Gene/Protein Disease Symptom Drug Enzyme Compound
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Query: UMLS:C0027960 (mole)
 21,279 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

3-Chloroacetylpyridine--adenine dinucleotide, which is active as a hydride acceptor (Km = 0.6 mM), inactivates and alkylates estradiol 17beta-dehydrogenase. The kinetics of inactivation by 3-chloroacetylpyridine--adenine dinucleotide and the absence of inactivation by 3-chloroacetylpyridine ribose phosphate show that the alkylation follows the formation of a binary complex (Kd = 4.5 X 10(-4) M). Studies of the labelling by 3-chloro[2-14C]acetylpyridine--adenine dinucleotide and the rate of alkylation as a function of pH, give evidence to the alkylation of a cysteine, the stoichiometry being one mole per subunit. The 14C label is distributed between three chymotryptic peptides, one of which accounts for about 50% of the radioactive label.
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PMID:Alkylation of estradiol 17beta-dehydrogenase from human placenta with 3-chloroacetylpyridine--adenine dinucleotide. 0 23

An automated AutoAnalyzer method using 5:5'-dithiobis-2-nitrobenzoic acid is described for determining whole blood glutathione reductase (BGR) activity and for measuring in vitro activation of BGR with flavin adenine dinucleotide (FAD). BGR activity is expressed as mumoles glutathione regenerated from oxidized glutathione per ml of whole blood (WB) or per g of hemoglobin. The stimulatory effect of FAD on BGR activity divided by the activity without FAD determined the activity coefficient (AC). We found that NADPH and oxidized glutathione assay concentrations of 0.100 mmole/liter and 0.250 mmole/liter, respectively, in 0.1 mole/liter phosphate buffer, pH 7.4, gave consistent results when WB, before assay, was diluted 20-fold. WB samples to be stored are initially diluted 10-fold with distilled water and frozen. Prior to assay, two aliquots of the sample are diluted 2-fold, one aliquot with distilled water and another with 46 mumole/liter FAD. With sample and manifold dilutions the assay FAD concentrations is 1.0 mumole/liter: assay concentrations greater than 5.0 mumole FAD/liter were shown to be inhibitory. We examined blood samples from 617 children in the age range 6 to 60 months and determined the normal AC range to be between 1.00 and 1.35. Six weaned rats (23 days of age), maintained on a riboflavin-deficient diet, showed a mean AC of 1.23, 1.54, 2.02, and 2.41 at 23, 26, 30, and 36 days of age, respectively. Six control rats maintained an AC of 1.23 +/- 0.05 (SD) during the same period.
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PMID:An automated flavin adenine dinucleotide-dependent glutathione reductase assay for assessing riboflavin nutriture. 0 81

The effect of temperature on the binding of thyroxine and triiodothyronine to thyroxine-binding globulin has been studied by equilibrium dialysis. Inclusion of ovalbumin in the dialysis mixture stabilized thyroxine-binding globulin against losses in binding activity which had been found to occur during equilibrium dialysis. Ovalbumin by itself bound the thyroid hormones very weakly and its binding could be neglected when analyzing the experimental results. At pH 7.4 and 37 degrees in 0.06 M potassium phosphate/0.7 mM EDTA buffer, thyroxine was bound to thyroxine-binding globulin at a single binding site with apparent association constants: at 5 degrees, K = 4.73 +/- 0.38 X 10(10) M-1; at 25 degrees, K = 1.55 +/- 0.17 X 10(10) M-1; and at 37 degrees, K = 9.08 +/- 0.62 X 10(9) M-1. Scatchard plots of the binding data for triiodothyronine indicated that the binding of this compound to thyroxine-binding globulin was more complex than that found for thyroxine. The data for triiodothyronine binding could be fitted by asuming the existence of two different classes of binding sites. At 5 degrees and pH 7.4 nonlinear regression analysis of the data yielded the values n1 = 1.04 +/- 0.10, K1 = 3.35 +/- 0.63 X 10(9) M-1 and n2 = 1.40 +/- 0.08, K2 = 0.69 +/- 0.20 X 10(8) M-1. At 25 degrees, the values for the binding constants were n1 = 1.04 +/- 0.38, K1 = 6.5 +/- 2.8 X 10(8) M-1 and n2 = 0.77 +/- 0.22, K2 = 0.43 +/- 0.62 X 10(8) M-1. At 37 degrees where less curvature was observed, the estimated binding constants were n1 = 1.02 +/- 0.06, K1 = 4.32 +/- 0.59 X 10(8) M-1 and n2K2 = 0.056 +/- 0.012 X 10(8) M-1. When n1 was fixed at 1, the resulting values obtained for the other three binding constants were at 25 degrees, K1 = 6.12 +/- 0.35 X 10(8) M-1, n2 = 0.72 +/- 0.18, K2 = 0.73 +/- 0.36 X 10(8) M-1; and at 37 degrees K1 = 3.80 +/- 0.22 X 10(8) M-1, n2 = 0.44 +/- 0.22, and K2 = 0.43 +/- 0.38 X 10(8) M-1. The thermodynamic values for thyroxine binding to thyroxine-binding globulin at 37 degrees and pH 7.4 were deltaG0 = -14.1 kcal/mole, deltaH0 = -8.96 kcal/mole, and deltaS0 = +16.7 cal degree-1 mole-1. For triiodothyronine at 37 degrees, the thermodynamic values for binding at the primary binding site were deltaG0 = -12.3 kcal/mole, deltaH0 = -11.9 kcal/mole, and deltaS0 = +1.4 cal degree-1 mole-1. Measurement of the pH dependence of binding indicated that both thyroxine and triiodothyronine were bound maximally in the region of physiological pH, pH 6.8 to 7.7.
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PMID:Thyroxine-protein interactions. Interaction of thyroxine and triiodothyronine with human thyroxine-binding globulin. 0 58

1. L-asparaginase from M. phlei was purified about 170-fold with an 11% yield. The purification procedure consisted of: fractionation with ammonium sulphate; adsorption of contaminating proteins on calcium phosphate gel; chromatography on Sephadex G-150 and DEAE-cellulose. The specific activity of the final preparation was 32.6 i.u./mg protein. 2. Molecular weight of the enzyme as determined by Sephadex G-100 filtration amounted to 126 000. Optimum pH was 8.8-9.2. The enzyme did not hydrolyse L-glutamine over the pH range 4-9, and was inhibited by D-asparagine. The apparent Michaelis constant for L-asparagine was 0.7 mM; energy of activation, 9800 cal/mole. 3. On polyacrylamide-gel electrophoresis the final preparation revealed two protein bands, one of which was coincident with the enzyme activity.
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PMID:Purification and properties of L-asparaginase from Mycobacterium phlei. 0 91

Evidence is presented to show that an optical isomer of 5-phenylhydantoin is subject to racemization (interconversion) in different buffer systems. With phosphate buffers in the pH range of 6.0-7.5, it appears that the buffer-catalyzed racemization reaction is due solely to catalysis by divalent phosphate (general base catalysis). Other buffers studied include arsenate, imidazole, triethanolamine, and pyrophosphate. When 5-phenylhydantoin, the N-de-ethylated metabolite of ethotoin, was administered to dogs in an earlier investigation, the observation was made that somewhat more than the theoretical quantity (50 mole percent of the dose) of the substances recovered from urine had the R-configuration. The principal metabolite was (R)-(-)-2-phenylhydantoic acid, formed stereo-specifically in a ring-opening reaction of (R)-5-phyenylhydantoin by dihydropyrimidinase (EC 3.5.2.2). The results of the present in vitro study support the hypothesis that in vivo the interconversion of the optical isomers of 5-phenylhydantoin can be catalyzed by buffering components of the mammalian physiological system, and that the catalytic activities of the endogenous buffer components can account for the racemization and ultimate metabolism of the (S)-isomer of 5-phenylhydantoin by dihydropyrimidinase.
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PMID:Buffer catalysis of the racemization reaction of some 5-phenylhydantoins and its relation to the in vivo metabolism of ethotoin. 0 88

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

Alkaline phosphatase of Escherichia coli, isolated by procedures which do not alter its intrinsic metal content, contains 4.0 +/- 0.3 g-atoms of tightly bound zinc per mole (Kd less than 1 muM) and 1.3 +/- 0.2 g-atoms of magnesium per mole (Bosron, W.F., Kennedy, F.S., and Vallee, B.L. (1975), Biochemistry 14, 2275-2282). Importantly, the binding of magnesium is dependent both upon pH and zinc content. Hence, the failure to assign the maximal magnesium stoichiometry to enzyme isolated by conventional procedures may be considered a consequence of the conditions chosen for optimal bacterial growth and purification of the enzyme which are not the conditions for optimal binding of magnesium to alkaline phosphatase. Under the conditions employed for the present experimental studies, a maximum of six metal sites are available to bind zinc and magnesium, i.e., four for zinc and two for magnesium. Magnesium alone does not activate the apoenzyme, but it regulates the nature of the zinc-dependent restoration of catalytic activity to apophosphatase, increasing the activity of enzyme containing 2-g-atoms of zinc five-fold and that of enzyme containing 4-g-atoms of zinc 1.4-fold. Moreover, hydrogen-tritium exchange reveals the stabilizing effects of magnesium on the structural properties of phosphatase. However, neither the KM for substrate nor the phosphate binding stoichiometry and Ki are significantly altered by magnesium. Hence, magnesium, which is specificially bound to the enzyme, both stabilizes the dynamic protein structure and regulates the expression of catalytic activity by zinc in alkaline phosphatase.
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PMID:Effect of magnesium on the properties of zinc alkaline phosphatase. 1 22

The oxygen-binding characteristics and the multiplicity of the stripped hemoglobiin from active lungfish Protopterus amphibius, are the same as in specimens that have been estivating for about 30 months, showing that alteration in the hemoglobin molecules is not involved in the earlier reported increase in oxygen affinity of whole blood during estivation (Johansen et al., '76). At pH 7.0 and 26 degrees C the hemolysates show a high oxygen affinity (P50 = 3.1 Torr), a Bohr factor (delta log P50/delta pH) of - 0.33, and a cooperativity coefficient (n) of 1.7. Between 15 and 26 degrees C, the apparent heat of oxygenation (delta H) is - 8.6 Kcal-mole-1 at pH 7.0, corresponding with data for other fish. A low sensitivity of oxygen affinity to urea appears to be adaptive to the high urea concentrations in estivating lungfish. The salt sensitivity is, however, similar to human hemoglobin. The hemoglobin consists of two major (electrophoretically anodal) components, which differ slightly in oxygen affinity but are both sensitive to pH and nucleoside triphosphates (NTP). Guanosine triphosphate (GTP), the major erythrocytic organic phosphate, however, depresses the oxygen affinity of the composite and separated hemoglobins more effectively than ATP suggesting that GTP is the primary modulator of oxygen affinity. Comparative measurements reveal only one major hemoglobin component in P. annectens which has a markedly lower oxygen affinity and phosphate sensitivity than P. amphibius hemoglobins and thus seems less pliable to phosphate-mediated variation in oxygen affinity. The data are discussed in relation to the hemoglobin systems of other fish.
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PMID:Oxygen-binding properties of hemoglobins from estivating and active African lungfish. 1 21

(NADPH)-cytochrome P-450 reductase was purified to apparent homogeneity by a procedure utilizing nicotinamide adenine dinucleotide phosphate (NADP)-Sepharose affinity column chromatography. The purified flavoprotein has a molecular weight of 79 700 and catalyzes cytochrome P-450 dependent drug metabolism, as well as reduction of exogenous electron acceptors. Aerobic titration of cytochrome P-450 reductase with NADPH indicates that an air-stable reduced form of the enzyme is generated by the addition of 0.5 mol of NADPH per mole of flavin, as judged by spectral characteristics. Further addition of NADPH causes no other changes in the absorbance spectrum. A Km value for NADPH of 5 micron was observed when either cytochrome P-450 or cytochrome c was employed as electron acceptor. A Km value of 8 +/- 2 micron was determined for cytochrome c and a Km of 0.09 +/- 0.01 micron was estimated for cytochrome P-450.
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PMID:NADPH-cytochrome P-450 reductase from rat liver: purification by affinity chromatography and characterization. 1 71


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