Purification and properties of 6-phosphogluconate dehydrogenase from sheep liver.
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ABSTRACT:
SUBMITTER: Villet RH
PROVIDER: S-EPMC1185188 | biostudies-other | 1969 Dec
REPOSITORIES: biostudies-other
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Project description:1. 6-Phosphogluconate dehydrogenase from rabbit mammary gland was purified to homogeneity by the criterion of polyacrylamide-gel electrophoresis in the presence of sodium dodecyl sulphate. The molecular weight of the subunit is 52 000. The enzyme was purified 150-fold with a final specific activity of 20 mumol of NADP+ reduced/min per mg of protein and overall yield of 3%. The molecular weight of the native enzyme is estimated to be 104 000 from gel-filtration studies. The final purification step was carried out by affinity chromatography with NADP+-Sepharose. 2. The Km values for 6-phosphogluconate and NADP+ are approx. 54 muM and 23 muM respectively. 3. Citrate and pyrophosphate are competitive inhibitors of the enzyme with respect to both 6-phosphogluconate and NADP+. 4. MgCl2 affects the apparent Km for NADP+ at saturating concentrations of 6-phosphogluconate.
| S-EPMC1172355 | biostudies-other
Project description:Two crystal structures of recombinant Geobacillus stearothermophilus 6-phosphogluconate dehydrogenase (Gs6PDH) in complex with the substrate 6-phosphogluconate have been determined at medium resolution. Gs6PDH shares significant sequence identity and structural similarity with the enzymes from Lactococcus lactis, sheep liver and the protozoan parasite Trypanosoma brucei, for which a range of structures have previously been reported. Comparisons indicate that amino-acid sequence conservation is more pronounced in the two domains that contribute to the architecture of the active site, namely the N-terminal and C-terminal domains, compared with the central domain, which is primarily involved in the subunit-subunit associations required to form a stable dimer. The active-site residues are highly conserved, as are the interactions with the 6-phosphogluconate. There is interest in 6PDH as a potential drug target for the protozoan parasite T. brucei, the pathogen responsible for African sleeping sickness. The recombinant T. brucei enzyme has proven to be recalcitrant to enzyme-ligand studies and a surrogate protein might offer new opportunities to investigate and characterize 6PDH inhibitors. The high degree of structural similarity, efficient level of expression and straightforward crystallization conditions mean that Gs6PDH may prove to be an appropriate model system for structure-based inhibitor design targeting the enzyme from Trypanosoma species.
| S-EPMC2675582 | biostudies-literature
Project description:1. The activity of phosphofructokinase in sheep liver was found to be dependent on the composition and molarity of the buffer used in extraction. Under optimum conditions a value of 4-7mumoles/min./g. wet wt. of tissue was obtained. 2. The enzyme was purified 480-fold by a combination of ammonium sulphate fractionation, heat treatment in the presence of ethanol, DEAE-cellulose chromatography and Sephadex G-200 gel filtration. The final specific activity was 18.5mumoles/min./mg. of protein. 3. The purified enzyme was inhibited by ATP and citrate, the degree of inhibition depending on the concentration of fructose 6-phosphate, magnesium chloride and ammonium sulphate, as well as on the pH. ATP and citrate inhibition was overcome by AMP and fructose 1,6-diphosphate. 4. The enzyme was also inhibited by NADH and NADPH in a manner largely independent of other components of the assay medium. AMP and fructose 1,6-diphosphate were not able to overcome this type of inhibition. 5. Octanoate was not an inhibitor of phosphofructokinase. 6. Differences between these results and those of other workers are discussed.
| S-EPMC1184628 | biostudies-other
Project description:[2-18O]Ribulose 5-phosphate was prepared and shown to be converted enzymically by 6-phosphogluconate dehydrogenase from sheep liver into 6-phosphogluconate with complete retention of the heavy isotope. This finding unequivocally excludes the possibility of a Schiff-base mechanism for the enzyme. The involvement of metal ions has already been excluded, and other possible mechanisms are discussed. The enzyme was purified by an improved large-scale procedure, which is briefly described.
| S-EPMC1146624 | biostudies-other
Project description:Aldehyde dehydrogenase from sheep liver mitochondria was purified to homogeneity as judged by electrophoresis on polyacrylamide gels, and by sedimentation-equilibrium experiments in the analytical ultracentrifuge. The enzyme has a molecular weight of 198000 and a subunit size of 48000, indicating that the molecule is a tetramer. Fluorescence and spectrophotometric titrations indicate that each subunit can bind 1 molecule of NADH. Enzymic activity is completely blocked by reaction of 4mol of 5,5'-dithiobis-(2-nitrobenzoate)/mol of enzyme. Excess of disulfiram or iodoacetamide decreases activity to only 50% of the control value, and only two thiol groups per molecule are apparently modified by these reagents.
| S-EPMC1164692 | biostudies-other
Project description:The kinetics of the NAD+-dependent oxidation of aldehydes, catalysed by aldehyde dehydrogenase purified from sheep liver mitochondria, were studied in detail. Lag phases were observed in the assays, the length of which were dependent on the enzyme concentration. The measured rates after the lag phase was over were directly proportional to the enzyme concentration. If enzyme was preincubated with NAD+, the lag phase was eliminated. Double-reciprocal plots with aldehyde as the variable substrate were non-linear, showing marked substrate activation. With NAD+ as the variable substrate, double-reciprocal plots were linear, and apparently parallel. Double-reciprocal plots with enzyme modified with disulfiram (tetraethylthiuram disulphide) or iodoacetamide, such that at pH 8.0 the activity was decreased to 50% of the control value, showed no substrate activation, and the plots were linear. At pH 7.0, the kinetic parameters Vmax. and Km NAD+- for the oxidation of acetaldehyde and butyraldehyde by the native enzyme are almost identical. Formaldehyde and propionaldehyde show the same apparent maximum rate. Aldehyde dehydrogenase is able to catalyse the hydrolysis of p-nitrophenyl esters. This esterase activity was stimulated by both NAD+ and NADH, the maximum rate for the NAD+ stimulated esterase reaction being roughly equal to the maximum rate for the oxidation of aldehydes. The mechanistic implications of the above behaviour are discussed.
| S-EPMC1186151 | biostudies-other
Project description:Alcohol dehydrogenase (EC 1.1.1.1) from the rat liver supernatant fraction has been purified 200-fold and partially characterized. The isolation procedure involved ammonium sulphate fractionation, DEAE-Sephadex chromatography and gel filtration. The purified enzyme behaved as a homogeneous preparation as evaluated by cellulose acetate and polyacrylamide-gel disc electrophoresis. Sulphoethyl-Sephadex chromatography and immunoelectrophoresis with rabbit antiserum indicated the presence of a minor component. Rat liver alcohol dehydrogenase appears to contain 4mol of zinc/mol, has an estimated molecular weight of 65000 and consists of two subunits of similar molecular weight. Heavy-metal ions, thiol-blocking reagents, urea at concentrations below 8m, low pH (5.5) and chelating agents deactivate the enzyme but do not dissociate it into subunits. Deactivated enzyme could not be reactivated. The enzyme is strictly specific for NAD(+) and has a broad specificity for alcohols, which are bound at a hydrophobic site. Inhibition occurred with the enzyme equilibrated with Zn(2+) at concentrations above 0.1mm.
| S-EPMC1178266 | biostudies-other
Project description:1. A procedure for the purification of the cytoplasmic isoenzyme of aspartate aminotransferase from sheep liver is described. 2. The purified isoenzyme shows a single component in the ultracentrifuge at pH7.6 and forms a single protein band on agar-gel electrophoresis at pH6.3 or 8.6, as well as when stained for protein or activity after polyacrylamide-gel or cellulose acetate electrophoresis at pH8.8. 3. Immunoelectrophoresis on agar gel yields only one precipitin arc associated with the protein band, with rabbit antiserum to the purified isoenzyme. By immunodiffusion, cross-reaction was detected between the cytoplasmic isoenzymes from sheep liver and pig heart, but not between the cytoplasmic and mitochondrial sheep liver isoenzymes. 4. The s(20,w) of the enzyme is 5.69S and the molecular weight determined by sedimentation equilibrium is 88900; 19313 molecules of oxaloacetate were formed/min per molecule of enzyme at pH7.4 and 25 degrees C. 5. The amino acid composition of the isoenzyme is presented. It has about 790 residues per molecule. 6. The holoenzyme has a maximum of absorption at 362nm at pH7.6 and 25 degrees C. 7. A value of 2.1 was found for the coenzyme/enzyme molar ratio. 8. The purified enzyme revealed two bands of activity on polyacrylamide-gel electrophoresis at pH7.4 and an extra, faster, band in some circumstances. These bands occurred even when dithiothreitol was present throughout the isolation procedure. 9. Three main bands were obtained by electrofocusing on polyacrylamide plates with pI values 5.75, 5.56 and 5.35. 10. Structural similarities with cytoplasmic isoenzymes from other organs are discussed.
| S-EPMC1165884 | biostudies-other
Project description:BackgroundAs the third enzyme of the pentose phosphate pathway, 6-phosphogluconate dehydrogenase (6PGDH) is the main generator of cellular NADPH. Both thioredoxin reductase and glutathione reductase require NADPH as the electron donor to reduce oxidized thioredoxin or glutathione (GSSG). Since thioredoxin and GSH are important antioxidants, it is not surprising that 6PGDH plays a critical role in protecting cells from oxidative stress. Furthermore the activity of 6PGDH is associated with several human disorders including cancer and Alzheimer's disease. The 3D structural investigation would be very valuable in designing small molecules that target this enzyme for potential therapeutic applications.ResultsThe crystal structure of 6-phosphogluconate dehydrogenase (6PGDH/Gnd1) from Saccharomyces cerevisiae has been determined at 2.37 A resolution by molecular replacement. The overall structure of Gnd1 is a homodimer with three domains for each monomer, a Rossmann fold NADP+ binding domain, an all-alpha helical domain contributing the majority to hydrophobic interaction between the two subunits and a small C-terminal domain penetrating the other subunit. In addition, two citrate molecules occupied the 6PG binding pocket of each monomer. The intact Gnd1 had a Km of 50 +/- 9 microM for 6-phosphogluconate and of 35 +/- 6 microM for NADP+ at pH 7.5. But the truncated mutants without the C-terminal 35, 39 or 53 residues of Gnd1 completely lost their 6PGDH activity, despite remaining the homodimer in solution.ConclusionThe overall tertiary structure of Gnd1 is similar to those of 6PGDH from other species. The substrate and coenzyme binding sites are well conserved, either from the primary sequence alignment, or from the 3D structural superposition. Enzymatic activity assays suggest a sequential mechanism of catalysis, which is in agreement with previous studies. The C-terminal domain of Gnd1 functions as a hook to further tighten the dimer, but it is not necessary for the dimerization. This domain also works as a lid on the substrate binding pocket to control the binding of substrate and the release of product, so it is indispensable for the 6PGDH activity. Moreover, the co-crystallized citrate molecules, which mimic the binding mode of the substrate 6-phosphogluconate, provided us a novel strategy to design the 6PDGH inhibitors.
| S-EPMC1919378 | biostudies-literature
Project description:The kinetic properties of highly purified preparations of sheep liver cytoplasmic aldehyde dehydrogenase (preparations that had been shown to be free from contamination with the corresponding mitochondrial enzyme) were investigated with both propionaldehyde and butyraldehyde as substrates. At low aldehyde concentrations, double-reciprocal plots with aldehyde as the variable substrate are linear, and the mechanism appears to be ordered, with NAD+ as the first substrate to bind. Stopped-flow experiments following absorbance and fluorescence changes show bursts of NADH production in the pre-steady state, but the observed course of reaction depends on the pre-mixing conditions. Pre-mixing enzyme with NAD+ activates the enzyme in the pre-steady state and we suggest that the reaction mechanism may involve isomeric enzyme--NAD+ complexes. High concentrations of aldehyde in steady-state experiments produce significant activation (about 3-fold) at high concentrations of NAD+, but inhibition at low concentrations of NAD+. Such behaviour may be explained by postulating the participation of an abortive complex in product release. Stopped-flow measurements at high aldehyde concentrations indicate that the mechanism of reaction under these conditions is complex.
| S-EPMC1158276 | biostudies-other