丙酮酸脱氢酶多酶复合体研究进展

2 氧 (代 )酸脱氢酶复合体 (ＯＡＤＨｃ)是众多多酶复合体中的一个典型代表 ,该复合体家族包括 3种多酶复合体———丙酮酸脱氢酶多酶复合体 (ＰＤＨｃ)、支链 2 氧 (代 )酸脱氢酶多酶复合体和 2 酮戊二酸脱氢酶多酶复合体。其中ＰＤＨｃ催化糖代谢中丙酮酸的不可逆地氧化脱羧产生乙酰ＣｏＡ。1 .丙酮酸脱氢酶多酶复合体的组成ＰＤＨｃ广泛地分布于微生物、植物和哺乳动物中。在真核生物中 ,组成ＰＤＨｃ的所有蛋白质都是由核基因编码 ,且它们主要都位于线粒体上 (在一些生物的质体中也发现了它们的同工酶 )。ＰＤＨｃ的核心结构是由 3个…     

日本血吸虫苹果酸脱氢酶的研究

本文报告了日本血吸虫存在苹果酸脱氢酶与延胡索酸酶。在采用的实验条件下(苹果酸钠,0.02M;细胞色素c,1×10~(-5)M;磷酸盐缓冲液,0.01M,pH 7.4)测定了血吸虫匀浆的酶活力。合抱成虫的酶活力为:氧耗量Q_o(?)=4.5微升氧/毫克氮量/小时;草酰乙酸产生量(以丙酮酸测定值表示)Q_p=O.26微克分子丙酮酸/毫克氮量/小时;延胡索酸产生量Q_F=2.5微克分子延胡索酸/毫克氮量/小时。当雌雄虫分别测定时,在等氮量基础上,雌虫酶活力较雄虫者为高。当苹果酸钠用作底物时,合抱成虫的氧耗量可因加入谷氨酸钠及NAD而增高110%。于此同时,用纸层析法证明血吸虫之苹果酸脱氢酶可与谷-草转氨酶相互联系而产生天门冬氨酸。加入NAD可使合抱成虫的氧耗量增高30%,同时发现有相当量乳酸产生(Q_L=0.44微克分子乳酸/毫克氮量/小时)。以上结果表明在血吸虫匀浆存在苹果酸脱氢酶与乳酸脱氢酶的交互作用。此外,研究了虫苹果酸脱氢酶的可逆作用。借助于虫自身的乳酸脱氢酶使加入的NAD还原为NADH,然后此NADH又可将底物草酰乙酸还原。在以上情况下,用纸层析法鉴定其产物为苹果酸及延胡索酸。二巯基丁二酸锑钠(Sb-58)在最后浓度为10~(-3)M及10~(-4)M时,分别抑制血吸虫苹果酸脱氢酶活力约65%及30%。甲状腺素及中酒石酸对该酶均呈抑制作用。酒石酸锑钾在10~(-3)M时对酶活力无显著影响。     

膜状急纤虫(原生动物,纤毛门)休眠期包囊与营养期细胞同工酶组成和活性比较

应用聚丙烯酰胺凝胶电泳酶化学技术显示,腹毛目纤毛虫膜状急纤虫（Tachysoma pellionella）休眠包囊和营养细胞中乳酸脱氢酶、α磷酸甘油脱氢酶、醇脱氢酶、细胞色素氧化酶、葡萄糖-6-磷酸脱氢酶、过氧化物酶和过氧化氢酶等7种同工酶的酶谱组成有明显差异,并且在休眠包囊中其同工酶成分少、活性低,部分同工酶酶谱表现出趋于简单的趋势。ATP酶、苹果酸脱氢酶和谷氨酸脱氢酶等3种同工酶在休眠包囊与营养细胞中有相同的酶谱,但在休眠期包囊酶的活性低于营养期细胞。     

排硫硫杆菌D6中硫化氢脱氢酶的纯化及性质

从烟气生物脱硫系统的好氧产硫磁性稳态流化床反应器中,经反复纯化分离出脱硫优势菌排硫硫杆菌菌株D6,采用四步工艺纯化出膜结合型硫化氢脱氢酶。SDS-PAGE测定显示其由α1β1亚基组成,光谱分析表明含有1 mol FAD/mol酶,血红素染色揭示小亚基上结合有1 mol血红素c/mol酶,该酶属于氧还蛋白家族。该酶的最适pH为8.6,对马心细胞色素c和硫化物的表观Km分别为2.5μmol/L和6.1μmol/L,反应计量实验表明其氧化产物为元素硫。硫化氢脱氢酶受到硫和亚硫酸盐的抑制,100μmol/L的氰化钾对该酶抑制率达72%。     

保加利亚乳杆菌ATCC11842 L-乳酸脱氢酶同工酶基因的克隆与表达分析

通过对保加利亚乳杆菌(Lactobacillus delbrueckii subsp.bulgaricus)L-乳酸脱氢酶(L-lactate dehydrogenase, L-LDH)同工酶基因的异源表达、酶活测定和摇瓶发酵研究L-LDH在乳酸合成中的作用。将保加利亚乳杆菌ATCC11842中L-乳酸脱氢酶基因ldb0120和ldb0094分别克隆至载体pET28a(+)中,构建重组表达载体pET28aldb0120和pET28aldb0094,并转化到大肠埃希菌(Escherichia coli) BL21(DE3)中进行表达。进一步对重组蛋白进行Ni-NTA柱亲和层析和酶学活性测定,结果显示,LDB0120和LDB0094的比活力分别为0和25 U/mg,表明LDB0094是具有低活性的L-乳酸脱氢酶,而LDB0120不具有活性。对两株重组菌分别进行好氧和微好氧发酵,重组菌E.coli BL21/pET28aldb0094在好氧和微好氧条件可以合成L-乳酸,浓度分别为41.9和227.9 mg/L,而菌株E.coli BL21/pET28aldb0120在两种培养条件下均基本不合...     

TCA循环中间产物对酿酒酵母胞内代谢关键酶活性的影响

对酿酒酵母在添加苹果酸、柠檬酸和琥珀酸的混合培养基与其在YEPD培养基中胞内丙酮酸激酶、葡萄糖-6-磷酸脱氢酶、异柠檬酸脱氢酶、苹果酸脱氢酶、乙醇脱氢酶的酶活力差异进行了对比分析。结果表明:添加苹果酸使胞内丙酮酸激酶、异柠檬酸脱氢酶、苹果酸脱氢酶、乙醇脱氢酶的酶活分别下降34.82%、57.23%、39.15%、12.10%;添加柠檬酸使胞内丙酮酸激酶、异柠檬酸脱氢酶、苹果酸脱氢酶的酶活分别下降50.17%、42.20%、48.40%;添加琥珀酸使胞内丙酮酸激酶、葡萄糖-6-磷酸脱氢酶、异柠檬酸脱氢酶、苹果酸脱氢酶、乙醇脱氢酶的酶活分别下降34.16%、34.16%、50.87%、50.87%、12.37%。丙酮酸激酶、异柠檬酸脱氢酶和苹果酸脱氢酶对3种有机酸的耐受性较差,葡萄糖-6-磷酸脱氢酶、乙醇脱氢酶对3种有机酸的耐受具有选择性。     

长期饥饿下熊本牡蛎生理代谢和酶活性变化

为了探讨熊本牡蛎(Crassostrea sikamea)在长期饥饿胁迫下的生理代谢响应，测定了长期饥饿(0～80 d)以及再投喂(90～118 d)状态下熊本牡蛎的耗氧率、排氨率以及肝胰腺组织乳酸脱氢酶、琥珀酸脱氢酶、丙酮酸激酶、超氧化物歧化酶、碱性磷酸酶和溶菌酶的活性。结果表明：长期饥饿下，熊本牡蛎的耗氧率和排氨率极显著降低(P<0.01);随着饥饿时间的延长，熊本牡蛎乳酸脱氢酶、琥珀酸脱氢酶、丙酮酸激酶、超氧化物歧化酶和碱性磷酸酶活性均呈现波动下降的趋势，溶菌酶活性呈现波动上升的趋势；再投喂后熊本牡蛎的耗氧率、排氨率均极显著升高(P<0.01);长期饥饿对熊本牡蛎代谢和免疫功能造成损伤，熊本牡蛎通过降低代谢率和代谢酶的活性应对长期饥饿胁迫。本研究可为海水贝类饥饿胁迫下代谢调节机制的研究提供参考。     

张杂谷苹果酸脱氢酶和过氧化物酶同工酶研究

以张杂谷‘3号’、‘5号’、‘6号’及其亲本为材料,通过聚丙烯酰胺垂直板凝胶电泳技术研究不同材料不同生育期苹果酸脱氢酶和过氧化物酶同工酶酶谱特性,同时对总酶活进行了测定,探讨谷子杂种优势的形成机理。结果表明:(1)张杂谷及其亲本叶片苹果酸脱氢酶共有5条酶带,有共同酶带和偏母本型酶带;过氧化物酶同工酶共有14条,属于共同酶带;从负极到正极苹果酸脱氢酶同工酶Rf分别为0.611、0.626、0.642、0.684和0.716,而过氧化物酶同工酶Rf分别为0.08、0.21、0.24、0.30、0.36、0.44、0.49、0.58、0.66、0.69、0.72、0.75、0.85和0.89。(2)杂种苹果酸脱氢酶同工酶和母本酶谱同型,过氧化物酶同工酶酶谱和父母本一致;供试品种苹果酸脱氢酶酶谱相对简单,酶带集中,而过氧化物酶同工酶酶带数量、活性和宽度差异性较大,酶谱比较复杂。(3)所有品种苹果酸脱氢酶总活性在抽穗期最高,过氧化物酶总活性在灌浆初期最高。杂种苹果酸脱氢酶活性在苗期和抽穗期表现出不同程度的超亲优势,而过氧化物酶在抽穗和灌浆初期均表现出不同程度的超亲优势,这可能与杂种优势有关。     

聚乙二醇处理大豆种子子叶中几种酶活性和可溶性蛋白含量的变化

聚乙二醇处理的大豆种子的异柠檬酸裂解酶、苹果酸脱氢酶、过氧化氢酶、超氧物歧化酶、酸性磷酸酶、碱性磷酸酶的活性明显高于受低温吸胀冷害的种子子叶的活性,相关的酶活性协同地增长,而蛋白质的含量没有明显的变化。这些酶活性的提高可能是渗透调控处理对细胞膜系统修补的结果。     

对硝基苯氨甲酰甲基溴对甘油醛-3-磷酸脱氢酶的抑制

甘油醛-3-磷酸:NAD氧还酶(甘油醛-3-磷酸脱氢酶,酶学委员会编号1.2.1.12)是一个典型需要巯基的酶。许多巯基试剂如碘代乙酸或对氯汞苯甲酸等皆能完全抑制酶的活力。近年来一些作者曾利用标记的碘代乙酸研究了酶和抑制剂的结合当量。我们发现对硝基苯氨甲酰甲基溴(以下简     

Enzymatic Activity in Mitochondria from Orobanche

Mitochondria from Orobanche were analysed for the activities of aconitate hydratase, isocitrate dehydrogenase, succinate dehydro-genase, fumarate hydratase, malate dehydrogenase, NADH oxidase, substrate-cytochrome c oxidoreductases, glutamate dehydrogenase, aminotransferases, ATPase and “malic” enzyme. The specific activities of isocitrate dehydrogenase, NADH oxidase, substrate-cytochrome c oxidoreductases and glutamate dehydrogenase in the mitochondria) fraction from parasite tissue compared favourably with those reported for most of the mitochondria from growing and storage tissues. Succinate dehydrogenase, fumarate hydratase and aspartate aminotransferase were of intermediate activity, while aconitate hydratase and malate dehydrogenase had rather low activity, and “malic” enzyme had very low activity in comparison with other preparations. The relevance of these findings in relation to mitochondrial metabolism in the parasite is discussed. No evidence was obtained to suggest any basic abnormality in the biochemical properties of the mitochondria from Orobanche centua which may be correlated with its obligatorily parasitic existence.     

Regulation of the dicarboxylic acid part of the citric acid cycle in Bacillus subtilis.

The regulation of alpha-ketogluterate dehydrogenase, succinate dehydrogenase, fumarase, malate dehydrogenase, and malic enzyme has been studied in Bacillus subitilis. The levels of these enzymes increase rapidly during late exponential phase in a complex medium and are maximal 1 to 2 h after the onset of sporulation. Regulation of enzyme synthesis has been studied in the wild type and different citric acid cycle mutants by adding various metabolites to the growth medium. Alpha-ketoglutarate dehydrogenase is induced by glutamate or alpha-ketoglutarate; succinate dehydrogenase is repressed by malate; and fumarase and malic enzyme are induced by fumarate and malate, respectively. The addition of glucose leads to repression of the citric acid cycle enzymes whereas the level of malic enzyme is unaffected. Studies on the control of enzyme activities in vitro have shown that alpha-ketoglutarate dehydrogenase and succinate dehydrogenase are inhibited by oxalacetate. Enzyme activities are also influenced by the energy level, expressed as the energy charge of the adenylate pool. Isocitrate dehydrogenase, alpha-ketoglutarate dehydrogenase, succinate dehydrogenase, and malic enzyme are inhibited at high energy charge values, whereas malate dehydrogenase is inhibited at low energy charge. A survey of the regulation of the citric acid cycle in B.subtilis, based on the present work and previously reported results, is presented and discussed.     

Kinetics and regulation of hepatoma mitochondrial NAD(P) malic enzyme.

Kinetic studies of Morris 7777 hepatoma mitochondrial NAD(P) malic enzyme were consistent with an ordered mechanism where NAD adds to the enzyme before malate and dissociation of NADH from the enzyme is rate-limiting. In addition to its active site, malate apparently also associates with a lower affinity with an activator site. The activator fumarate competes with malate at the activator site and facilitates dissociation of NADH from the enzyme. The ratio of NAD(P) malic enzyme to malate dehydrogenase activity in the hepatoma mitochondrial extract was found to be too low, even in the presence of known inhibitors of malate dehydrogenase, to account for the known ability of NAD(P) malic enzyme to intercept exogenous malate from malate dehydrogenase in intact tumor mitochondria (Moreadith, R.W., and Lehninger, A.L. (1984) J. Biol. Chem. 259, 6215-6221). However, NAD(P) malic enzyme may be able to intercept exogenous malate because according to the present results, it can associate with the pyruvate dehydrogenase complex, which could localize NAD(P) malic enzyme in the vicinity of the inner mitochondrial membrane. The activity levels of some key metabolic enzymes were found to be different in Morris 7777 mitochondria than in liver or mitochondria of other rapidly dividing tumors. These results are discussed in terms of differences among tumors in their ability to utilize malate, glutamate, and citrate as respiratory fuels.     

Determination of NAD Malic Enzyme in Leaves of C(4) Plants : EFFECTS OF MALATE DEHYDROGENASE AND OTHER FACTORS

Malate dehydrogenase may interfere with the assay of NAD malic enzyme, as NADH is formed during the conversion of malate to oxaloacetate. During the present study, two additional effects of malate dehydrogenase were investigated; they are evident only if the malate dehydrogenase reaction is allowed to reach equilibrium prior to initiating the malic enzyme reaction. One of these (Outlaw, Manchester 1980 Plant Physiol 65: 1136-1138) might cause an underestimation of NAD reduction by malic enzyme due to the oxidation of NADH during reversal of the malate dehydrogenase reaction. A second effect may result in overestimation of malic enzyme activity, as Mn²⁺-catalyzed oxaloacetate decarboxylation causes continuing net NADH formation via malate dehydrogenase. These effects were studied by assaying the activity of a partially purified preparation of Amaranthus retroflexus NAD malic enzyme in the presence or absence of purified NAD malate dehydrogenase.     

Changes in enzyme pattern of ehrlich ascites tumor cells following serial cultivation in media with increased (hypertonic) NaCl content

Adaptation of Ehrlich ascites tumor cells to serial cultivation in media with progressively elevated (hypertonic) NaCl content (“high NaCl”-tolerant cells) has resulted in progressive increases of the cellular activities of NAD-dependent glycerol-3-phosohate dehydrogenase (EC 1.1.1.8), NAD-dependent malate dehydrogenase (EC 1.1.1.37), glutamate—oxalacetate transaminase (EC 2.6.1.1.), NAD(P)-dependent glutamate dehydrogenase (EC 1.4.1.3), NADP-dependent malate dehydrogenase (EC 1.1.1.40, “malic enzyme”) and NADP-dependent isocitrate dehydrogenase (EC 1.1.1.42). The activities of glutamate—pyruvate transaminase (EC 2.6.1.2.) and of glycolytic enzymes as phosphofructokinase (EC 2.7.1.11), glyceradehydephosphate dehydrogenase (EC 1.2.1.12) and lactate dehydrogenase (EC 1.1.1.27) were only slightly and not in progressive manner (in response to the progressive increase of the environmental NaCl concentration) affected. These changes are discussed with respect to a metabolic pattern of these “high NaCl”-tolerant cells which is compatible with increased energy requirements, especially for active cation transport. It is suggested that these increased cellular enzyme activitees reflect an increased transfer of reducing equivalents across mitochondrial membranes (via the “glycerophosphate cycle and the malate—aspartate shuttle”) and possibly a stimulated lipid metabolism. These alterations in the level of enzyme activities must be regarded as an adaptive cellular response to the “high NaCl” enviromment, since readaptation to growth in regular isotonic media resulted in a reversion to the enzyme pattern characteristic of the parent cells.     

Selective Inhibition of Bacterial Enzymes by Free Fatty Acids

Octanoic acid inhibits, in vitro, the bacterial enzymes glucose-6-phosphate dehydrogenase, phosphofructokinase, pyruvate kinase, fumarase, lactate dehydrogenase, and the malic enzyme of Arthrobacter crystallopoietes. The free fatty acid appears to act as an inhibitor of lipogenesis, although it does not affect the rate of gluconeogenesis. To demonstrate that this inhibition may be of physiological significance in vivo, those enzymes not involved in lipogenesis, such as fructose-1, 6-diphosphatase, phosphoglucomutase, phosphohexoisomerase, aconitase, nicotinamide adenine dinucleotide phosphate (NADP) isocitrate dehydrogenase, NADP glutamate dehydrogenase, malate dehydrogenase, and isocitrate lyase, were assayed and found not to be inhibited by the free fatty acid.     

Dietary induction of hepatic malic enzyme activity: differentiation of the induction process

The activity of rat liver malic enzyme shows a marked increase when the animals are maintained on a restricted protein diet. Of the NADP-linked dehydrogenases tested (malic enzyme, glucose-6-phosphate dehydrogenase, and isocitrate dehydrogenase), the response is confined only to malic enzyme. Dietary sucrose is not required for the increase in activity, but elevated dietary levels of this disaccharide increase hepatic malic enzyme regardless of dietary protein. Glucose-6-phosphate dehydrogenase activity is increased by dietary sucrose provided adequate dietary protein is supplied. The specificity of the response to lowered dietary protein shown by malic enzyme suggests that the control of the hepatic enzyme is mediated by processes different from those controlling the activity of glucose-6-phosphate dehydrogenase.     

泡沙参同工酶基因位点的遗传分析

利用聚丙烯酰胺凝胶电泳技术 ,对来自天然群体 (居群 )的泡沙参 (Adenophora potaninii Korsh.)及其人工杂交子代进行了 8种同工酶的电泳检测和谱带遗传分析 ,以确定编码这些酶系统的基因位点和等位基因。选用 4种不同的凝胶缓冲系统 ,对下列不同酶系统进行了酶谱的遗传分析 :天冬氨酸转氨酶 (AAT)、酯酶 (EST)、甲酸脱氢酶 (FDH)、谷氨酸脱氢酶 (GDH)、异柠檬酸脱氢酶 (IDH)、乳酸脱氢酶(LDH)、苹果酸酶 (ME)和超氧化物歧化酶 (SOD)。结果表明 ,这 8种酶系统至少由 1 8个基因位点编码 ,其中 1 2个位点为遗传稳定的等位酶位点 ,是可靠的遗传标记。酶谱的分离式样表明 ,EST为单聚体结构 ,AAT、FDH、IDH、SOD为二聚体结构 ,GDH为六聚体结构。最后对同工酶的器官和发育特异性以及同工酶基因位点的遗传分析进行了讨论     

Distribution of Nitrate-assimilating Enzymes between Mesophyll Protoplasts and Bundle Sheath Cells in Leaves of Three Groups of C(4) Plants

Intercellular distribution of enzymes involved in amino nitrogen synthesis was studied in leaves of species representing three C₄ groups, i.e. Sorghum bicolor, Zea mays, Digitaria sanguinalis (NADP malic enzyme type); Panicum miliaceum (NAD malic enzyme type); and Panicum maximum (phosphoenolpyruvate carboxykinase type). Nitrate reductase, nitrite reductase, glutamine synthetase, and glutamate synthase were predominantly localized in mesophyll cells of all the species, except in P. maximum where nitrite reductase had similar activity on a chlorophyll basis, in both mesophyll and bundle sheath cells. NADH-glutamate dehydrogenase was concentrated in the bundle sheath cells, while NADPH-glutamate dehydrogenase was localized in both mesophyll and bundle sheath cells. The activities of nitrate-assimilating enzymes, except for nitrate reductase, were high enough to account for the proposed in vivo rates of nitrate assimilation.     

Genetic Analysis of Isozyme Loci in Adenophora potaninii Korsh.

Enzyme polymorphism in Adenophora potaninii Korsh. was investigated using vertical slab polyacrylamide gel electrophoresis. Genetic analysis of the population samples and the progeny of intraspecific crosses allowed the verification of the isozyme loci from eight enzyme systems. The system studies included aspartate aminotransferase (AAT), esterase (EST). formate dehydrogenase (FDH), glutamate dehydrogenase (GDH), isocitrate dehydrogenase (IDH), lactate dehydrogenase (LDH), malic enzyme (ME) and superoxide dismutase (SOD). The results indicated that the eight enzyme systems are specified by at least 18 loci, 12 of which behaved as al|ozyme loci. Zymogram patterns showed that EST is monomeric and GDH is hexameric. AAT, FDH, IDH and SOD are apparently dimeric. The tissue and developmental variability are also discussed along with the genetic analysis of isozymes.