scholarly journals Identification and characterization of the natural electron donor ferredoxin and of FAD as a possible prosthetic group of benzoyl-CoA reductase (dearomatizing), a key enzyme of anaerobic aromatic metabolism

1998 ◽  
Vol 251 (3) ◽  
pp. 946-954 ◽  
Author(s):  
Matthias Boll ◽  
Georg Fuchs
Keyword(s):  
Electron Donor ◽  
Prosthetic Group ◽  
Aromatic Metabolism ◽  
Anaerobic Aromatic Metabolism ◽  
Key Enzyme ◽  
Coa Reductase ◽  
Identification And Characterization

scholarly journals Identification and characterization of Sulfolobus solfataricusD-gluconate dehydratase: a key enzyme in the non-phosphorylated Entner–Doudoroff pathway

2005 ◽  
Vol 387 (1) ◽  
pp. 271-280 ◽  
Author(s):  
Seonghun KIM ◽  
Sun Bok LEE
Keyword(s):  
Molecular Mass ◽  
Gel Filtration ◽  
Maximal Activity ◽  
Genome Database ◽  
Sds Page ◽  
Key Enzyme ◽  
Bivalent Metal Ions ◽  
Ammonium Sulphate Fractionation ◽  
Identification And Characterization

The extremely thermoacidophilic archaeon Sulfolobus solfataricus utilizes D-glucose as a sole carbon and energy source through the non-phosphorylated Entner–Doudoroff pathway. It has been suggested that this micro-organism metabolizes D-gluconate, the oxidized form of D-glucose, to pyruvate and D-glyceraldehyde by using two unique enzymes, D-gluconate dehydratase and 2-keto-3-deoxy-D-gluconate aldolase. In the present study, we report the purification and characterization of D-gluconate dehydratase from S. solfataricus, which catalyses the conversion of D-gluconate into 2-keto-3-deoxy-D-gluconate. D-Gluconate dehydratase was purified 400-fold from extracts of S. solfataricus by ammonium sulphate fractionation and chromatography on DEAE-Sepharose, Q-Sepharose, phenyl-Sepharose and Mono Q. The native protein showed a molecular mass of 350 kDa by gel filtration, whereas SDS/PAGE analysis provided a molecular mass of 44 kDa, indicating that D-gluconate dehydratase is an octameric protein. The enzyme showed maximal activity at temperatures between 80 and 90 °C and pH values between 6.5 and 7.5, and a half-life of 40 min at 100 °C. Bivalent metal ions such as Co2+, Mg2+, Mn2+ and Ni2+ activated, whereas EDTA inhibited the enzyme. A metal analysis of the purified protein revealed the presence of one Co2+ ion per enzyme monomer. Of the 22 aldonic acids tested, only D-gluconate served as a substrate, with Km=0.45 mM and Vmax=0.15 unit/mg of enzyme. From N-terminal sequences of the purified enzyme, it was found that the gene product of SSO3198 in the S. solfataricus genome database corresponded to D-gluconate dehydratase (gnaD). We also found that the D-gluconate dehydratase of S. solfataricus is a phosphoprotein and that its catalytic activity is regulated by a phosphorylation–dephosphorylation mechanism. This is the first report on biochemical and genetic characterization of D-gluconate dehydratase involved in the non-phosphorylated Entner–Doudoroff pathway.

Identification and Characterization of Novel Human Glucose-6-Phosphate Dehydrogenase Inhibitors

2012 ◽  
Vol 18 (3) ◽  
pp. 286-297 ◽  
Author(s):  
Janina Preuss ◽  
Adam D. Richardson ◽  
Anthony Pinkerton ◽  
Michael Hedrick ◽  
Eduard Sergienko ◽  
...  
Keyword(s):  
High Throughput Screening ◽  
Basic Research ◽  
Pentose Phosphate ◽  
Screening Assay ◽  
High Throughput Screening Assay ◽  
Key Enzyme ◽  
Small Molecule Compound ◽  
Glucose 6 Phosphate Dehydrogenase ◽  
Identification And Characterization

Glucose-6-phosphate dehydrogenase (G6PD) is the key enzyme of the pentose phosphate pathway, converting glucose-6-phosphate to 6-phosphoglucono-δ-lactone with parallel reduction of NADP+. Several human diseases, including cancer, are associated with increased G6PD activity. To date, only a few G6PD inhibitors have been available. However, adverse side effects and high IC50 values hamper their use as therapeutics and basic research probes. In this study, we developed a high-throughput screening assay to identify novel human G6PD (hG6PD) inhibitors. Screening the LOPAC (Sigma-Aldrich; 1280 compounds), Spectrum (Microsource Discovery System; 1969 compounds), and DIVERSet (ChemBridge; 49 971 compounds) small-molecule compound collections revealed 139 compounds that presented ≥50% hG6PD inhibition. Hit compounds were further included in a secondary and orthogonal assay in order to identify false-positives and to determine IC50 values. The most potent hG6PD inhibitors presented IC50 values of <4 µM. Compared with the known hG6PD inhibitors dehydroepiandrosterone and 6-aminonicotinamide, the inhibitors identified in this study were 100- to 1000-fold more potent and showed different mechanisms of enzyme inhibition. One of the newly identified hG6PD inhibitors reduced viability of the mammary carcinoma cell line MCF10-AT1 (IC50 ~25 µM) more strongly than that of normal MCF10-A cells (IC50 >50 µM).

scholarly journals Identification and Characterization of Human Activation-Induced ChAT+CD4+ T Cells

2021 ◽  
Author(s):  
L Tarnawski ◽  
AL Gallina ◽  
EJ Kort ◽  
VS Shavva ◽  
Z Zhuge ◽  
...  
Keyword(s):  
T Cells ◽  
No Synthase ◽  
Functional Studies ◽  
Human T Cells ◽  
Single Cell Rna Sequencing ◽  
Key Enzyme ◽  
Muscarinic Ach Receptor ◽  
Identification And Characterization ◽  
High Relative Frequency

AbstractVasodilation is a cornerstone of inflammation physiology. By regulating vasodilation and tissue entry of T cells, CD4+ T lymphocytes expressing choline acetyltransferase (ChAT), a key enzyme for biosynthesis of the vasorelaxant acetylcholine (ACh), critically link immunity with vascular biology in mice. However, the characterization of primary human ChAT+ T cells remained elusive. Here, we identified human ChAT+ T cells and report that ChAT mRNA was induced by activation. Functional studies demonstrated that T cell-derived ACh increased muscarinic ACh-receptor dependent NO-synthase activity and vasorelaxation. Further, single-cell RNA-sequencing revealed ChAT+CD4+ T cells in blood from patients with severe circulatory failure and a high relative frequency of ChAT+CD4+ T cells correlated with better 30-day survival in this cohort. Our findings provide the first insights into ChAT biology in primary human T cells, linking ChAT+ T cells with vasorelaxation as well as survival in a cohort of critically ill patients.

scholarly journals Identification and characterization of a novel gene of grouper iridovirus encoding a purine nucleoside phosphorylase

2004 ◽  
Vol 85 (10) ◽  
pp. 2883-2892 ◽  
Author(s):  
Jing-Wen Ting ◽  
Min-Feng Wu ◽  
Chih-Tung Tsai ◽  
Ching-Chun Lin ◽  
Ing-Cherng Guo ◽  
...  
Keyword(s):  
Purine Nucleoside Phosphorylase ◽  
Enzymic Activity ◽  
Host Cells ◽  
Purine Nucleoside ◽  
Nucleoside Phosphorylase ◽  
Egfp Reporter ◽  
Key Enzyme ◽  
Egfp Fusion Protein ◽  
Identification And Characterization

Purine nucleoside phosphorylase (PNP) is a key enzyme in the purine salvage pathway. It catalyses the reversible phosphorolysis of purine (2′-deoxy)ribonucleosides to free bases and (2′-deoxy)ribose 1-phosphates. Here, a novel piscine viral PNP gene that was identified from grouper iridovirus (GIV), a causative agent of an epizootic fish disease, is reported. This putative GIV PNP gene encodes a protein of 285 aa with a predicted molecular mass of 30 332 Da and shows high similarity to the human PNP gene. Northern and Western blot analyses of GIV-infected grouper kidney (GK) cells revealed that PNP expression increased in cells with time from 6 h post-infection. Immunocytochemistry localized GIV PNP in the cytoplasm of GIV-infected host cells. PNP–EGFP fusion protein was also observed in the cytoplasm of PNP–EGFP reporter construct-transfected GK and HeLa cells. From HPLC analysis, the recombinant GIV PNP protein was shown to catalyse the reversible phosphorolysis of purine nucleosides and could accept guanosine, inosine and adenosine as substrates. In conclusion, this is the first report of a viral PNP with enzymic activity.

scholarly journals The known purified mammalian 2,4-dienoyl-CoA reductases are mitochondrial isoenzymes.

1989 ◽  
Vol 37 (12) ◽  
pp. 1863-1867 ◽  
Author(s):  
E H Hakkola ◽  
H I Autio-Harmainen ◽  
R T Sormunen ◽  
I E Hassinen ◽  
J K Hiltunen
Keyword(s):  
Protein A ◽  
Subcellular Location ◽  
Liver Mitochondria ◽  
Rat Liver Mitochondria ◽  
Affinity Column ◽  
Beta Oxidation ◽  
Key Enzyme ◽  
Coa Reductase ◽  
Monospecific Antibodies

The aim of this work was to determine the subcellular location of mammalian 2,4-dienoyl-CoA reductase, a key enzyme for degradation of polyunsaturated fatty acids by beta-oxidation. The enzyme was purified according to Kimura et al. (J Biochem 96:1463, 1984), and antibodies were raised in rabbits. Monospecific antibodies were obtained via purification on an affinity column. Immunoblotting of isolated rat liver mitochondria and peroxisomes with the monospecific reductase antibody showed that the antigen was located only in mitochondria. Immunocytochemical experiments with liver tissue, using the protein A-gold labeling technique, confirmed this result. The similarity of their characteristics suggests that the purified reductases described in the literature are the same isoenzyme. Consequently, since the rat enzyme was localized here to the mitochondria, purification and characterization of peroxisomal mammalian reductases remain to be achieved in the future. In addition, a significant induction also of mitochondrial reductase by clofibrate was observed in the immunoblotting experiments.

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