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)

In addition to the phosphate residues contained in the acid-dissociable FAD and the molybdenum cofactor moieties, milk xanthine oxidase contains one mole of covalently bound phosphorus per active-center molybdenum. Acid hydrolysis of the apoprotein moiety and subsequent analysis by high-voltage thin-layer electrophoresis has identified the phosphorylated amino acid residue to be phosphoserine. 31P NMR data show the phosphopeptide to be monosubstituted, in agreement with the chemical analysis. A pH-dependent chemical shift of the phosphorus residue in the molybdenum cofactor moiety is also observed which provides unequivocal support for suggestions in the literature that this cofactor contains a monosubstituted phosphate. 31P NMR studies on the intact enzyme show phosphorus resonances at about -3 ppm, +1 ppm, +8.8 ppm and at +13.5 ppm. The resonances at +8.8 ppm and at +13.5 ppm are assigned to those of the pyrophosphate linkage of the FAD moiety by analogy with chemical shift data of the FAD on glucose oxidase [James, T.L., Edmondson, D.E., and Husain, M. (1981) Biochemistry 20, 617] and from the absence of any resonances in this region upon examination of preparations of deflavo xanthine oxidase. The intensity and resolution of the resonance at about -3 ppm is dependent on the degree of functionality of the enzyme. This resonance has a small amplitude relative to the FAD resonances in 50-60% functional enzyme, but increases dramatically in intensity in the desulpho enzyme. This resonance is the only one exposed to solvent as it is the only one susceptible to paramagnetic line-broadening on the addition of Mn(II) to the enzyme solution. Treatment of the enzyme with allopurinol leads to alteration of the approximately equal to -3-ppm resonance, but does not significantly affect the other resonances. Formation of the stable Mo(V) 'inhibited' form of the enzyme with ethylene glycol results in extensive line-broadening of the resonances at -3 ppm and +1 ppm, but has no observable affect on the FAD resonances. These data suggest that in addition to the phosphate on the molybdenum cofactor, the phosphoserine residue in xanthine oxidase is also in close proximity to the activesite molybdenum center of this enzyme. These results are discussed with respect to possible implications on the catalytic mechanism of the enzyme.
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PMID:31P nuclear magnetic resonance and chemical studies of the phosphorus residues in bovine milk xanthine oxidase. 654 6

The stoichiometry of the copper requirement for the dopamine beta-monooxygenase-catalyzed conversion of dopamine to norepinephrine has been investigated by rapid chemical-quench techniques. This approach, which employs concentrated samples of enzyme, overcomes ambiguities of interpretation arising from levels of trace copper in excess of enzyme concentrations normally added to steady state kinetic assays. Low turnover numbers are observed when rapid quench kinetic studies are performed under conditions in which enzyme concentrations (2.5-7.1 microM) are in excess over trace copper levels (about 0.7 microM). The addition of exogenous Cu(II) results in full restoration of activity, which is maximal at a stoichiometry of 2 mol of copper/mol of enzyme subunit. From the dependence of catalytic activity on copper levels we conclude that both coppers are required for catalysis. No stimulation of activity was observed upon addition of the following metal ions: Ni(II), Co(II), Mn(II), Fe(III), and Zn(II). In addition, the magnitude of the tritium isotope effect for [2-3H]dopamine hydroxylation is invariant over a large range of enzyme activities accompanying changes in the ratio of copper to enzyme concentration. These results appear to rule out an effector role for the second mole of copper/subunit, implicating both copper atoms in active site redox chemistry.
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PMID:Evidence for two copper atoms/subunit in dopamine beta-monooxygenase catalysis. 670 64

Superoxide dismutase from the anaerobe Bacteroides fragilis has been purified to apparent homogeneity. The protein, Mr 42,000, is a dimer of equally sized subunits joined by noncovalent interactions. Metal analysis of the native enzyme revealed 1.8-1.9 g-atoms Fe, 0.2 g-atoms Zn, and less than 0.05 g-atoms Mn per mole dimer in a preparation whose specific activity was 1200 U/mg. Exposure of the enzyme to guanidinium chloride plus 8-hydroxyquinoline (T. Kirby, J. Blum, I. Kahane, and I. Fridovich, 1980, Arch. Biochem. Biophys. 201, 551-555) resulted in complete loss of enzymatic activity. Activity could be restored by dialysis of the denatured apoprotein against Tris buffer containing either ferrous ammonium sulfate or manganous chloride. The Fe-reconstituted enzyme was inhibited by 1 mM azide and inactivated by H2O2 in a manner similar to the native enzyme. Mn-reconstituted enzyme was inhibited by azide but resisted inactivation by H2O2 comparable to other purified manganese-containing superoxide dismutases. The manganese reconstituted protein contained approximately 1 gm-atom Mn/mol dimer. Zn ion potently inhibited reconstitution of the denatured apoprotein by either Mn or Fe and bound to the protein with a stoichiometry of 2-3 g-atoms/mol dimer.
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PMID:Isolation of iron-containing superoxide dismutase from Bacteroides fragilis: reconstitution as a Mn-containing enzyme. 683 Feb 40

The effect of degree of saturation of concanavalin A with Mn2+ or Ca2+, or both, on its thermal denaturation was investigated by differential scanning calorimetry. Acid-demetallized concanavalin A was partly or fully remetallized in acetate buffer (pH 5.0) containing 0.4 to 0.5 M NaCl. Under these conditions, native dimeric concanavalin A is highly stable, undergoing heat denaturation at 101 degrees C, with an enthalpy of denaturation of 7.4 cal/g. Removal of metal ions lowered stability considerably; concanavalin A with 0.06 Mn2+/monomer and 0.23 Ca2+/monomer (mol/mol) was denatured at 74 degrees C with an enthalpy of denaturation of 3.2 cal/g. Added Mn2+ stabilized demetallized concanavalin A, but added Ca2+ alone (up to 2 mol/mol monomer) did not. The Ca2+/ concanavalin A ratio influenced stabilization by Mn2+. In the presence of 1 to 2 Mn2+/ monomer and 0.5 or less Ca2+/monomer (mol/mol), stabilized concanavalin A was denatured at 85-88 degrees C and at 94-97 degress C, indicating presence of two stabilized metallo-concanavalin A species. At 1.0 or more mole each of Mn2+ and Ca2+ per monomer, one endotherm was observed at or above 98 degrees C and the enthalpy of denaturation was increased to 5.3 cal/g from less than 3.6 cal/g at lower metal ion/protein ratios. Stabilization was greater with Mn2+ plus Ca2+ than with Mn2+ alone, consistent with intrasubunit cooperativity in metal ion-induced stabilization of concanavalin A.
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PMID:Effects of manganese and calcium on conformational stability of concanavalin A: a differential scanning calorimetric study. 727 37

Polychlorinated biphenyls (PCBs), cis-chlordane, oxychlordane, heptachlor epoxide, mirex, hexachlorobenzene (HCB), lindane, octachlorostyrene (OCS), p,p'-DDE,p,p'-DDT, dieldrin, triphenylphosphate (TPP), polybrominated biphenyls (PBBs), and polybrominated diphenyl ethers (PB-DPEs) were measured in the blubber, and five metals (mercury, lead, cadmium, chromium, and manganese) and selenium were measured in the liver of bottlenose dolphins (Tursiops truncatus) obtained from the Gulf of Mexico during an unusual mortality event in 1990. The collection of animals included fetuses, sucklings (< 1 year old), immature dolphins (2-5 years old), and adults of both sexes. PCBs, p,p'-DDE, HCB, and PBDPEs were detected in the blubber of each animal. Mean concentrations of organic contaminants were generally highest in adult males. p,p'-DDE was the single component analyte measured at the highest concentration. Immature females had greater concentrations of most chlorinated organics than adult females. Mercury and cadmium concentrations in liver increased with increasing age-class. The correlation between mercury and selenium in all animals was r = 0.96, with a mole ratio of 0.90. Concentrations of lead, manganese, cadmium, and chromium did not follow any particular age-class trend.
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PMID:Organochlorine, organobromine, metal, and selenium residues in bottlenose dolphins (Tursiops truncatus) collected during an unusual mortality event in the Gulf of Mexico, 1990. 775 3

The propensity of a large number of metal ions to induce cooperative conformational or structural transitions in double-stranded poly d(G-C) was assessed by UV and CD spectrometry. This ability was seen to be an intrinsic property of most metal ions. The observed (metal ion)/(polydeoxynucleotide) mole ratio calculated per G-C base pair and corresponding to the midpoints of the principal transition ranged from 0.3 (Ag(II) to 100 (Al(III)). A strong correlation was seen [y = -1.01(log x) + 3.26, r = 0.95, n = 20] between the (metal ion)/(poly d(G-C)) mole ratio required for the transition midpoint (x) and a covalent index to complex stability (y) of the metal ions. This relationship was independent of the types of transitions observed (monophasic or biphasic) or of specific conformations (e.g., B, Z, psi). The y index measures the ability of metal ions to bind to nitrogen and/or sulphur donor atoms in ligands compared to oxygen centers; equilibrium analysis indicates that the mole-ratio x decreases with increasing affinity of metal ions for poly d(G-C). Thus the observed relationship suggests that base-nitrogen binding facilitates the induced transitions. In general, metal ions designated as Class B or nitrogen/sulphur seeking (Ag(I), Hg(II), and Ru(III)) induced monophasic transitions, whereas Class A or oxygen seeking ions (La(III), Ce(III), Tb(III), Dy(III)) induced biphasic transitions. Transitions generated by ions of more ambivalent ligand preference (Borderline ions) were either monophasic (Mn(II), Fe(III), Cu(II), Cd(II), In(III), and Pb(II)) or biphasic (Cr(III), Co(II), Ni(II) and Zn(II)). Poorly defined transition-curve profiles were observed for Pt(II), Pd(II), and Al(III). Specific conformational assignments were made for some of the observed transitions. For a limited number of metal ions (Ni(II), Cu(II), Cd(II), Ag(I), Hg(II)), interaction with calf thymus DNA was similarly examined. In these instances, the susceptibility to DNase I digestion of both the DNA and polydeoxynucleotide complexes was assessed.
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PMID:The interaction of metal ions with synthetic DNA: induction of conformational and structural transitions. 802 40

Calcium-activated phosphoenolpyruvate carboxykinase from Escherichia coli is not inactivated by a number of sulfhydryl-directed reagents [5,5'-dithiobis(2-nitrobenzoate), iodoacetate, N-ethylmaleimide, N-(1-pyrenyl)maleimide or N-(iodoacetyl)-N'-(5-sulfo-1-naphthylethylenediamine)], unlike phosphoenolpyruvate carboxykinase from other organisms. On the other hand, the enzyme is rapidly inactivated by the arginyl-directed reagents 2,3-butanedione and 1-pyrenylglyoxal. The substrates, ADP plus PEP in the presence of Mn2+, protect the enzyme against inactivation by the diones. Quantitation of pyrenylglyoxal incorporation indicates that complete inactivation correlates with the binding of one inactivator molecule per mole of enzyme. Chemical modification by pyridoxal 5'-phosphate also produces inactivation of the enzyme, and the labeled protein shows a difference spectrum with a peak at 325 nm, characteristic of a pyridoxyl derivative of lysine. The inactivation by this reagent is also prevented by the substrates. Binding stoichiometries of 1.25 and 0.30 mol of reagent incorporated per mole of enzyme were found in the absence and presence of substrates, respectively. The results suggest the presence of functional arginyl and lysyl residues in or near the active site of the enzyme, and indicate lack of reactive functional sulfhydryl groups.
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PMID:Reactivity of cysteinyl, arginyl, and lysyl residues of Escherichia coli phosphoenolpyruvate carboxykinase against group-specific chemical reagents. 814 99

We describe here a new procedure permitting rapid (12-13 h) isolation of a pure oxygen-evolving photosystem II (PSII) core complex from the cyanobacterium Synechocystis PCC 6803. This procedure involves dodecyl maltoside extraction of thylakoid membranes followed by single-step column chromatography using a weak anion-exchanger. SDS-PAGE and immunoblotting show that the complex consists of five intrinsic membrane proteins (CP47, CP43, D1, D1, and cyt b559), one extrinsic protein (MSP), and one unknown protein with a molecular mass of approximately 26 kDa. A chemical and functional analysis, normalized to 2 molecules of pheophytin a, indicates that this PSII core complex contains 1 photoactive plastoquinone, QA, 4 manganese atoms, 38 chlorophyll a molecules, 1 cytochrome b559, 2 plastoquinone-9, and 9-10 beta-carotenes. The complex exhibits high rates of oxygen evolution, typically 2400-2600 mumol of O2 (mg of Chl)-1 h-1 in the presence of 2,5-dichlorobenzoquinone as an artificial electron acceptor with a pH optimum of 6.5. A strong light minus dark multiline EPR signal, arising from the S2 state of the oxygen-evolving complex (OEC), is observed at 10 K following illumination at 198 K. The determination of the absolute oxygen yield per saturating microsecond flash indicates that essentially all of the PSII centers contain functional oxygen-evolving complexes. This point is further supported by the absence of photoaccumulation, upon room temperature illumination, of the immediate oxidant of the OEC, redox-active tyrosine, YZ.. On the basis of EPR spectra, oxidized minus reduced difference spectra, and SDS-PAGE, the preparation contains on a per mole basis with PSII only trace amounts of PSI (approximately 0.04), cytochrome b6/f complex (< or = 0.01), and ATPase (< or = 0.05). All of these results indicate that this PSII preparation is to date the most highly purified oxygen-evolving core complex from Synechocystis 6803 that retains all of the reaction centers active for oxygen evolution. As Synechocystis 6803 is being used extensively for site-directed mutagenesis of PSII, this preparation is particularly valuable for spectroscopic and biochemical analyses of PSII from wild-type and from site-directed mutants.
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PMID:Biochemical and spectroscopic characterization of a new oxygen-evolving photosystem II core complex from the cyanobacterium Synechocystis PCC 6803. 816 15

A manganese-dependent peroxidase (MnP) from Phanerochaete chrysosporium catalyzed the reduction of cytochrome c in a reaction mixture containing H2O2, Mn(II)-tartrate, and p-hydroquinone. Electron spin resonance studies have shown that the hydroquinone-dependent reductive activity of MnP is due to the benzosemiquinone formed upon the one-electron oxidation of p-hydroquinone by Mn(III)-tartrate, which is formed upon the oxidation of Mn(II) by MnP. The reductive activity increased linearly with an increase in the concentration of p-hydroquinone. The reductive activity was also observed using other hydroquinones such as methylhydroquinone, 2,5-dimethylhydroquinone, and trimethylhydroquinone. The apparent Km values for Mn(II) and H2O2 for the hydroquinone-dependent reductive activity were similar to those for oxidative reactions of MnP. A stoichiometry study showed that about 1.5 mol of cytochrome c was reduced per mole of H2O2 consumed. The stoichiometry decreased with an increase in the concentration of H2O2. The optimal pH for the reductive activity was 5.0, approximately the physiological pH of the fungus. The reduction of cytochrome c was also observed using a quinone and cellobiose:quinone oxidoreductase isolated from the extracellular medium of the fungus.
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PMID:Reductive activity of a manganese-dependent peroxidase from Phanerochaete chrysosporium. 821 23

Reactive oxygen metabolites (ROMs) are thought to play a key role in the pathogenesis of the adult respiratory distress syndrome (ARDS). Accordingly, the use of ROM scavengers, such as N-acetyl-cysteine or dimethylthiourea, as therapeutic adjuncts to prevent oxidant-mediated damage to the lung have been evaluated extensively in animal models of ARDS. Results with this approach have been quite variable among studies. Another strategy that has been examined in animal models of ARDS is the administration of various enzymes, particularly superoxide dismutase (SOD) or catalase (CAT), in an effort to promote the conversion of ROMs to inactive metabolites. In theory, this strategy should be more effective than the use of ROM scavengers since a single molecule of a catalytically active molecule can neutralize a large number of molecules of a reactive species, whereas most scavengers act in a stoichiometric fashion to neutralize radicals on a mole-for-mole basis. This notion is supported by studies showing that prophylactic treatment with CAT provides impressive protection against acute lung injury induced in experimental animals by the administration of lipopolysaccharide (LPS). Results with SOD have been more variable. Recently, we have utilized a porcine model of LPS-induced ARDS to investigate the therapeutic potential of EUK-8, a novel, synthetic, low molecular salen-manganese complex that exhibits both SOD-like and CAT-like activities in vitro. Using both pre- and post-treatment designs, we have documented that treatment with EUK-8 significantly attenuates many of the features of LPS-induced acute lung injury, including arterial hypoxemia, pulmonary hypertension, decreased dynamic pulmonary compliance, and pulmonary edema. These findings support the view that salen-manganese complexes warrant further evaluation as therapeutic agents for treatment or prevention of sepsis-related ARDS in humans.
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PMID:Role of oxidant stress in the adult respiratory distress syndrome: evaluation of a novel antioxidant strategy in a porcine model of endotoxin-induced acute lung injury. 882 94


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