Purification and immunological properties of an NAD(H) dependent glutamate dehydrogenase from soybean cells (Glycine max L.)

Studies were carried out on glutamate dehydrogenase (GDH, EC 1.4.1.2) isolated from the SB1 and SB3 soybean (Glyciene max L. cv. Mandarin) cell cultures. The NAD(H) dependent enzyme from SB1 and SB3 cells was purified to homogeneity, and that from the SB3 cells studied in detail. It was shown to be activated by calcium. The molecular weight of the native enzyme was found to be 263 000 ± 12 000. The molecular weight of the subunits was shown to be 41 000 ± 2000, which indicates that the enzyme has a hexameric structure. Anti-GDH antibodies were produced in rabbits, to GDH purified to homogeneity from both cell cultures. Each antibody preparation reacted with the purified enzyme produced from either cell culture. Antibodies to GDH from SB3 cells were utilized to study the apparent induction of GDH, which occurs when these cells are grown in a medium with ammonium ions as the sole nitrogen source. The increase in GDH activity was shown to be due to de-novo protein synthesis. The anti-SB3-GDH antibody preparation was also tested for cross reactivity with crude GDH preparations from a number of plant sources, and purified GDH from a number of other organisms. The antibody was shown to cross react with a number of the GDH preparations.     

Apparent unbalance between the activities of 6-phosphogluconate and glucose-6-phosphate dehydrogenases in rat liver

The ratio of activities of 6-phosphogluconate dehydrogenase/glucose-6-phosphate dehydrogenase measured in liver extracts of rats in lipogenic nutritional conditions is only 0.2, suggesting an apparent physiological unbalance between the two dehydrogenases of the hexosemonophosphate shunt. This potential unbalance is enhanced by the fact that TPNH is a more powerful competitive inhibitor of 6-phosphogluconate dehydrogenase than of glucose-6-phosphate dehydrogenase. Accordingly, a strong activation of 6-phosphogluconate dehydrogenase would be required for efficient functioning of this pathway, unless there is an alternative outlet for 6-phosphogluconate so far unrecognized in animal tissues.     

Carrier mediated GABA translocation into rat brain mitochondria

GABA added to rat brain mitochondria causes oxidation of intramitochondrial NAD(P)H as well as inducing glutamate efflux from the mitochondrial matrix. The rate of NAD(P)H oxidation shows saturation characteristics, depends on GABA transport across the mitochondrial membrane and is inhibited by non-penetrant compounds and by the metal-complexing agent bathophenanthroline. These results show the existence of a specific GABA carrier. Inhibition studies strongly suggest the existence of two separate binding sites, namely the GABA binding site and the dicarboxylates binding site, as well as suggest the presence of a metal ion (ions) at GABA binding site. The occurrence of a GABA/GLUTAMATE antiport is proposed which allows a cyclical route to account for GABA synthesis and degradation in brain.     

不同条件下两株溶磷菌溶磷量及葡萄糖脱氢酶基因表达与酶活分析北大核心CSCD

【目的】获得大豆根际土壤中溶磷能力较强的菌株,明确在菌株溶磷过程中葡萄糖脱氢酶(GDH)的作用特点及其基因的表达水平。【方法】利用溶磷圈方法分离与纯化溶磷菌株,采用Vitek 2系统和16S r RNA序列分析菌株的分类地位;测定2菌株的溶磷量、GDH活性,并根据GDH基因的保守区序列设计引物,克隆GDH基因,利用实时荧光定量PCR测定不同条件下基因的相对表达量。【结果】筛选出2株具有较强溶磷能力的溶磷菌,分别鉴定为Pseudomonas sp.和Enterobacter sp.,2菌株最高溶磷量分别为558μg/m L和478μg/m L;成功地克隆了2株溶磷菌的GDH基因,片段大小分别为2007 bp和2066 bp;2菌株在不同磷源、不同p H值培养基中GDH活性及基因表达量不同,菌株wj1在高磷条件下基因表达量最高,磷胁迫条件下基因表达量较低,而wj3在不同磷源条件下GDH基因表达量都较低。且GDH基因表达量及酶活的变化与wj3菌株溶磷量没有直接的关系。【结论】从大豆根际土壤中分离获得溶磷能力较强的菌株Pseudomonas sp.wj1和Enterobacter sp.Wj3,GDH活性及基因表达在2株菌溶磷过程中具有不同的作用特点,2菌株溶磷机制不完全相同。     

Asymmetric synthesis of (S)-ethyl-4-chloro-3-hydroxy butanoate using a Saccharomyces cerevisiae reductase: Enantioselectivity and enzyme–substrate docking studies

Ethyl (S)-4-chloro-3-hydroxy butanoate (ECHB) is a building block for the synthesis of hypercholesterolemia drugs. In this study, various microbial reductases have been cloned and expressed in Escherichia coli. Their reductase activities toward ethyl-4-chloro oxobutanoate (ECOB) have been assayed. Amidst them, Baker's yeast YDL124W, YOR120W, and YOL151W reductases showed high activities. YDL124W produced (S)-ECHB exclusively, whereas YOR120W and YOL151W made (R)-form alcohol. The homology models and docking models with ECOB and NADPH elucidated their substrate specificities and enantioselectivities. A glucose dehydrogenase-coupling reaction was used as NADPH recycling system to perform continuously the reduction reaction. Recombinant E. coli cell co-expressing YDL124W and Bacillus subtilis glucose dehydrogenase produced (S)-ECHB exclusively.     

Mitochondrial free fatty acid β-oxidation supports oxidative phosphorylation and proliferation in cancer cells

Oxidative phosphorylation (OxPhos) is functional and sustains tumor proliferation in several cancer cell types. To establish whether mitochondrial β-oxidation of free fatty acids (FFAs) contributes to cancer OxPhos functioning, its protein contents and enzyme activities, as well as respiratory rates and electrical membrane potential (ΔΨm) driven by FFA oxidation were assessed in rat AS-30D hepatoma and liver (RLM) mitochondria. Higher protein contents (1.4–3 times) of β-oxidation (CPT1, SCAD) as well as proteins and enzyme activities (1.7–13-times) of Krebs cycle (KC: ICD, 2OGDH, PDH, ME, GA), and respiratory chain (RC: COX) were determined in hepatoma mitochondria vs. RLM. Although increased cholesterol content (9-times vs. RLM) was determined in the hepatoma mitochondrial membranes, FFAs and other NAD-linked substrates were oxidized faster (1.6–6.6 times) by hepatoma mitochondria than RLM, maintaining similar ΔΨm values. The contents of β-oxidation, KC and RC enzymes were also assessed in cells. The mitochondrial enzyme levels in human cervix cancer HeLa and AS-30D cells were higher than those observed in rat hepatocytes whereas in human breast cancer biopsies, CPT1 and SCAD contents were lower than in human breast normal tissue. The presence of CPT1 and SCAD in AS-30D mitochondria and HeLa cells correlated with an active FFA utilization in HeLa cells. Furthermore, the β-oxidation inhibitor perhexiline blocked FFA utilization, OxPhos and proliferation in HeLa and other cancer cells. In conclusion, functional mitochondria supported by FFA β-oxidation are essential for the accelerated cancer cell proliferation and hence anti-β-oxidation therapeutics appears as an alternative promising approach to deter malignant tumor growth.     

Metabolic flux analysis of CHO cells at growth and non-growth phases using isotopic tracers and mass spectrometry

Chinese hamster ovary (CHO) cells are the main platform for production of biotherapeutics in the biopharmaceutical industry. However, relatively little is known about the metabolism of CHO cells in cell culture. In this work, metabolism of CHO cells was studied at the growth phase and early stationary phase using isotopic tracers and mass spectrometry. CHO cells were grown in fed-batch culture over a period of six days. On days 2 and 4, [1,2-¹³C] glucose was introduced and the labeling of intracellular metabolites was measured by gas chromatography-mass spectrometry (GC–MS) at 6, 12 and 24 h following the introduction of tracer. Intracellular metabolic fluxes were quantified from measured extracellular rates and ¹³C-labeling dynamics of intracellular metabolites using non-stationary ¹³C-metabolic flux analysis (¹³C-MFA). The flux results revealed significant rewiring of intracellular metabolic fluxes in the transition from growth to non-growth, including changes in energy metabolism, redox metabolism, oxidative pentose phosphate pathway and anaplerosis. At the exponential phase, CHO cell metabolism was characterized by a high flux of glycolysis from glucose to lactate, anaplerosis from pyruvate to oxaloacetate and from glutamate to α-ketoglutarate, and cataplerosis though malic enzyme. At the stationary phase, the flux map was characterized by a reduced flux of glycolysis, net lactate uptake, oxidative pentose phosphate pathway flux, and reduced rate of anaplerosis. The fluxes of pyruvate dehydrogenase and TCA cycle were similar at the exponential and stationary phase. The results presented here provide a solid foundation for future studies of CHO cell metabolism for applications such as cell line development and medium optimization for high-titer production of recombinant proteins.     

Dimeric Crystal Structure of Rabbit l-Gulonate 3-Dehydrogenase/λ-Crystallin: Insights into the Catalytic Mechanism

l-Gulonate 3-dehydrogenase (GDH) is a bifunctional dimeric protein that functions not only as an NAD⁺-dependent enzyme in the uronate cycle but also as a taxon-specific λ-crystallin in rabbit lens. Here we report the first crystal structure of GDH in both apo form and NADH-bound holo form. The GDH protomer consists of two structural domains: the N-terminal domain with a Rossmann fold and the C-terminal domain with a novel helical fold. In the N-terminal domain of the NADH-bound structure, we identified 11 coenzyme-binding residues and found 2 distinct side-chain conformers of Ser124, which is a putative coenzyme/substrate-binding residue. A structural comparison between apo form and holo form and a mutagenesis study with E97Q mutant suggest an induced-fit mechanism upon coenzyme binding; coenzyme binding induces a conformational change in the coenzyme-binding residues Glu97 and Ser124 to switch their activation state from resting to active, which is required for the subsequent substrate recruitment. Subunit dimerization is mediated by numerous intersubunit interactions, including 22 hydrogen bonds and 104 residue pairs of van der Waals interactions, of which those between two cognate C-terminal domains are predominant. From a structure/sequence comparison within GDH homologues, a much greater degree of interprotomer interactions (both polar and hydrophobic) in the rabbit GDH would contribute to its higher thermostability, which may be relevant to the other function of this enzyme as λ-crystallin, a constitutive structural protein in rabbit lens. The present crystal structures and amino acid mutagenesis studies assigned the role of active-site residues: catalytic base for His145 and substrate binding for Ser124, Cys125, Asn196, and Arg231. Notably, Arg231 participates in substrate binding from the other subunit of the GDH dimer, indicating the functional significance of the dimeric state. Proper orientation of the substrate-binding residues for catalysis is likely to be maintained by an interprotomer hydrogen-bonding network of residues Asn196, Gln199, and Arg231, suggesting a network-based substrate recognition of GDH.     

Involvement of GDH3-encoded NADP+-dependent Glutamate Dehydrogenase in Yeast Cell Resistance to Stress-induced Apoptosis in Stationary Phase Cells

Glutamate metabolism is linked to a number of fundamental metabolic pathways such as amino acid metabolism, the TCA cycle, and glutathione (GSH) synthesis. In the yeast Saccharomyces cerevisiae, glutamate is synthesized from α-ketoglutarate by two NADP⁺-dependent glutamate dehydrogenases (NADP-GDH) encoded by GDH1 and GDH3. Here, we report the relationship between the function of the NADP-GDH and stress-induced apoptosis. Gdh3-null cells showed accelerated chronological aging and hypersusceptibility to thermal and oxidative stress during stationary phase. Upon exposure to oxidative stress, Gdh3-null strains displayed a rapid loss in viability associated with typical apoptotic hallmarks, i.e. reactive oxygen species accumulation, nuclear fragmentation, DNA breakage, and phosphatidylserine translocation. In addition, Gdh3-null cells, but not Gdh1-null cells, had a higher tendency toward GSH depletion and subsequent reactive oxygen species accumulation than did WT cells. GSH depletion was rescued by exogenous GSH or glutamate. The hypersusceptibility of stationary phase Gdh3-null cells to stress-induced apoptosis was suppressed by deletion of GDH2. Promoter swapping and site-directed mutagenesis of GDH1 and GDH3 indicated that the necessity of GDH3 for the resistance to stress-induced apoptosis and chronological aging is due to the stationary phase-specific expression of GDH3 and concurrent degradation of Gdh1 in which the Lys-426 residue plays an essential role.