Maternal effect and embryonic gene expression for lactate dehydrogenase B (LDH-B), glucose-6-phosphate dehydrogenase (G6PDH), and peptidase (PEP-1) during the development of Pleurodeles waltl (urodele amphibian)

The polymorphism of three enzymes [lactate dehydrogenase B (LDH-B), glucose-6-phosphate dehydrogenase, (G6PDH), and peptidase-1 (PEP-1)] in Pleurodeles waltl has allowed the expression of the corresponding loci to be followed during the development of spawnings arising from various crosses. A maternal effect lasting up to the late tail-bud stage (approx. stage 28) was shown for PEP-1. A similar situation was observed for LDH-B and G6PDH. The embryonic alleles present retarded expression: from about stage 28 for PEP-1 and G6PDH and from about stages 22 to 24 (the young tail-bud stage) for LDH-B.     

Ion exchange purification of G6PDH from unclarified yeast cell homogenates using expanded bed adsorption

The use of expanded beds of STREAMLINE ion exchange adsorbents for the direct extraction of an intracellular enzyme glucose-6-phosphate dehydrogenase (G6PDH) from unclarified yeast cell homogenates has been investigated. It has been demonstrated that such crude feedstocks can be applied to the bed without prior clarification steps. The purification of G6PDH from an unclarified yeast homogenate was chosen as a model system containing the typical features of a direct extraction technique. Optimal conditions for the purification were determined in small scale, packed bed experiments conducted with clarified homogenates. Results from these experiments were used to develop a preparative scale separation of G6PDH in a STREAMLINE 50 EBA apparatus. The use of an on-line rotameter for measuring and controlling the height of the expanded bed when operated in highly turbid feedstocks was demonstrated. STREAMLINE DEAE has been shown to be successful in achieving isolation of G6PDH from an unclarified homogenate with a purification factor of 12 and yield of 98% in a single step process. This ion exchange adsorbent is readily cleaned using simple cleaning-in-place procedures without affecting either adsorption or the bed expansion properties of the adsorbent after many cycles of operation. The ability of combining clarification, capture, and purification in a single step will greatly simplify downstream processing flowsheets and reduce the costs of protein purification. (c) 1996 John Wiley & Sons, Inc.     

Integration of mechanical cell disruption and fluidised bed recovery of G3PDH from unclarified disrupted yeast: a comparative study of the performance of unshielded and polymer shielded dye-ligand chromatography systems

The development of a simplified process for the simultaneous disruption and direct selective purification of intracellular proteins from unclarified yeast disruptate has been investigated. The recovery of glyceraldehyde 3-phosphate dehydrogenase (G3PDH) from baker's yeast was selected as a potential demonstration of the generic applicability and practical feasibility of this integrated technique. The application of an adsorbent characterised by high density (UpFront steel-agarose; rho=2.65g ml(-1)) facilitated the combining of cell disruption operation (bead milling of 50% ww/v of yeast suspension at 7.2 lh(-1)) with fluidised bed dye-ligand (Cibacron Blue 3GA) adsorption operated immediately downstream of the disrupter. The adoption of a polymer shielded, dye-ligand technique advanced recovery efficiency. It was demonstrated that G3PDH could be recovered with a yield of 67.5% bound activity and a specific activity of 40.2IU mg(-1), after a single step elution with 0.15M NaCl. The generic application of this approach has been evaluated.     

Expanded bed affinity chromatography of dehydrogenases from bakers' yeast using dye-ligand perfluoropolymer supports

Malate dehydrogenase (MDH) and glucose 6-phosphate dehydrogenase (G6PDH) have been partially purified from preparations of homogenized yeast cells using Procion Yellow H-E3G and Procion Red H-E7B, respectively, immobilized on solid perfluoropolymer supports in an expanded bed. A series of pilot experiments were carried out in small packed beds using clarified homogenate to determine the optimal elution conditions for both MDH and G6PDH. Selective elution of MDH using NADH was effective but the yields obtained were dependent on the concentration of NADH used. Selective elution was found to be most effective when a low concentration of NaCl (0.1 M) was present. MDH could be recovered in 84% yield with a purification factor of 94 when this strategy was adopted. In the case of G6PDH, specific elution using NADP(+) was successful in purifying G6PDH 178-fold in 96% yield. The dynamic capacity of both affinity supports was estimated by frontal analysis, in an expanded bed with unclarified homogenate, and corresponded to 17 U MDH/mL of settled Procion Yellow H-E3G perfluoropolymer support and 7.7 U H6PDH/mL of settled Procion Red H-E7B perfluoropolymer support. Expanded bed affinity chromatography of MDH resulted in an eluted fraction containing 89% of the applied activity with a purification factor of 113. Expanded bed affinity chromatography of G6PDH resulted in an eluted fraction containing 84% of the applied activity with a purification factor of 172. With both enzymes, the overall recovery of enzyme activity was greater than 94%, showing that the expanded bed approach to purification was nondenaturing. (c) 1995 John Wiley & Sons, Inc.     

Ammonium induction of a novel isoform of glucose-6P dehydrogenase in barley roots

The effects of ammonium and glutamine supply on amino acid levels and the activity of glucose-6P dehydrogenase (G6PDH EC 1.1.1.49), the main regulated enzyme of the oxidative pentose phosphate pathway, were investigated in barley roots ( Hordeum vulgare cv. Alfeo). Feeding ammonium to barley plants increased the contents of glutamine, asparagine and G6PDH in roots. These effects were abolished by using inhibitors of glutamine synthetase. Glutamine-fed barley roots showed a similar increase in G6PDH activities to ammonium-fed plants. Two G6PDH enzymes (G6PDH 1 and 2) were partially purified and characterized from ammonium-fed and glutamine-fed roots. The isozymes had different pH optima and apparent Km values for glucose-6P. G6PDH 2 showed similar kinetic parameters to the G6PDH present in root extracts of barley grown without any nitrogen source, while G6PDH 1 exhibited different kinetic parameters, suggesting the appearance of a second G6PDH isoform in response to ammonium. Western blot analysis demonstrated the existence of two G6PDH subunits of different molecular mass in barley roots grown in the presence of ammonium or glutamine, while only one isoform could be detected in roots grown without any nitrogen source. The results suggest a primary role of ammonium and/or glutamine in the appearance of a novel G6PDH isoform; this enzyme (G6PDH 1) shows kinetic parameters similar to those measured previously for chloroplastic and plastidic isoforms and seems to be induced by changes in glutamine content or a related compound(s) in the roots.     

Metabolic background for establishment of the subpopulation structure in early ontogenesis in the Atlantic salmon (the case of energy of carbohydrate metabolism)

The activity of some enzymes involved in energy and carbohydrate metabolism was studied in Atlantic salmon embryos at the eyed egg stage and in salmon fingerlings (0+) from two trophic–ecological groups: the Varzuga River bed and two tributaries, the Pyatka and Sobachii rivers (Kola Peninsula). It has been demonstrated that heterogeneity of embryos was most evident in the case of cytochrome c oxidase (CO), malate dehydrogenase (MDH), glycerol-1-phosphate dehydrogenase (G1PDH), and glucose-6-phosphate dehydrogenase (G6PDH), while the lowest level of heterogeneity was observed for lactate dehydrogenase (LDH) and aldolase. A positive correlation was revealed between the activities of CO, LDH, MDH, and G1PDH. It was noted that G6PDH showed a negative correlation with almost all enzymes under study. It was found that salmon juveniles inhabiting the tributaries were characterized by high LDH, aldolase, and G1PDH activity and lower activity of G6PDH compared to the juveniles inhabiting the main river bed. Notably, the differences in the activity of the enzymes involved in aerobic metabolism between the two groups of fingerlings under analysis were observed only in the autumn.     

米曲霉6-磷酸葡萄糖脱氢酶基因gsdA的克隆及生物信息学分析

6-磷酸葡萄糖脱氢酶催化6-磷酸葡萄糖生成6-磷酸葡萄糖酸,并生成NADPH,是微生物胞内磷酸戊糖途径（PPP）的关键酶。本研究以食品安全菌米曲霉CICC2012为材料,克隆获得6-磷酸葡萄糖脱氢酶基因(GenBank登录号：JN123468)。序列分析表明,该酶是由222个氨基酸组成的亲水性蛋白;128～134位氨基酸序列DHYLGKE为活性区域;170～176位氨基酸序列GTEGRGG可能为辅因子结合位点。进化树分析表明,米曲霉6-磷酸葡萄糖脱氢酶同其他丝状真菌及酵母的G6PDH较相似。     

Ancestral lipid biosynthesis and early membrane evolution

Archaea possess unique membrane phospholipids that generally comprise isoprenoid ethers built on sn-glycerol-1-phosphate (G1P). By contrast, bacterial and eukaryal membrane phospholipids are fatty acid esters linked to sn-glycerol-3-phosphate (G3P). The two key dehydrogenase enzymes that produce G1P and G3P, G1PDH and G3PDH, respectively, are not homologous. Various models propose that these enzymes originated during the speciation of the two prokaryotic domains, and the nature (and even the very existence) of lipid membranes in the last universal common ancestor (cenancestor) is subject to debate. G1PDH and G3PDH belong to two separate superfamilies that are universally distributed, suggesting that members of both superfamilies existed in the cenancestor. Furthermore, archaea possess homologues to known bacterial genes involved in fatty acid metabolism and synthesize fatty acid phospholipids. The cenancestor seems likely to have been endowed with membrane lipids whose synthesis was enzymatic but probably non-stereospecific.     

Control of autolysis of Bacillus subtilis for selective production of the intracellular enzyme glucose-6-phosphate dehydrogenase in aqueous two-phase systems

A method for the release of intracellular enzyme by autolysis of Bacillus subtilis cells is presented. Both the growth and lysis processes were further applied to aqueous two-phase systems (ATPS). Lysis induced by the addition of Triton X-100 and by low-temperature treatment facilitated the release of cytoplasmic enzyme glucose-6-phosphate dehydrogenase (G6PDH) in ATPS. The release selectivity increased when lysis was regulated by the addition of 50 μM or 100 μM Triton X-100. Cardiolipin efficiently inhibited the autolytic process. Control of the autolytic system promoted the selective release of G6PDH. B. subtilis cells could be grown and lysed in aqueous two-phase systems in a similar fashion to the conventional single-phase medium solutions. The released enzymes were partitioned according to their surface properties. G6PDH were extracted to the top phase in a PEG1540/Dex100K-200K sytem. Cells were partitioned to the bottom phase or the interface, and could be recycled into the fermentor. The selectivity of enzyme production was also increased in two-phase systems by the addition of cardiolipin.     

Selection of developmentally competent oocytes through brilliant cresyl blue stain enhances blastocyst development rate after bovine nuclear transfer

The aim of the present investigation was to study the effect of oocyte selection on the efficiency of bovine nuclear transfer in terms of increased blastocyst production. For this purpose, prior to in vitro maturation (IVM), oocytes were selected for their developmental competence on the basis of glucose-6-phosphate dehydrogenase (G6PDH) activity indicated by brilliant cresyl blue (BCB) staining. It has been hypothesized that growing oocytes have a higher level of active G6PDH in comparison to the mature oocytes. Compact cumulus oocyte complexes (COCs) were recovered from slaughterhouse-collected bovine ovaries and classified either as control group, which were placed immediately into culture without exposure to BCB stain, or treatment group, which were stained with BCB for 90min before culture. Treated oocytes were then divided into BCB- (colourless cytoplasm, increased G6PDH) and BCB+ (coloured cytoplasm, low G6PDH) based on their ability to metabolize the stain. After IVM, oocytes were subjected to nuclear transfer procedure for the production of cloned embryos which were then cultured for a period of 8 days to determine the blastocyst rate. The BCB+ oocytes yielded a significantly higher blastocyst rate (39%) than the control (21%) or BCB- oocytes (4%). These results show that the staining of bovine cumulus-oocyte complexes with BCB before in vitro maturation could be used to select developmentally competent oocytes for nuclear transfer. In addition, G6PDH activity could prove to be a useful marker for determining the oocyte quality in future.     

高等植物葡萄糖-6-磷酸脱氢酶与6-磷酸葡萄糖酸脱氢酶基因的不同进化起源

葡萄糖-6-磷酸脱氢酶与6-磷酸葡萄糖酸脱氢酶是植物戊糖磷酸途径中的两个酶.在克隆了水稻质体葡萄糖-6-磷酸脱氢酶基因OsG6PDH2和质体6-磷酸葡萄糖脱氢酶基因Os6PGDH2基础上,分析比较了水稻胞质和质体葡萄糖-6-磷酸脱氢酶基因和6-磷酸葡萄糖酸脱氢酶基因的基因结构、表达特性和进化地位.结合双子叶模式植物拟南芥两种酶基因的分析结果,认为高等植物葡萄糖-6-磷酸脱氢酶基因和6-磷酸葡萄糖酸脱氢酶基因在进化方式上截然不同,葡萄糖-6-磷酸脱氢酶的胞质基因与动物和真菌等真核生物具有共同的祖先;6-磷酸葡萄糖酸脱氢酶的胞质酶和质体酶基因都起源于原核生物的内共生.讨论了植物葡萄糖-6-磷酸脱氢酶与6-磷酸葡萄糖酸脱氢酶基因可能的进化模式,为高等植物及质体的进化起源提供了新的资料.     

高等植物葡萄糖-6- 磷酸脱氢酶与6- 磷酸葡萄糖酸脱氢酶基因的不同进化起源

葡萄糖-6-磷酸脱氢酶与6-磷酸葡萄糖酸脱氢酶是植物戊糖磷酸途径中的两个关键酶。在克隆了水稻质体葡萄糖-6-磷酸脱氢酶基因OsG6PDH2和质体6-磷酸葡萄糖脱氢酶基因Os6PGDH2基础上,分析比较了水稻胞质和质体葡萄糖-6-磷酸脱氢酶基因和6-磷酸葡萄糖酸脱氢酶基因的基因结构、表达特性和进化地位。结合双子叶模式植物拟南芥两种酶基因的分析结果,认为高等植物葡萄糖-6-磷酸脱氢酶基因和6-磷酸葡萄糖酸脱氢酶基因在进化方式上截然不同,葡萄糖-6-磷酸脱氢酶的胞质基因与动物和真菌等真核生物具有共同的祖先;6-磷酸葡萄糖酸脱氢酶的胞质酶和质体酶基因都起源于原核生物的内共生。讨论了植物葡萄糖-6-磷酸脱氢酶与6-磷酸葡萄糖酸脱氢酶基因可能的进化模式,为高等植物及质体的进化起源提供了新的资料。     

Development of an expanded bed technique for an affinity purification of G6PDH from unclarified yeast cell homogenates

The use of an expanded bed of STREAMLINE Red H-7B for the purification of the intracellular glycolytic enzyme glucose 6-phosphate dehydrogenase (G6PDH) directly from untreated preparations of disrupted yeast cells has been investigated. Small-scale experiments, carried out in packed beds, have shown that the optimal pH for adsorption is 6.0 and have enabled optimization of elution conditions using a series of eluents. The dynamic capacity of the adsorbent for G6PDH was determined in a small expanded bed to be 28 units/mL. These results were used to develop a preparative scale separation of G6PDH in a STREAMLINE 50 expanded bed column. G6PDH was purified directly from an unclarified yeast homogenate in 99% yield with an average purification factor in the eluted fraction of 103. Cleaning-in-place (CIP) procedures using 0.5 M NaOH and 4M urea in 60% (v/v) ethanol have demonstrated that the adsorbent can be regenerated with no loss of adsorption capacity of alteration of bed expansion characteristics after many cycles of operation. (c) 1995 John Wiley & Sons, Inc.     

The substrate of the glucose-6-phosphate dehydrogenase of Pseudomonas aeruginosa provides structural stability

In general, eukaryotic glucose-6-phosphate dehydrogenases (G6PDHs) are structurally stabilized by NADP⁺. Here we show by spectrofluorometric analysis, thermal and urea denaturation, and trypsin proteolysis, that a different mechanism stabilizes the enzyme from Pseudomonas aeruginosa (PaG6PDH) (EC 1.1.1.363). The spectrofluorometric analysis of the emission of 8-anilino-1-naphthalenesulfonic acid (ANS) indicates that this stabilization is the result of a structural change in the enzyme caused by G6P. The similarity between the K_d values determined for the PaG6PDH-G6P complex (78.0 ± 7.9 μM) and the K_0.5 values determined for G6P (57.9 ± 2.5 and 104.5 ± 9.3 μM in the NADP⁺- and NAD⁺-dependent reactions, respectively) suggests that the structural changes are the result of G6P binding to the active site of PaG6PDH. Modeling of PaG6PDH indicated the residues that potentially bind the ligand. These results and a phylogenetic analysis of the amino acid sequences of forty-four G6PDHs, suggest that the stabilization observed for PaG6PDH could be a characteristic that distinguishes this and other G6PDHs that use NAD⁺ and NADP⁺ from those that use NADP⁺ only or preferentially, such as those found in eukaryotes. This characteristic could be related to the metabolic roles these enzymes play in the organisms to which they belong.     

Some properties of glucose-6-phosphate dehydrogenase from rat and chick brain: a comparative study.

1. Glucose-6-phosphate dehydrogenase (G6PDH) has been purified to homogeneity from rat and chick brain by affinity chromatography with Sepharose bound 2',5' ADP. 2. Some properties of the two enzymes are studied and the effects of hydrogen ion concentration, Mg2+ ions, temperature and urea on the initial rate of enzyme are described. 3. G6PDH from chick brain differs from the rat enzyme in affinity for 2',5' ADP Sepharose, in pH optimum, in heat stability and it is differently affected by Mg2+ ions. No effect is detectable after urea treatment on enzymes from both sources.     

Involvement of G6PDH in heat stress tolerance in the calli from Przewalskia tangutica and Nicotiana tabacum

Glucose-6-phosphate dehydrogenase (G6PDH) has been implicated in supplying reduced nicotine amide cofactors for biochemical reactions and in modulating the redox state of cells. In this study, the role of G6PDH in thermotolerance of the calli from Przewalskia tangutica and tobacco (Nicotiana tabacum L.) was investigated. Results showed that Przewalskia tangutica callus was more sensitive to heat stress than tobacco callus. The activity of G6PDH and antioxidant enzymes (ascorbate peroxidase, catalase, peroxidase and superoxide dismutase) in calli from Przewalskia tangutica and tobacco increased after 40 °C treatment, although two calli exhibited a difference in the degree and timing of response to heat stress. When G6PDH was partially inhibited by glucosamine pretreatment, the antioxidant enzyme activities and thermotolerance in both calli significantly decreased. Simultaneously, the heat-induced H₂O₂ content and the plasma membrane NADPH oxidase activity were also reduced. Application of H₂O₂ increased the activity of G6PDH and antioxidant enzymes in both calli. Diphenylene iodonium, a NADPH oxidase inhibitor, counteracted heatinduced H₂O₂ accumulation and reduced the heat-induced activity of G6PDH and antioxidant enzymes. Moreover, exogenous H₂O₂ was effective in restoring the activity of G6PDH and antioxidant enzymes after glucosamine pretreatment. Western blot analysis showed that G6PDH gene expression in both calli was also stimulated by heat and H₂O₂, and blocked by DPI and glucosamine under heat stress. Taken together, under heat stress G6PDH promoted H₂O₂ accumulation via NADPH oxidase and the elevated H₂O₂ was involved in regulating the activity of antioxidant enzymes, which in turn facilitate to maintain the steady-state H₂O₂ level and protect plants from the oxidative damage.     

磷酸戊糖途径的调节对红豆杉细胞生长及紫杉醇合成的影响

在正常的红豆杉细胞悬浮培养过程,葡萄糖-6-磷酸脱氢酶(G6PDH)活性的变化趋势与生物量的基本相似。而在chitosan处理的细胞中G6PDH活性升高而生物量下降。100 mg·L^-1 chitosan和500mg·L^-1 chitosan均对细胞G6PDH具有诱导作用,且后者的诱导强度较前者的高。乙二醇双2-氨基乙基醚四乙酸(EGTA)的加入降低chitosan对细胞G6PDH的诱导程度,显示chitosan对G6PDH的诱导需要Ca²⁺的参与。谷胱甘肽(GHS)的处理可反馈抑制chitosan对细胞G6PDH的诱导。通过分析调节后G6PDH的各种活性与细胞中紫杉醇产量的关系,认为采用合适的处理方法调节磷酸戊糖途径,有利于红豆杉细胞合成紫杉醇。     

Thioredoxins and the redox modulation of glucose-6-phosphate dehydrogenase in Anabaena sp. strain PCC 7120 vegetative cells and heterocysts.

Glucose-6-phosphate dehydrogenase (G6PDH) was isolated from heterocysts and vegetative cells of Anabaena sp. strain PCC 7120. Both enzyme preparations proved to be more active in their oxidized than in their reduced forms. At least one protein with thioredoxin activity was found in Anabaena sp. which, if reduced with dithiothreitol, deactivated the G6PDH preparations. The deactivated heterocyst G6PDH could be reactivated neither by O2 nor by oxidized thioredoxin. Reactivation of the enzyme was, however, achieved by oxidized glutathione or H2O2. The active form of Anabaena G6PDH was readily deactivated by heterologous thioredoxin(s). The Anabaena thioredoxin(s) modulated heterologous enzymes.