence of the formate dehydrogenase genes from Methanobacterium formicicum

The genes for the two subunits of the formate dehydrogenase from Methanobacterium formicicum were cloned and their sequences determined. When expressed in Escherichia coli, two proteins were produced which had the appropriate mobility on an SDS gel for the two subunits of formate dehydrogenase and cross-reacted with antibodies raised to purified formate dehydrogenase. The genes for the two formate dehydrogenase subunits overlap by 1 base pair and are preceded by DNA sequences similar to both eubacterial and archaebacterial promoters and ribosome-binding sites. The amino acid sequences deduced from the DNA sequence were analyzed, and the arrangement of putative iron-sulfur centers is discussed.     

Complementation of succinate dehydrogenase mutants in fused Bacillus subtilis protoplasts

Abstract Succinate dehydrogenase (SDH) and cytochrome b ₅₅₈ are present in Bacillus subtilis cytoplasmic membranes as a complex consisting of three different subunits. Complementation of membrane-bound SDH activity was studied in fused protoplasts of two SDH-negative mutants mutated in different subunits of the complex. The complementation was found to be rapid, temperature dependent and not sensitive to inhibitors of respiration. It is concluded that the complementation predominantly results from the assembly of preformed SDH subunits.     

A new type of a multifunctional beta-oxidation enzyme in euglena

The biochemical and molecular properties of the beta-oxidation enzymes from algae have not been investigated yet. The present study provides such data for the phylogenetically old alga Euglena (Euglena gracilis). A novel multifunctional beta-oxidation complex was purified to homogeneity by ammonium sulfate precipitation, density gradient centrifugation, and ion-exchange chromatography. Monospecific antibodies used in immunocytochemical experiments revealed that the enzyme is located in mitochondria. The enzyme complex is composed of 3-hydroxyacyl-coenzyme A (-CoA) dehydrogenase, 2-enoyl-CoA hydratase, thiolase, and epimerase activities. The purified enzyme exhibits a native molecular mass of about 460 kD, consisting of 45.5-, 44.5-, 34-, and 32-kD subunits. Subunits dissociated from the complete complex revealed that the hydratase and the thiolase functions are located on the large subunits, whereas two dehydrogenase functions are located on the two smaller subunits. Epimerase activity was only measurable in the complete enzyme complex. From the use of stereoisomers and sequence data, it was concluded that the 2-enoyl-CoA hydratase catalyzes the formation of L-hydroxyacyl CoA isomers and that both of the different 3-hydroxyacyl-CoA dehydrogenase functions on the 32- and 34-kD subunits are specific to L-isomers as substrates, respectively. All of these data suggest that the Euglena enzyme belongs to the family of beta-oxidation enzymes that degrade acyl-CoAs via L-isomers and that it is composed of subunits comparable with subunits of monofunctional beta-oxidation enzymes. It is concluded that the Euglena enzyme phylogenetically developed from monospecific enzymes in archeons by non-covalent combination of subunits and presents an additional line for the evolutionary development of multifunctional beta-oxidation enzymes.     

Purification and properties of pyruvate dehydrogenase kinase from bovine kidney

Pyruvate dehydrogenase kinase was purified about 2,700-fold to apparent homogeneity from extracts of bovine kidney mitochondria. The kinase consists of two subunits (alpha beta) with molecular weights of 48,000 (alpha) and 45,000 (beta) as estimated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Kinase activity resides in the alpha subunit. The alpha subunit is sensitive to proteolysis by chymotrypsin, whereas the beta subunit is selectively modified by trypsin. These observations, together with the results of peptide mapping, indicate that the two subunits are distinctly different proteins. It is proposed that the beta subunit is a regulatory subunit.