Identification and Characterization of MAE1, the Saccharomyces cerevisiae Structural Gene Encoding Mitochondrial Malic Enzyme

Pyruvate, a precursor for several amino acids, can be synthesized from phosphoenolpyruvate by pyruvate kinase. Nevertheless, pyk1 pyk2 mutants of Saccharomyces cerevisiae devoid of pyruvate kinase activity grew normally on ethanol in defined media, indicating the presence of an alternative route for pyruvate synthesis. A candidate for this role is malic enzyme, which catalyzes the oxidative decarboxylation of malate to pyruvate. Disruption of open reading frame YKL029c, which is homologous to malic enzyme genes from other organisms, abolished malic enzyme activity in extracts of glucose-grown cells. Conversely, overexpression of YKL029c/MAE1 from the MET25 promoter resulted in an up to 33-fold increase of malic enzyme activity. Growth studies with mutants demonstrated that presence of either Pyk1p or Mae1p is required for growth on ethanol. Mutants lacking both enzymes could be rescued by addition of alanine or pyruvate to ethanol cultures. Disruption of MAE1 alone did not result in a clear phenotype. Regulation of MAE1 was studied by determining enzyme activities and MAE1 mRNA levels in wild-type cultures and by measuring β-galactosidase activities in a strain carrying a MAE1::lacZ fusion. Both in shake flask cultures and in carbon-limited chemostat cultures, MAE1 was constitutively expressed. A three- to fourfold induction was observed during anaerobic growth on glucose. Subcellular fractionation experiments indicated that malic enzyme in S. cerevisiae is a mitochondrial enzyme. Its regulation and localization suggest a role in the provision of intramitochondrial NADPH or pyruvate under anaerobic growth conditions. However, since null mutants could still grow anaerobically, this function is apparently not essential.     

Gene Dosage and the Expression of Electrophoretic Patterns in Heteroploid Mouse Cell Lines

Spontaneously transformed mouse cell lines heterozygous for electrophoretic markers have been studied to determine the relationship between gene dosage and phenotype. It is shown that a clone with an electrophoretic pattern for glucosephosphate isomerase of three bands in a ratio of 4A:4AB:1B contains three copies of chromosome 7, which carries the gene for this enzyme. A clone from a different line with a pattern of three bands in a ratio of 1A:6AB:9B for NADP-isocitrate dehydrogenase has four copies of the chromosome carrying this gene or three copies plus a rearrangement which apparently involves this chromosome. These results show that all of the alleles for each enzyme are expressed to an equal extent in these cells.     

pH Effects on the Activity and Regulation of the NAD Malic Enzyme

The NAD malic enzyme shows a pH optimum of 6.7 when complexed to Mg²⁺ and NAD⁺ but shifts to 7.0 when the catalytically competent enzyme-substrate (E-S) complex forms upon binding malate⁻². This is characteristic of an induced conformational change. The slope of the V_max or V_max/K_m profiles is steeper on the alkaline side of the pH optimum. The K_m for malate increases markedly under alkaline conditions but is not greatly affected by pH values below the optimum. The loss of catalysis on the acidic side is due to protonation of a single residue, pK 5.9, most likely histidine. Photooxidation inactivation with methylene blue showed that a histidine is required for catalytic activity. The location of this residue at or near the active site is revealed by the protection against inactivation offered by malate. Three residues, excluding basic residues such as lysine (which have also been shown to be vital for catalytic activity, must be appropriately ionized for malate decarboxylation to proceed optimally. Two of these residues directly participate in the binding of substrates and are essential for the decarboxylation of malate. A pK of 7.6 was determined for the two residues required by the E-S complex to achieve an active state, this composite value representing both histidine and cysteine suggests that both have decisive roles in the operation of the enzyme. A major change in the enzyme takes place as protonation nears the pH optimum, this is recorded as a change in the enzyme's intrinsic affinity for malate (K_{m pH6.7} = 9.2 millimolar, K_{m pH7.7} = 28.3 millimolar). Similar changes in K_m have been observed for the NAD malic enzyme as it shifts from dimer to tetramer. It is most likely that the third ionizable group (probably a cysteine) revealed by the V_max/K_m profile is needed for optimal activity and is involved in the association-dissociation behavior of the enzyme.     

Diversity of Enzyme Electrophoretic Patterns in the Eggplant Complex

Studies on electrophoretic patterns of five enzymes, aspartate aminotransferase (AAT), glucose-phosphate isomerase (GPI), glutamate dehydrogenase (GDH), shikimate dehydrogenase (SKDH) and triose-phosphate isomerase (TPI) resulted in the identification of 17 phenotypes in 21 accessions of Solanum melongena L, S. insanum L and S. incanum L. The results provided evidence of 10 isozyme loci represented by 20 alleles involved in the control of the above enzymes. GPI and AAT showed high number of phenotypes, while GDHpresented a single morpho The most frequent phenotypes were identical in S. melongena, S. insanum and S. incanum indicating close genetic proximity of the three taxa. S. melongena accessions showed high degree of zymogram homogeneity, while the other two species were more diverse.     

红冬孢酵母苹果酸酶基因RKME1的克隆与表达

苹果酸酶(malic enzyme,ME)普遍存在于各种生物体中,在二价阳离子(Mg~(2+)或者Mn~(2+))的存在下,它可以催化L-苹果酸进行氧化脱羧反应,产生丙酮酸、CO_2和NADPH。前期分析结果预测红冬孢酵母YM25235菌株具有2个苹果酸酶同功酶基因RKME1和RKME2,而且RKME1基因在15℃低温条件下mRNA转录水平显著提高。为了验证RKME1的结构与功能,以红冬孢酵母YM25235 cDNA为模板,PCR扩增得到大小为1 623 bp的开放阅读框,共编码540个氨基酸。序列分析结果显示该序列含有苹果酸酶保守的4个结构域(Ⅰ~Ⅳ),同源建模结果显示该序列的三级结构和蛔虫中的苹果酸酶晶体结构有较高相似性且高度保守。进一步将RKME1插入到载体pET32a(+)中构建重组表达质粒pET32a-RKME1,然后导入大肠杆菌BL21中进行表达。经IPTG诱导,获得了相对分子质量约为70 kD的蛋白质条带。将该蛋白质进行镍柱亲和层析纯化后,酶活分析的结果表明,重组表达的RKME1蛋白可催化苹果酸脱氢生成丙酮酸,同时生成NADPH,酶活为160 U/mg以上。上述结果表明,RKME1是一个新的苹果酸酶基因,这为深入研究苹果酸酶与红冬孢酵母低温条件下生长适应性之间的关系奠定了基础。     

Patterns of Gene Duplication in Lepidopteran Pheromone Binding Proteins

We have isolated and characterized cDNAs representing two distinct pheromone binding proteins (PBPs) from the gypsy moth, Lymantria dispar. We use the L. dispar protein sequences, along with other published lepidopteran PBPs, to investigate the evolutionary relationships among genes within the PBP multigene family. Our analyses suggest that the presence of two distinct PBPs in genera representing separate moth superfamilies is the result of relatively recent, independent, gene duplication events rather than a single, ancient, duplication. We discuss this result with respect to the biochemical diversification of moth PBPs. Received: 19 March 1997 / Accepted: 11 July 1997     

Slow Transients in the Activity of the NAD Malic Enzyme from Crassula

The NAD malic enzyme from Crassula argentea shows a slow reaction transient in the form of a lag before reaching a steady-state rate in assays. This lag, which has a half-time or τ ranging from seconds to many minutes under various conditions, poses problems in the interpretation of kinetic data with this enzyme. The NAD malic enzyme from Kalanchoë daigremontiana has a similar lag.     

Cloning of the Malic Enzyme Gene from Corynebacterium glutamicum and Role of the Enzyme in Lactate Metabolism

Malic enzyme is one of at least five enzymes, known to be present in Corynebacterium glutamicum, capable of carboxylation and decarboxylation reactions coupling glycolysis and the tricarboxylic acid cycle. To date, no information is available concerning the physiological role of the malic enzyme in this bacterium. The malE gene from C. glutamicum has been cloned and sequenced. The protein encoded by this gene has been purified to homogeneity, and the biochemical properties have been established. Biochemical characteristics indicate a decarboxylation role linked to NADPH generation. Strains of C. glutamicum in which the malE gene had been disrupted or overexpressed showed no detectable phenotype during growth on either acetate or glucose, but showed a significant modification of growth behavior during lactate metabolism. The wild type showed a characteristic brief period of exponential growth on lactate followed by a linear growth period. This growth pattern was further accentuated in a malE-disrupted strain (ΔmalE). However, the strain overexpressing malE maintained exponential growth until all lactate had been consumed. This strain accumulated significantly larger amounts of pyruvate in the medium than the other strains.     

Patterns of Gene Duplication and Their Contribution to Expansion of Gene Families in Grapevine

Grapevine is an important fruit crop that has undergone a long history of evolution. Analysis of the whole genome sequence of grapevine has revealed presence of an early palaeo-hexaploid along with three complements. Thus, gene duplication and genome expansion are common in this genome. In this study, we identified 17,922 duplicated genes in the whole grapevine genome. Among these, 2,039; 628; 1,428; 722; and 2,942 were identified respectively as produced by genome-wide, tandem, proximal, retrotransposed, and DNA-based transposed duplications. Analyses of the evolutionary patterns for different types of duplication using non-synonymous and synonymous substitution rates uncovered a series of underlying rules. Thereafter, all the grapevine genes were classified into families, and the contributions of different types of duplication to the expansion of large families were revealed. No duplication type was solely responsible for the formation of any large gene family, but some families showed enrichment of a special type of duplication. On the basis of this study, we believe that uncovering the underlying rules for gene duplications, expansions of gene families, and their evolutionary styles will contribute significantly to a comprehensive understanding of the features of the grapevine genome.     

Gene Activity Patterns and Cellular Differentiation

Puffing in giant chromosomes ot Diptera is considered to reflectthe pattern of active gene loci in these chromosomes. In anyone tissue only a relatively small portion of the total bands(about 10 to 20%) have been observed to form a puff at sometime or another in larval development. These patterns of "potentiallyactive" loci are tissue specific, though greatly overlapping.The actual rate of activity at these loci is controlled independentlyof each other and independently in each tissue by factors ofthe extranuclear metabolism. Puffing at some loci seems to berelated to specific cellular functions, such as secretion ofthe salivary glands. The activity of others may be related tomore basic metabolic processes. In relation to larval development,puffing patterns may change with changing cell functions orwith developmental processes in the cells themselves. In salivaryglands ofChironomus activity of DNAase and of acid phosphataseseems to change in relation to cell breakdown at the end ofthe pupal molt. Changes of acid phosphatase activity begin earlyin the last larval ins tar, but the enzyme is bound to lysosomesuntil metamorphosis. This suggests that the genes specificallyactive during metamorphosis have to interact with a longtermcontrol-system of development. The induction of metamorphosisis a sequential process, gene activations being among the firststeps in this sequence. The activation of these genes by ecdysoneis independent of protein synthesis. It is only the reactionof these genes that leads to the subsequent events in the cell,including the subsequent puff activations. This is shown bythe fact that they depend on early RNA synthesis as well ason protein synthesis. These results on puffing are discussedwith regard to the general problem of the relationships betweenpatterns of gene activity and differentiation.     

Isopentenyl-Diphosphate Isomerases in Human and Mouse: Evolutionary Analysis of a Mammalian Gene Duplication

Isopentenyl diphosphate isomerase (IDI) activates isopentenyl diphosphate (IPP) for polymerization by converting it to its highly nucleophilic isomer dimethylallyl diphosphate (DMAPP). In plants, this central reaction of isoprenoid biosynthesis is catalyzed by various highly conserved isozymes that differ in expression pattern and subcellular localization. Here we report the identification of an IDI duplication in mammals. In contrast to the situation in plants, only one of the two isoforms (IDI1) is highly conserved, ubiquitously expressed and most likely responsible for housekeeping isomerase activity. The second isoform (IDI2) is much more divergent. We demonstrate that after the initial duplication IDI2 underwent a short phase of apparently random change, during which its active center became modified. Afterwards, IDI2 was exapted for a novel function and since then has been under strong purifying selection for at least 70 million years. Molecular modeling shows that the modified IDI2 is still likely to catalyze the isomerization of IPP to DMAPP. In humans, IDI2 is expressed at high levels only in skeletal muscle, where it may be involved in the specialized production of isoprenyl diphosphates for the posttranslational modification of proteins. The significant positive fitness effect of IDI2, revealed by the pattern of sequence conservation, as well as its specific expression pattern underscores the importance of the IDI gene duplication in mammals.     

热休克对小白鼠组织中抗氧化酶活性的影响

热休克诱导小白鼠心、肝、肾、肺、脑等组织细胞SOD活性非常显著或显著升高,其中主要诱导CuZn-SOD的生物合成;42℃诱导小白鼠各组织细胞CAT活性极显著下降,正常情况下,脑细胞几乎不存在CAT活性,42℃热休克20min诱导脑CAT活力升高;热休克亦诱导各组织cSH-Px活性变化,心、脑细胞GSH-Px极显著升高,对肺的影响较小。SOD、CAT、GSH-Px协同作用,维持机体正常生理机能。还探讨了热休克应答、热耐受能力的获得与抗氧化酶活性变化存在的对应关系。     

Utilization of Oxalacetate by Acinetobacter calcoaceticus: Evidence for Coupling Between Malic Enzyme and Malic Dehydrogenase

Growth of Acinetobacter calcoaceticus strain BD413 in malate-mineral medium resulted in the excretion of large quantities of oxalacetate. Malate was virtually depleted by the time the cell density reached 60% of its final value; most of the remaining growth took place at the expense of oxalacetate. Experiments in which oxalacetate was used as the initial substrate showed that pyruvate was not utilized until most of the oxalacetate disappeared. The generation time for growth on malate or oxalacetate was approximately 40 min; the generation time for growth on pyruvate was 62 min, which implies that pyruvate transport may be rate limiting. Oxalacetate and pyruvate, however, supported approximately the same growth yield. These observations suggested that the first step in the utilization of oxalacetate as an energy source consisted of an enzymatic decarboxylation of the keto acid to pyruvate and CO(2). Three enzyme reactions that carry out this decarboxylation have been detected in extracts of A. calcoaceticus. The first, which functioned maximally at pH 4.8, was attributable to the oxalacetate decarboxylase activity of oxidized diphosphopyridine nucleotide-malic enzyme. The second and third, which functioned in the neutral pH range, resulted from coupling of oxidized diphosphopyridine nucleotide-malic enzyme to reduced diphosphopyridine nucleotide-dependent malic dehydrogenase, and oxidized triphosphopyridine nucleotide-malic enzyme to a reduced triphosphopyridine nucleotide-dependent malic dehydrogenase. The efficiency of these coupled reactions was high enough so that the overall reaction could be physiologically significant.     

Subcellular Localization of Folate Turnover and Folate-Dependent Enzyme Activity in Mouse Brain

Abstract: Using 5-[methyl-¹⁴C]methyltetrahydrofolate and 5-[2-¹⁴C]methyl-tetrahydrofolate, the rates of uptake and turnover of the methyl and folate moieties in mouse brain were compared. The methyl moiety was taken up and retained more readily than the folate moiety. Intracerebral injections of labeled folate were used to study the retention and subcellular turnover of folate. The injected folate exchanged slowly with the endogenous pool, stabilizing after 2 days. Analyses of the subcellular distribution of label with time showed that folate underwent intercompartmental exchange in all fractions except the synaptosomal particulate fractions, which appeared to contain a folate pool inert to exchange. The mitochondria] and synaptosomal soluble fractions exhibited a comparatively high content of labeled folate. The folate turnover rate was extremely slow. Subcellular localization of two folate-dependent enzymes showed that /V5, Arlo-methylenetetrahydrofolate reductase and N ⁵-methyltetrahydrofolate homocysteine methyltransferase activities were largely soluble, the latter exhibiting a very high specific activity in the synaptosomal lysate. The implications of these findings with respect to one-carbon metabolism and the compartmentalization of folate function in mouse brain are discussed.     

Increase of Specific Activity, Electrophoretic Type-Transition and Gene Expression of Alkaline Phosphatase during Endodermal Differentiation of F9 Mouse Embryonal Carcinoma Cells

In F9 mouse embryonal carcinoma cells, the specific activity of alkaline phosphatase (ALPase) increases markedly during endodermal differentiation induced by retinoic acid (RA) treatment, but the specific 5'-nucleotidase activity of a similar ecto-phosphatase increases only temporally. Polyacrylamide disc gel electrophoresis showed that F9 cells express only type I ALPase, whereas RA-treated F9 cells express both type I and type II ALPases. Type II ALPase is a minor form on day 1 of RA treatment and becomes the major form on day 4. RA-treated F9 cells also expressed mRNAs for endoderm cell-specific molecules, such as α-fetoprotein, type IV collagen and laminin B1 chain, but their expression of M₂-type pyruvate kinase mRNA of an essential non-ectoenzyme remains constant throughout endodermal differentiation. Northern blot analyses showed that type I ALPase was encoded by a liver (L)/bone (B)/kidney (K)/placenta (P)-type mRNA. The expression of L/B/K/P-type ALPase mRNA was induced in RA-treated F9 cells, but its increase preceded that of ALPase specific activity. These results suggest that the expression of L/B/K-type ALPase is regulated at the translational and/or post-translational level. The differential inhibition of ALPases by L-phenylalanine/L-homoarginine and the thermal inactivation (56°C for 60 min) inferred that type II ALPase was also an L/B/K-type isozyme.     

小鼠超高硫角蛋白基因启动子的克隆及其表达活性分析

目的筛选在小鼠毛囊中具有高表达活性的内源启动子,为建立小鼠被毛特异表达外源蛋白的转基因技术奠定基础。方法以小鼠基因组为模板,克隆得到超高硫角蛋白基因启动子超高硫角蛋白(UHS),将其分别与pβgal-Basic和pAcGFP1-N1载体连接,构建了重组真核表达载体。采用阳离子脂质体法转染胎鼠组织块,分析其表达活性。结果转染后48 h,在蓝光激发条件下可以检测到绿色荧光蛋白(GFP)在小鼠毛囊区高表达,转染96 h后,表达减弱;此外,转染后48 h,βgal染色结果显示在皮肤块的毛囊区存在蓝色点状区域。结论 UHS启动子在小鼠毛囊中具有表达特异性。     

Utilization of Carbon Substrates, Electrophoretic Enzyme Patterns, and Symbiotic Performance of Plasmid-Cured Clover Rhizobia

Plasmids in Rhizobium spp. are relatively large, numerous, and difficult to cure. Except for the symbiotic plasmid, little is known about their functions. The primary objective of our investigation was to obtain plasmid-cured derivatives of Rhizobium leguminosarum bv. trifolii by using a direct selection system and to determine changes in the phenotype of the cured strains. Three strains of rhizobia were utilized that contained three, four, and five plasmids. Phenotypic effects observed after curing of plasmids indicated that the plasmids were involved in the utilization of adonitol, arabinose, catechol, glycerol, inositol, lactose, malate, rhamnose, and sorbitol and also influenced motility, lipopolysaccharide production, and utilization of nitrate. Specific staining of 26 enzymes electrophoretically separated on starch gels indicated that superoxide dismutase, hexokinase, and carbamate kinase activities were affected by curing of plasmids. Curing of cryptic plasmids also influenced nodulation and growth of plants on nitrogen-deficient media. The alteration in the ability to utilize various substrates after curing of plasmids suggests that the plasmids may encode genes that contribute significantly to the saprophytic competence of rhizobia in soil.     

转解毒酶基因工程菌的构建及酶活性分析

实验构建了能高效降解有机磷、有机氯等四大类农药的转解毒酶基因工程菌。将抗性库蚊解毒酶酯酶B1基因片段引入融合表达载体pThioHisA中,转化入大肠杆菌DH5α,在IPTG诱导下,经过8 h,酯酶B1在大肠杆菌中的获得融合高效表达。研究了工程菌的酶酯活性,重组质粒pThioHisA-B1表达的酯酶融合蛋白具有较高的酯酶B1活性,能高效降解酯酶的特异性底物α-乙酸萘酯(-αNA)和β-乙酸萘酯(-βNA),该工程菌将为农药污染的生物治理提供新手段。