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)

An overexpression system (pCR105) for DAHP synthase (Tyr) was constructed by cloning the aroF gene at the NdeI site of the pET-22b(+) translation vector, a plasmid expression vector that contains the T7 lac promoter. The enzyme was overexpressed, purified to > 90% purity (by SDS-polyacrylamide gel electrophoresis), and characterized. The protein was overexpressed at a level of 58% the total soluble cell protein (based on enzymatic activities). About 244 mg of pure enzyme was obtained from a 2-liter cell culture. So far, this is the highest yield reported for the isozyme DAHP synthase (Tyr). The enzyme showed a bell-shaped pH-activity profile, with a pH optimum at pH 7.0-7.5 and pK values of 6.10 and 8.92. Inhibition of the enzyme by tyrosine was specific with 50% inhibition observed at 9 microM tyrosine, pH 7.0. The specific activity of the enzyme increased with added metal and metal sensitivity increased with purity of the enzyme. Only substoichiometric amounts of Cu, Fe, and Zn were found in the pure enzyme and this result is consistent with sensitivity of the enzyme to added metal. Although treatment with EDTA inactivated the enzyme almost completely, the activity of the apoenzyme was restored to differing extents by a variety of metals including Mn2+, Cd2+, Co2+, Fe2+, Cu2+, Mg2+, and Zn2+. Both Fe2+ and Cu2+ only partially reactivated EDTA-treated enzyme. Reconstitution of EDTA-treated enzyme with either Cd2+ or Mn2+ gave 1 mol of metal per mole of enzyme monomer. KCN inactivated the enzyme to only 80% and added metals reactivated the CN-treated enzyme only to a small extent. These results confirm the importance of the metal in the enzymatic reaction.
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PMID:Overexpression, purification, and characterization of tyrosine-sensitive 3-deoxy-D-arabino-heptulosonic acid 7-phosphate synthase from Escherichia coli. 905 91

The gating and conduction properties of a channel activated by intracellular Na+ were studied by recording unitary currents in inside-out patches excised from lobster olfactory receptor neurons. Channel openings to a single conductance level of 104 pS occurred in bursts. The open probability of the channel increased with increasing concentrations of Na+. At 210 mm Na+, membrane depolarization increased the open probability e-fold per 36.6 mV. The distribution of channel open times could be fit by a single exponential with a time constant of 4.09 msec at -60 mV and 90 mm Na+. The open time constant was not affected by the concentration of Na+, but was increased by membrane depolarization. At 180 mm Na+ and -60 mV, the distribution of channel closed times could be fit by the sum of four exponentials with time constants of 0.20, 1.46, 8.92 and 69.9 msec, respectively. The three longer time constants decreased, while the shortest time constant did not vary with the concentration of Na+. Membrane depolarization decreased all four closed time constants. Burst duration was unaffected by the concentration of Na+, but was increased by membrane depolarization. Permeability for monovalent cations relative to that of Na+ (PX/PNa), calculated from the reversal potential, was: Li+ (1.11) > Na+ (1.0) > K+ (0.54) > Rb+ (0.36) > Cs+ (0.20). Extracellular divalent cations (10 mm) blocked the inward Na+ current at -60 mV according to the following sequence: Mn2+ > Ca2+ > Sr2+ > Mg2+ > Ba2+. Relative permeabilities for divalent cations (PY/PNa) were Ca2+ (39.0) > Mg2+ (34.1) > Mn2+ (15.5) > Ba2+ (13.8) > Na+ (1.0). Both the reversal potential and the conductance determined in divalent cation-free mixtures of Na+ and Cs+ or Li+ were monotonic functions of the mole fraction, suggesting that the channel is a single-ion pore that behaves as a multi-ion pore when the current is carried exclusively by divalent cations. The properties of the channel are consistent with the channel playing a role in odor activation of these primary receptor neurons.
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PMID:Gating and conduction properties of a sodium-activated cation channel from lobster olfactory receptor neurons. 907 48

The catalase-peroxidase of Mycobacteria smegmatis exhibits Mn(II)-peroxidase activity characterized by a low Km for Mn(II) (5 microM) and a high Km for t-butyl hydroperoxide (100 mM). This activity, monitored by the formation of Mn(III)-malate or -malonate, is inhibited by Co(II) but not by superoxide dismutase. Optical evidence for binding of Mn(II) to the resting (ferric) enzyme is found in a change in intensity of the Soret peak upon titration with Mn(II). A potential role for Mn(III) in the antimycobacterial action of the antibiotic isoniazid is suggested by the rapid reduction of Mn(III)-malonate by this drug. The stoichiometry of the reaction is consistent with two single electron transfer steps per mole of isoniazid.
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PMID:The role of Mn(II)-peroxidase activity of mycobacterial catalase-peroxidase in activation of the antibiotic isoniazid. 908 4

Carboxymethylcellulase (CMCase) was extracted and purified from an angiosperm parasite Cuscuta reflexa free from beta-glucosidase and other enzyme activities. The molecular mass and Stokes' radius of the purified enzyme are 144 kDa and 44 A, respectively. The diffusion coefficient and frictional ratio of the enzyme were 5.15 x 10(-7) cm2/sec and 1.27. The SDS-PAGE revealed homotetrameric nature of the enzyme with a subunit molecular mass of 35 +/- 1 kDa. Titration against DTNB and NBS revealed 19 sulfhydryl groups and 8 tryptophan groups, respectively, per mole of the enzyme. A sharp pH optimum at 5.0 was obtained. Cuscuta CMCase activity is unique amongst plant endoglucanases in being stimulated by Mg2+ and Mn2+ ions and by various thiols. Reaction product analysis, mode of enzyme action and substrate specificity test suggest the endo- nature of the purified CMCase. The enzyme showed K(m) value of 26 +/- 1 mg/ml for carboxymethylcellulose (sodium salt).
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PMID:Physico-chemical and functional characterization of a high molecular weight carboxymethylcellulase from Cuscuta reflexa. 949 45

Electron spin-echo envelope modulation (ESEEM) spectroscopy is widely used to investigate the active sites of biological molecules in frozen solutions. Various cryoprotection techniques, particularly the addition of co-solvents, are commonly employed in the preparation of such samples. In conjunction with ESEEM studies of Mn(II) guanosine nucleotide complexes of p21 ras, we have investigated the effects of cryoprotection on the spectroscopy, the structure, and the activity of this protein. Echo decay times, which typically govern ESEEM spectral resolution, were found to vary linearly with the concentration of glycerol or methyl alpha-D-glucopyranoside (MG), with both additives equally effective on a per-mole basis. The effect of glycerol and MG on the ESEEM amplitudes of various protein nucleiwas studied in ras p21.Mn(II). 5'guanylylimido-diphosphate(p21.Mn(II)-GMPPNP) complexes: these additives did not alter the distances of these nuclei from the Mn(II) ion. In particular, in p21 incorporating [2H-3]Thr, the Mn(II)-[2H-3]Thr35 distance was found to be unaffected by the concentration of cryoprotectant or the rate of freezing. The proximity of the cryoprotectants to the Mn(II) ion was probed by 2H ESEEM in solutions made with d5-glycerol and d7-methyl alpha-D-glucopyranoside (d7-MG). In p21.Mn(II)GMPPNP, the large deuterium modulations from the d5-glycerol exhibit saturation behavior with increasing d5-glycerol concentration, implying that glycerol, a widely used cryoprotectant, replaces the aquo ligands of the Mn(II) ion. The interaction between the Mn(II) ion of p21 and MG, however, is less intimate: the deuterium ESEEM amplitudes are much smaller for samples prepared with d7-MG than with d5-glycerol. Several polyhydroxylic compounds were found to have essentially no effect on the ability of the guanosine 5'-triphosphate (GTP) hydrolysis activating protein, GAP334, to catalyze hydrolysis of p21. guanosine 5'-triphosphate. This observation implies that the introduction of cryoprotectant does not significantly perturb the structure of p21 and gives insight into the mechanism of the GTPase reaction.
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PMID:The effects of cryoprotection on the structure and activity of p21 ras: implications for electron spin-echo envelope modulation spectroscopy. 974 Jul 40

A new procedure for loading doxorubicin into large unilamellar vesicles (LUVs) is characterized. It is shown that doxorubicin can be loaded into LUVs composed of sphingomyelin/cholesterol (55:45 mole/mole) in response to a transmembrane MnSO4 gradient in the absence of a transmembrane pH gradient. Complex formation between doxorubicin and Mn2+ is found to be a driving force for doxorubicin uptake. Uptake levels approaching 100% can be achieved up to a drug-to-lipid molar ratio of 0.5 utilizing an encapsulated MnSO4 concentration of 0.30 M. In vitro leakage assays show excellent retention properties over a 24 h period. The possible advantages of a liposomal formulation of doxorubicin loaded in response to entrapped MnSO4 are discussed.
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PMID:Loading of doxorubicin into liposomes by forming Mn2+-drug complexes. 980 55

Dantrolene sodium is a medically important hydantoin derivative that interferes with release of Ca2+ from intracellular stores of skeletal muscle by an unknown mechanism. Identification of the molecular target of dantrolene would greatly aid in understanding both the mechanism of action of the drug and the dynamics of intracellular Ca2+ release in muscle. [3H]Azidodantrolene was designed and synthesized as a photoaffinity analogue in order to identify a putative dantrolene receptor in skeletal muscle. Introduction of 1 mole-atom of tritium into aldehyde 5b was required during radioligand synthesis in order to ensure high enough specific activity for detection of photo-cross-linked proteins by fluorographic methods. This was accomplished by reduction of ester 3 with custom synthesized, 100% tritium-labeled lithium triethylborotritide, followed by oxidation to 5b by manganese(IV) oxide. Compound 6b was demonstrated to be >/=95% tritium-labeled at the imine position by NMR spectroscopy, and the specific radioactivity of [3H]azidodantrolene sodium was empirically determined by HPLC and liquid scintillation counting to be 24.4 Ci/mmol, approximately 85% of theoretical maximum. [3H]Azidodantrolene was found to be pharmacologically active in ligand-receptor binding studies with skeletal muscle sarcoplasmic reticulum membranes. Photo-cross-linking experiments analyzed by SDS-PAGE and tritium fluorography have identified a approximately 160-kDa specifically labeled protein as the putative, intracellular, skeletal muscle dantrolene receptor. This photolabeled protein comigrates with a protein in Western blots immunologically cross-reactive to a polyclonal anti-rabbit skeletal muscle ryanodine receptor antibody. Thus, the putative dantrolene receptor may be related to the skeletal muscle ryanodine receptor.
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PMID:[3H]Azidodantrolene: synthesis and use in identification of a putative skeletal muscle dantrolene binding site in sarcoplasmic reticulum. 1035 95

The B-Z transition of the synthetic oligonucleotide, (dG-dC)20, induced by Mn2+ ions at room temperature, was investigated by absorption and Vibrational Circular Dichroism (VCD) spectroscopy in the range of 1800-800 cm(-1). Metal ion concentration was varied from 0 to 0.73 M Mn2+ (0 to 8.5 moles of Mn2+ per mole of oligonucleotide phosphate, [Mn]/[P]). While both types of spectra showed considerable changes as the Mn2+ concentrations were raised, differences between the two were often complementary in their expression and extent, those displayed by VCD being more clearly evident due to the inversion of the opposite helical sense from the right-handed to the left-handed conformation. The main phase of the transition occurred in the metal ion concentration between 0.8-1.1 [Mn]/[P]. Gradual changes that took place in the spectra were interpreted in terms of simultaneous processes that depended on metal ion concentration, namely B-Z transformation, binding of Mn2+ to phosphates and to nitrogen bases, and partial denaturation. Below approximately 0.6 [Mn]/[P], only a small portion of the oligonucleotide adopted the Z conformation within a 3 hour period, whereas conversion was completed in the same time interval for concentrations between 0.9-1.2 [Mn]/[P]. At [Mn]/[P] >1.7, complete transition to the Z-form took place immediately on adding Mn2+. Applying VCD spectroscopy in combination with conventional infrared absorption proved most useful for corroborating changes in the absorption spectra, and for detecting in an unique manner, not attainable by absorption methods, conformational changes that lead to the inversion of the helical sense of the oligonucleotide.
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PMID:Complexes of (dG-dC)20 with Mn2+ ions: a study by vibrational circular dichroism and infrared absorption spectroscopy. 1063 89

It has recently been noted that a diversity of hyperthermophilic microorganisms have the ability to reduce Fe(III) with hydrogen as the electron donor, but the reduction of Fe(III) or other metals by these organisms has not been previously examined in detail. When Pyrobaculum islandicum was grown at 100 degrees C in a medium with hydrogen as the electron donor and Fe(III)-citrate as the electron acceptor, the increase in cell numbers of P. islandicum per mole of Fe(III) reduced was found to be ca. 10-fold higher than previously reported. Poorly crystalline Fe(III) oxide could also serve as the electron acceptor for growth on hydrogen. The stoichiometry of hydrogen uptake and Fe(III) oxide reduction was consistent with the oxidation of 1 mol of hydrogen resulting in the reduction of 2 mol of Fe(III). The poorly crystalline Fe(III) oxide was reduced to extracellular magnetite. P. islandicum could not effectively reduce the crystalline Fe(III) oxide minerals goethite and hematite. In addition to using hydrogen as an electron donor for Fe(III) reduction, P. islandicum grew via Fe(III) reduction in media in which peptone and yeast extract served as potential electron donors. The closely related species P. aerophilum grew via Fe(III) reduction in a similar complex medium. Cell suspensions of P. islandicum reduced the following metals with hydrogen as the electron donor: U(VI), Tc(VII), Cr(VI), Co(III), and Mn(IV). The reduction of these metals was dependent upon the presence of cells and hydrogen. The metalloids arsenate and selenate were not reduced. U(VI) was reduced to the insoluble U(IV) mineral uraninite, which was extracellular. Tc(VII) was reduced to insoluble Tc(IV) or Tc(V). Cr(VI) was reduced to the less toxic, less soluble Cr(III). Co(III) was reduced to Co(II). Mn(IV) was reduced to Mn(II) with the formation of manganese carbonate. These results demonstrate that biological reduction may contribute to the speciation of metals in hydrothermal environments and could account for such phenomena as magnetite accumulation and the formation of uranium deposits at ca. 100 degrees C. Reduction of toxic metals with hyperthermophilic microorganisms or their enzymes might be applied to the remediation of metal-contaminated waters or waste streams.
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PMID:Reduction of Fe(III), Mn(IV), and toxic metals at 100 degrees C by Pyrobaculum islandicum. 1069 70

1. The separation of 0.9-S and 10.8-S allantoicase with the aid of a 2H2O-H2O gradient was described. The resulting preparations were subjected to sedimentation equilibrium, optical rotatory dispersion (ORD), circular dichroism (CD) and infrared studies. 2. The molecular weight of 0.9-S allantoicase was determined to be about 1.1 x 10(4) g/mole in studies on the sedimentation behavior, the metal content and amino acid composition. The molecular weight of 10.8-S allantoicase was about 15.4 x 10(4) g/mole. 3. Optical rotatory dispersion, circular dichroism and infrared studies indicated that both molecules contain alpha-helix, beta conformation and random coil. A Cotton effect at 418 nm was ascribed to the asymmetric binding of Mn2+ to the enzyme. Competitive inhibitors decreased the absorption and circular dichroism bands at about 280 nm and 418 nm. These phenomena suggested that the aromatic groups may play an essential role in the binding of substrates and inhibitors by the Mn(2+)-enzyme complex. 4. Comparison of alpha-helical contents of metalloallantoicases showed that the enzymes with low helical contents exhibited high enzymic activities. 5. The nearly identical physicochemical behavior and specific enzymic activity of 0.9-S and 10.8-S allantoicase indicated that they are very similar in structure and conformation.
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PMID:Conformation of allantoicase in aqueous solution. 1145 82


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