UMLS:C0027960 - FACTA Search

Gene/Protein Disease Symptom Drug Enzyme Compound
Pivot Concepts: Target Concepts:

Query: UMLS:C0027960 (mole)
21,279 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

Sheep ovarian 17 beta HSDH has been purified about 1000 fold to a specific activity of 0.5 IU/mg protein, using DEAE cellulose chromatography, affinity chromatography on estrone-amino caproate-Sepharose and a second DEAE cellulose chromatography. The molecular weight is 70,000 ; the pH optimum for activity is 9.2 and the energy of activation is 16.5 Kcal/mole. The kinetics of the oxidation of estradiol and many analogues have been studied at various concentrations and in the presence of different amounts of coenzyme. The data are in agreement with a compulsory order mechanism with the binding of NAD+ as the first substrate. Sheep ovarian 17 beta HSDH accepts subtituents in position C3, C11, C13 ; the substrate binding site is open in this region. On the contrary, the binding requirements are strict for the region of C10 since the presence of a C19 methyl group impairs binding and (or) oxidation of the steroid. Sheep ovarian and human placental 17 beta HSDH have close analogies : molecular weight, pH optimum, substrate binding site requirements. Their reaction mechanisms are different : random for the placental 17 beta HSDH, compulsory order for the ovarian 17 beta HSDH : this can be explained by the effect of the coenzyme upon the binding of the substrate : without effect on placental enzyme, the coenzyme fixation enhances the affinity of the ovarian 17 beta HSDH for any substrate.
...
PMID:17 beta-Hydroxysteroid dehydrogenase of the sheep ovary : purification, properties and substrate binding site. 0 49

Structural and conformational organization of chicken liver fatty acid synthetase has been probed using its fluorescent coenzyme, NADPH. Three NADPH binding sites per mole of the enzyme complex, of apparently identical dissociation constant (KD = 0.6 muM) can be titrated at temperatures above 12 degrees. These results are in disagreement with the earlier studies of Hsu and Wagner (Hsu, R. Y., and Wagner, B. J. (1970) Biochemistry, 9, 245-251) in which four such sites could be titrated. At 12 degrees, the composite sites split into two subsets: a pair of sites with a KD of 0.3 muM and a third site with a Kd of 1.1 muM. At lower temperatures (5 degrees or 2 degrees), the site with weak affinity disappears, leaving a pair of sites with a Kd of 0.5 muM. Similar observations were made when the enzyme was modified with phenylmethylsulfonyl fluoride, a specific and selective inhibitor of fatty acyl-CoA deacylase (s) of the pigeon liver enzyme complex (Kumar, S. (1975) J. Biol. Chem. 250, 5150-5158). Partial modification with phenylmethylsulfonyl fluoride elicits a NADPH binding response similar to the binding observed at 12 degrees, i.e. two sets of binding sites with nonidentical dissociation constants. Further modification corresponding to the complete loss of deacylase function results in a set of two apparently identical binding sites, and the third site is not available for titration. The modified enzyme retains the two reductase functions as measured by the model substrates, acetoacetyl-N-acetylcysteamine and crotonyl-CoA. Furthermore, the addition of acetyl- and malonyl-CoA (100 muM each) to the modified enzyme lowers the NADPH binding affinity by a factor of 3. Other observations show that the quantum yield, as measured by the ratio of fluorescence intensity of bound and free NADPH, changes with temperature and ionic strength. Lowering the temperature from 30 degrees to 2 degrees increases the enhancement ratio by 50%, whereas increase in ionic strength from 0.05 to 0.2 M potassium phosphate lowers it to 50% of the original level. Measurement of NADPH binding in the presence of NADP+, NADH, NAD+ and adenosine-2'-monophospho-5'-diphosphoribose demonstrates that NADP+ shows competitive behavior for NADPH sites (KD = 10.6 muM), whereas NADH and NAD+ show noncompetitive (KD (apparent) = nearly 600 muM) and rather complicated interactions implicating nonspecific conformational alteration of the enzyme complex. The behavior of adenosine 2'-monophospho-5'-diphosphoribose is intermediate between NADP+ and NADH. These data are discussed in terms of substrate-mediated conformational changes and the moles of each of the reductase enzymes per mole of the enzyme complex, the polarity of the NADPH binding region, and the probable structure of the nicotinamide moiety when bound to the enzyme.
...
PMID:Reduced nicotinamide adenine dinucleotide phosphate, a structural and conformational probe of chicken liver fatty acid synthetase. 0 63

The binding of nicotinamide adenine dinucleotide (NAD+) to yeast glyceraldehyde-3-phosphate dehydrogenase (GPDH) has been studied at pH 6.5 and 8.5, at 5,25, and 40 degrees C, by calorimetry, fluorometry, spectrophotometry, equilibrium dialysis, and flow dialysis. As reported earlier for pH 7.3 (Velick S.F., Baggott, J.P., and Sturtevant, J.M. (1971), Biochemistry 10, 779), the binding is accompanied by enthalpy changes which become rapidly more negative as the temperature increases, with delta Cp = -500 to -750 cal deg-1 (mole of NAD+ bound)-1, and by entropy changes which also, as required by the large negative delta Cp, become rapidly more negative with increasing temperature. The binding data at pH 6.5 can be fitted on the basis of either four identical noninteracting sites, or of four sites showing a small degree of negative cooperativity. The data at pH 8.5, particularly at 40 degrees C, require the introduction of positive cooperativity, as was previously shown by Kirschner et al. (Kirschner, K., Eigen, M., Bittman, R., and Voigt, B. (1966), Proc. Natl. Acad. Sci. U.S.A. 56, 1661), and can be equally well fitted on the basis of a sequential model (Adair, G.S. (1925), J. Biol. Chem. 63, 529) or a concerted model (Monod, J., Wyman, J., and Changeux, J.P. (1965), J. Mol. Biol. 12, 88). It is proposed that the observed thermodynamic changes are largely the result of a hydrophobic effect due to a decrease in the exposure of nonpolar groups to the solvent, and of a tightening of the protein structure when the coenzyme is bound with concomitant decrease in the number of easily excitable internal degrees of freedom.
...
PMID:Energetics of the cooperative and noncooperative binding of nicotinamide adenine dinucleotide to yeast glyceraldehyde-3-phosphate dehydrogenase at pH 6.5 and pH 8.5. Equilibrium and calorimetric analysis over a range of temperature. 1 17

Formate dehydrogenase (EC 1.2.1.2) prepared from peas (Pisum sativum) was a two-subunit enzyme. The enzyme accelerated the formation of an NAD+-cyanide compound having an adsorption band at 330 nm. The enzyme was able to bind one NAD+ molecule per each subunit but only 1 mole of NAD+-cyanide compound was formed per two subunits. The complex of NAD+, cyanide, and the enzyme was very stable and had no catalytic activity. Azide inhibited the formate dehydrogenase reaction in two different ways. By incubation of the enzyme with azide in the presence of NAD+, half of its catalytic activity was lost. The remaining activity was also inhibited by azide but this inhibition was removed competively by formate. Contrary to the case of cyanide the inhibition by azide could be removed by dialysis and no spectral species due to the addition compound of NAD+ and azide could be observed. The data from double recipricol plots of the initial velocity and the formate concentration led to a conclusion that formate dehydrogenase has two sites with about equal catalytic activity. The Km for formate was different for the two catalytic sites (1.67 and 6.25 mM) but the difference was not noticeable in the case of the Km for NAD+.
...
PMID:Formate dehydrogenase. Subunit and mechanism of inhibition by cyanide and azide. 16 90

The UDP-apiose/UDP-xylose synthase from cell suspension cultures of parsley has been purified 1400-fold by an improved method. The ratio of apiose to xylose formed from UDP-D-glucuronic acid (UDP-GlcUA) remained constant throughout the purification procedure. Dodecylsulfate-gel electrophoresis and sedimentation equilibrium measurements showed that this enzyme preparation is composed of two proteins with molecular weights of 65000 and 86000. The two proteins which are present in a molar ratio of about 1:0.7 to 1:0.9 could not be separated by ammonium sulfate fractionation, chromatography on DEAE-cellulose at different pH-values, and on omega-aminoalkyl-Sepharose, and by gel filtration on Acrylex P-100. Each protein is composed of two apparently identical subunits. The presence of only two different subunits was confirmed by end group analysis in which glycine was found as N-terminal amino acid for the larger and lysine for the smaller protein. Crosslinking with dimethylsuberimidate gave dimers of the identical subunits but no hybrids. Separation of the two proteins was achieved on DEAE-cellulose in the presence of urea. After dialysis only the 86000-Mr protein showed enzyme activity with no significant change in the apiose/xylose ratio. However, in the absence of the 65000-Mr protein enzyme stability was decreased drastically. By equilibrium dialysis it was found that 0.5 mol UDP-GlcUA are bound per mole of 86000-Mr protein. NAD+ alone was not bound, but in the presence of UDP it was also bound in a ratio of 0.5 mol/mol catalytic protein. Experiments in which sodium borohydride was added to the enzyme incubation gave no indication that the 4-keto intermediate is bound as a Schiff base to the enzyme. Also no evidence for epimerization at C-3 of the 4-ulose intermediate prior to ring contraction to apiose was found.
...
PMID:UDP-apiose/UDP-xylose synthase. Subunit composition and binding studies. 19 51

1. Pyridoxal 5'-phosphate inhibits glucose-6-phosphate dehydrogenase from Leuconostoc mesenteroides reversibly which Ki equals 0.04-0.06 mM. 2. This inhibition is competitive with respect to glucose 6-phosphate and non-competitive with respect to NADP+ or NAD+. Interaction between enzyme and excess pyridoxal 5'-phosphate follows pseudo-first-order kinetics and indicates that one molecule of inhibitor reacts with each active unit of enzyme. 3. Substrate and coenzyme protect the enzyme from inhibition by pyridoxal 5'-phosphate. Dissociation constants for NADP+ and glucose 6-phosphate were determined from their effects on the kinetics of enzyme--inhibitor interaction. 4. Reaction of the enzyme with pyridoxal 5'-phosphate produces a typical Schiff-base absorbance peak at 430 nm. Subsequent reduction with sodium borohydride leads to spectral changes characteristic for the formation of a secondary amine. 5. The irreversibly inactivated enzyme thus produced contains two moles of inhibitor per mole of enzyme (two subunits per mole). After protein hydrolysis, N-6-pyridoxyllysine can be identified by paper chromatography. 6. The enzyme is inhibited irreversibly by 1-fluoro-2,4-dinitrobenzene, even in the presence of excess 2-mercaptoethanol. At least one dinitrophenyl group is bound per active unit of enzyme; 4 to 5 moles of dinitrophenyl group are bound per mole of enzyme. NADP+ AND GLUCOSE 6-PHOSPHATE PROTECT AGAINST INHIBITION BY 1-FLUORO-2,4-DINITROBENZENE. The absorption spectrum of dinitrophenyl-enzyme corresponds to that for dinitrophenylated amino groups. 7. These studies indicate that there is an essential lysine at the active site of the enzyme. It is suggested that the function of this lysine is to bind glucose 6-phosphate. 8. It is proposed that a group of "active lysine" proteins may exist (in analogy with the "active serine" enzymes), which share a common structural feature at their substrate-binding site and to which pyridoxal 5'-phosphate binds specifically.
...
PMID:Evidence for an essential lysine in glucose-6-phosphate dehydrogenase from Leuconostoc mesenteroides. 23 86

Rat and calf adrenal cortex homogenates were found to contain three different malic enzymes. Two were strictly NADP+-dependent and were localized, one each, in the cytosol and the mitochondrial fractions, respectively. These two enzymes appear to be identical to those described by Simpson and Estabrook (Simpson, E. R., and Estabrook, R. W. (1969) Arch. Biochem. Biophys. 129, 384-395). The third was NAD(P)+-linked and was present in the mitochondrial fraction only. All three malic enzymes separated as distinct bands during electrophoresis on 5 percent polyacrylamide slab gels at pH 9.0. Marker enzymes and the mitochondrial malic enzymes migrated together in intact mitochondria during sucrose density gradient centrifugations despite changes in the equilibrium position of the mitochondria promoted by energy-dependent calcium phosphate accumulation. In adrenal cortex mitochondria subfractionated by the method of Sottocasa et al. (SOTTOCASA, G.L., KUYLENSTIERNA, B., ERNSTER, L., and BERGSTAND, A. (1967) J. Cell Biol. 32, 415-438), both malic enzymes were associated with the inner membrane-matrix space. Sonication solubilized the two malic enzymes along with the matrix space marker enzymes. The NAD(P)+-dependent malic enzyme was purified 100-fold from calf adrenal cortex mitochondria. The final preparation was free of malic dehydrogenase, fumarase, the strictly NADP+-linked malic enzyme and adenylate kinase. Either Mn24 orMg2+ was required for activity and 1 mol of pyruvate was formed for each mole of NAD+ and NADP+ reduced. The pH optima with NAD+ and NADP+ were 6.5 tp 7.0 and 6.0 to 6.5, respectively. Michaelis-Menten kinetics were observed on the alkaline side. Fumarate, succinate, and isocitrate were positive and ATP and ADP were negative modulators of the regulatory enzyme. The modulators did not influence the stoichiometry and they were not metabolized during the reaction. Under Vmax conditions the ratios for the rate of NAD+:NADP+ reduction were 1.76 and 1.15 at pH 7.4 and 6.0, respectively. The apparent Michaelis constants also differed depending on the pH and the coenzyme. At pH 7.4 (in the presence of 5 mM fumarate) and at pH 6.0 (no fumarate) the Km values for (-)-malate, NAD+, and Mn2+ were 1.7, 0.16, and 0.15 mM, and 0.31, 0.06, and 0.09 mM, respectively. At pH 7.4 (5MM fumarate) and pH 6.0 (no fumarate), the Km values for (-)-malate, NADP+, and Mn2+ were 6.5, 0.62, and 0.59 mM, and 0.68. 0.12, and 0.31 mM, respectively. The apparent Ki values for ATP with NAD+ and NADP+ as coenzyme were 0.42 and 0.27 mM, respectively.
...
PMID:The mitochondrial malic enzymes. I. Submitochondrial localization and purification and properties of the NAD(P)+-dependent enzyme from adrenal cortex. 23 89

The effect of ethanol on hepatic respiration and glycolysis was studied in perfused rat livers. 1. Ethanol increased the rate of oxygen uptake in livers from fed rats, but decreased the rate in livers from fasted animals perfused in the absence of added substrates. 2. Addition of ethanol decreased the rate of lactate + pyruvate production reflecting an inhibition of glycolysis irrespective of whether glycogen or added glucose was the substrate. 3. Half-maximal stimulation of respiration and inhibition of glycolysis were observed at ethanol concentrations between 0.2 and 0.4 mM. 4. A stoichiometric relationship of one mole of stimulated oxygen uptake to 3.6 mol of decreased lactate + pyruvate production was observed under a variety of experimental conditions. 5. The effects of ethanol on oxygen uptake and lactate + pyruvate production were abolished by the addition of 4-methylpyrazole, an inhibitor of alcohol dehydrogenase, but were unaffected by aminooxyacetate, an inhibitor of hydrogen transport across the mitochondrial membrane. 6. Carboxyatractyloside, an inhibitor of adenine nucleotide translocase, largely abolished the increase in oxygen uptake due to ethnol, but had little effect on the inhibitory action of ethanol on glycolysis. These data indicate that the ethanol-stimulated oxygen uptake is due to an increased flux through the mitochondrial respiratory chain and that it involves the NAD+-dependent oxidation of ethanol by alcohol dehydrogenase. The data are consistent with the hypothesis that the ethanol-stimulated respiration results from an increased demand for mitochondrial oxidative phosphorylation as a consequence of the decreased extramitochondrial ATP generation following inhibition of glycolysis by ethanol.
...
PMID:Interaction of glycolysis and respiration in perfused rat liver. Changes in oxygen uptake following the addition of ethanol. 86 14

The pyruvate dehydrogenase complex from Axotobacter vinelandii was isolated in a five-step procedure. The minimum molecular weight of the pure complex is 600,000, as based on an FAD content of 1.6 nmol-mg protein-1. The molecular weight is 1.0-1.2 X 10(6), indicating 1 mole of lipoamide dehydrogenase dimer per complex molecule. Sodium dodecylsulphate gel electrophoretical patterns show that apart from pyruvate dehydrogenase (Mr89,000) and lipoamide dehydrogenase (Mrmonomer 56,000) two active transacetylase isoenzymes are present with molecular weight on the gel 82,000 and 59,000 but probably actually lower. The pure complex has a specific activity of the pyruvate-NAD+ reductase (overall) reaction of 10 units-mg protein-1 at 25 degrees C. The partial reactions have the following specific activities in units-mg protein-1 at 25 degrees C under standard conditions: pyruvate-K3Fe(CN)6 reductase 0.14, transacetylase 3.6 and lipoamide dehydrogenase 2.9. The properties of this complex are compared with those from other sources. NADPH reduced the FAD of lipoamide dehydrogenase as well in the complex as in the free form. NADP+ cannot be used as electron acceptor. Under aerobic conditios pyruvate oxidase reaction, dependent on Mg2+ and thiamine pyrophosphate, converts pyruvate into CO2 and acetate; V is 0.2 mumol 02-min-1-mg-1, Km(pyruvate)0.3 mM. The kinetics of this reaction shows a linear 1/velocity-1/[pyruvate] plot. K3Fe(CN)6 competes with the oxidase reaction. The oxidase activity is stimulated by AMP and sulphate and is inhibited by acetyl-CoA. The partially purified enzyme contains considerable phosphotransacetylase activity. The pure complex does not contain this activity. The physiological significance of this activity is discussed.
...
PMID:The pyruvate-dehydrogenase complex from Azotobacter vinelandii. 120 21

Lipoamide dehydrogenase is a flavoprotein which catalyzes the reversible oxidation of dihydrolipoamide, Lip(SH)2, by NAD+. The ping-pong kinetic mechanism involves stable oxidized and two-electron-reduced forms. We have investigated the rate-limiting nature of proton transfer steps in both the forward and reverse reactions catalyzed by the pig heart enzyme by using a combination of alternate substrates and solvent kinetic isotope effect studies. With NAD+ as the variable substrate, and at a fixed, saturating concentration of either Lip(SH)2 or DTT, inverse solvent kinetic isotope effects of 0.68 +/- 0.05 and 0.71 +/- 0.05, respectively, were observed on V/K. Solvent kinetic isotope effects on V of 0.91 +/- 0.07 and 0.69 +/- 0.02 were determined when Lip(SH)2 or DTT, respectively, was used as reductant. When Lip(SH)2 or DTT was used as the variable substrate, at a fixed concentration of NAD+, solvent kinetic isotope effects of 0.74 +/- 0.06 and 0.51 +/- 0.04, respectively, were observed on V/K for these substrates. Plots of the kinetic parameters versus mole fraction D2O (proton inventories) were linear in all cases. Solvent kinetic isotope effect measurements performed in the reverse direction using NADH as the variable substrate showed equivalent, normal solvent kinetic isotope effects on V/KNADH when oxidized lipoamide, lipoic acid, or DTT were present at fixed, saturating concentrations. Solvent kinetic isotope effects on V were equal to 1.5-2.1. When solvent kinetic isotope effect measurements were performed using the disulfide substrates lipoamide, lipoic acid, or DTT as the variable substrates, normal kinetic isotope effects on V/K of 1.3-1.7 were observed.(ABSTRACT TRUNCATED AT 250 WORDS)
...
PMID:Pig heart lipoamide dehydrogenase: solvent equilibrium and kinetic isotope effects. 155 95

1 2 3 4 Next >>