UMLS:C0027960 - FACTA Search

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

D-Erythrulose reductase of beef liver was crystallized from ammonium sulfate solution at pH 8.17. The crystals are needle-shaped. The enzyme protein contains 851 amino acid residues per mole of the enzyme: Lys28, His11, Arg52, Asp79, Thr58, Ser56, Glu68, Pro20, Gly80, Ala107, Val112, Met24, Ile31, Leu88, Tyr7, Phe22, Trp4, and Cys4. The enzyme is inactivated by exposure to temperatures below 12degrees. The inactivation is accelerated by increasing the salt concentration and decreasing the enzyme concentration. The pH of the medium also has a pronounced effect, the maximum stability of the enzyme is obtained at pH 8.5. NADP+ protected the enzyme from cold inactivation at all stages of the process and also afforded protection against inactivation by heat and pH. The cold inactivation of the enzyme is accompanied by dissociation of the enzyme protein to subunits.
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PMID:Studies on D-tetrose metabolism. VI. Crystallization and some properties of D-erythrulose reducatase from beef liver. 0 10

Thermostable NADP+ -specific isocitrate dehydrogenase (EC 1.1.1.42) was purified from crude extract of an extremely thermophilic bacterium Thermus flavus AT-62 through DEAE-cellulose column, acetone fractionation, DEAE-Sephadex A-50 column and isoelectric focussing. The enzyme was purified about 500-folds in its specific activity and purity was found to be about 96%. The enzyme was not inactivated after 60 min at 70 degrees C, but 20 and 80% of the activity were lost after 60 min at 80 degrees and 90 degrees C, respectively. Oxalacetate plus glyoxylate (each 1 nM) demonstrated 75% inhibition of the activity in concerted manner. The degree of the inhibition and the affinity of the enzyme for isocitrate and NADP+ decreased with the rise of temperature, especially above 60 degrees C. The activation energy below and above 60 degrees C were 14,500 and 8,000 cal per mole respectively. In CD spectra negative bands at 210 and 220nm were observed and alpha-helix content was calculated to be about 26%. In the course of heating up to 60 degrees practically no change in CD bands are observed, but above 60 degrees the depth of CD bands decreased gradually and remarkably above 80 degrees C. The effect of temperature on kinetic parameters and secondary structures of the enzyme was discussed in relation to the temperature adaptation of the organism.
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PMID:Purification and some properties of NADP+ -specific isocitrate dehydrogenase from an extreme thermophile, Thermus flavus AT-62. 0 66

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.
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PMID:Reduced nicotinamide adenine dinucleotide phosphate, a structural and conformational probe of chicken liver fatty acid synthetase. 0 63

Michaelis-Menten kinetics are observed in studies of highly purified bovine adrenal glucose-6-phosphate dehydrogenase at pH8.0 in 0.1 M bicine. The Km for NADP+ is 3.8 muM and for glucose-6-phosphate, 61 muM. At pH 6.9 Km for NADP+ increases to 6.5 muM. The enzyme is inhibited by NADPH both at pH 6.8 and at 8.0 with a Kip of 2.36 muM at pH 8.0. Inhibition is competitive with respect to both substrates implying that addition of substrates is random ordered. The data are also interpreted in terms of "reducing charge", the mole fraction of coenzyme in the reduced form. This appears to be the major mechanism for regulation of the pentose shunt. D-glucose, oxidized by the enzyme at a very slow rate, is also a competitive inhibitor for the natural substrate with a Ki of 0.29 M. Phosphate is a competitive inhibitor for glucose-6-phosphate oxidation but both phosphate and sulfate accelerate glucose oxidation suggesting a common binding site for the two anions and the phosphate of the natural substrate. While binding of ACTH to our enzyme preparations has been observed, we have not been able, in spite of repeated attempts, to demonstrate augmentation of the activity of the enzyme by the addition of ACTH.
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PMID:Kinetics and control of bovine adrenal glucose-6-phosphate dehydrogenase. 0 67

A FAD-containing monooxygenase isolated from pig liver microsomes migrates as a single band upon electrophoresis in polyacrylamide gels in the presence of dodecyl sulfate. The minimum molecular weight based on mass of amino acids per mole of flavin is 64,000. However, the catalytically active enzyme exists as aggregating units of the monomer. Neither oxygen nor organic substrates perturbed the spectrum of the oxidized flavoprotein and their binding to this form of the enzyme could not be detected. Anaerobically NADPH bleaches the flavoprotein, and in the presence of both NADPH and oxygen a remarkably stable intermediate form of the enzyme, with an absorption band at 375 nm, is observed. The spectrum of the intermediate resembles that of a peroxyflavin. The monooxygenase catalyzes NADPH- and oxygen-dependent oxygenations of nucleophilic nitrogen- or sulfur-containing compounds. Kinetic studies carried out with a model organic nitrogen substrate (trimethylamine) and a sulfur substrate (methimazole) gave similar patterns. The kinetic data are consistent with an ordered Ter-Bi mechanism with an irreversible step between the second and third substrate where NADPH is added first, followed by oxygen, and the oxidizable organic substrate is added last. If NADPH is the first substrate added, then NADP+ must be the last product released since NADP+ is competitive with NADPH.
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PMID:The liver microsomal FAD-containing monooxygenase. Spectral characterization and kinetic studies. 3 96

Two unrelated Senegalese patients, both native of the Matam province, were found to have the same deficient G6PD variant. One has no hematological history, the other had several induced acute hemolytic episodes. The deficiency was almost complete in red blood cells and 20-30 percent of the normal level in leukocytes and platelets; in leukocytes the deficiency was due to a decrease in the molecular specific activity of the enzyme to which a molecular instability was added, explaining the greater deficiency in red blood cells. The electrophoretic mobility was slightly fast in leukocytes and platelets but normal in red blood cells. This pattern was confirmed by electrofocusing in ampholine-acrylamide gel. From a kinetic point of view, these enzymes were characterized by a lowered Km (G6P) (13 to 20 muM) a normal Km (NADP+), a Ki (NADPH) increased about twice, a thermal instability, a biphasic pH curve and an increased activation energy (15 kcal/mole). The polymorphonuclear cells were functionally strictly normal: engulfment, nitroblue tetrazolium (NBT) reduction test, induced iodination, and oxygen consumption were normal. The authors discuss the importance of post-synthetic modifications of the muted enzymes and their repercussions on the enzyme characteristics.
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PMID:Gd(minus)Matam, an African glucose-6-phosphate dehydrogenase variant with enzyme deficiency. Biochemical and immunological properties in various hemopoietic tissues. 23 89

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.
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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.
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PMID:The mitochondrial malic enzymes. I. Submitochondrial localization and purification and properties of the NAD(P)+-dependent enzyme from adrenal cortex. 23 89

A two-year-old boy with a malignant tumor of the brain (medulloblastoma) excreted large amounts of thymine and uracil in his urine. The excretion was related to progress and regress of the disease, and reached a maximum of 3.0 mol of thymine per mole of creatinine and 2.6 mol of uracil per mole of creatinine. The excretion by 20 apparently normal children was less than 0.01 mol/mol of creatinine for each of the two pyrimidines. Three children with brain tumors, two with leukemias, and one with neuroblastoma were also studied; two of them had a moderate increase in urinary pyrimidine excretion, but only up to 0.07 mol/mol of creatinine. The activity of dihydrouracil dehydrogenase (NADP+) (EC 1.3.1.2) in cultured fibroblasts from the patient was somewhat lower than in control fibroblasts. The tumor was considered to be the likely cause of the increased excretion of pyrimidines, but an impaired degradation of pyrimidines in the liver could not be ruled out.
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PMID:Urinary excretion of thymine and uracil in a two-year-old child with a malignant tumor of the brain. 28 71

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.
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PMID:The pyruvate-dehydrogenase complex from Azotobacter vinelandii. 120 21

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