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Biomedical subjects

G D Watt

Publications and source records attributed to G D Watt.

At least 55 records · Page 3Linked to original sources
PubMed1986

Solubilization of the iron molybdenum cofactor of Azotobacter vinelandii nitrogenase in dimethylformamide and acetonitrile.

The iron molybdenum cofactor of Azotobacter vinelandii nitrogenase has been solubilized for the first time in dimethylformamide and acetonitrile. These solutions have the ability to reconstitute the inactive nitrogenase of the UW 45 mutant of A. vinelandii and exhibit an S = 3/2 EPR signal similar to that for the cofactor in N-methylformamide. Our ability to obtain solutions of FeMoco in these solvents seemingly refutes a previous hypothesis concerning the necessity of solvents with a dissociable proton for iron molybdenum cofactor solubility and should facilitate the spectroscopic characterization of this important species.

Acetonitriles↗
PubMed1986

Segregation analysis of bronchial response to methacholine inhalation challenge in families with and without asthma.

A segregation analysis was performed on the bronchial response to a standardized methacholine inhalation challenge obtained from members of 83 families that were part of a Natural History of Asthma study population. Each bronchial response was expressed as the area under the best fitting parabolic dose-response curve. Standard methods of statistical analysis demonstrated that age, sex, and recent respiratory infection had a significant effect on the bronchial response to methacholine inhalation. Segregation analysis indicated that, although a familial component exists in the transmission of bronchial response to methacholine, the bimodal distribution of the bronchial response is not due to segregation at a single autosomal locus.

Asthma↗
PubMed1986

Methacholine inhalation challenge studies in a selected pediatric population.

To determine bronchial reactivity patterns, 400 subjects, 5 to 21 yr of age, underwent a methacholine challenge in a Natural History of Asthma study. The diagnosis of asthma or allergy was based on a respiratory questionnaire. Subjects were nonsmokers and had had no respiratory infections for 1 month. Intradermal skin tests were done. The methacholine challenge response was expressed as the area beneath the dose-response curve (Area 35). Fifty-five asthmatics, 113 normal subjects from normal families, 103 normal subjects from asthma families, 60 normal twins, and 69 allergic subjects without asthma were studied. Overall, 52% of nonasthmatics and 47% of nonallergic subjects had an Area 35 less than 4,000 (800 breath units). There was a difference (p less than 0.05) in the distribution of methacholine Area 35 responses in normal subjects from that in normal families compared with normal subjects from asthma families. The age of the nonasthmatic subjects had an influence on the degree of bronchial reactivity. Methacholine challenge studies in pediatric patients must be interpreted with age, personal atopic status, and family asthma history in mind.

Adolescent↗
PubMed1985

Complex formation and O2 sensitivity of Azotobacter vinelandii nitrogenase and its component proteins.

The O2 stability of the MoFe protein, the Fe protein, a 1:1 mixture of these proteins, and a 1:1 mixture in the presence of the Azotobacter vinelandii FeS-II protein has been studied as a function of time under controlled O2 partial pressures. The Fe protein is much more sensitive to O2 exposure than is the MoFe protein. The presence of the FeS-II protein at a 1:1 ratio with the component proteins measurably increases the O2 stability of the MoFe and Fe proteins. O2 inactivation of the MoFe protein was studied in some detail and found to be quite complex. At least three partially overlapping reactions are suggested. The first is the reversible oxidation of the metal clusters of the MoFe protein to the combined extent of 12 electrons with full retention of activity. The second phase consists primarily of activity loss with little increase in the extent of reversible oxidation. The third phase continues to decrease the protein activity but is also accompanied by formation of a g = 2.0 EPR signal and more extensive oxidation. Ultracentrifugation studies of the FeS-II protein at a 1:1:1 ratio with the Fe and MoFe proteins do not support the formation of the Bulen complex. The formation of other O2-stable complexes is discussed.

Azotobacter↗
PubMed1985

Reduction of mammalian ferritin.

Mammalian ferritin from horse spleen undergoes an electrochemical or chemical reduction reaction in which each iron atom present is reduced by one electron (2300 electrons per ferritin molecule containing 2300 Fe3+ ions). Midpoint potentials of -190 mV, -310 mV, and -416 mV were determined at pH 7.0, 8.0, and 9.0. This variation of potential with pH indicates that approximately 2 H+ are transferred to the core for each Fe3+ reduced to Fe2+. Mössbauer measurements of partially reduced ferritin give spectra that consist of a ferric quadrupole doublet with a superposed ferrous quadrupole doublet. The relative intensities of these doublets are consistent with the electrochemically determined degree of reduction.

Animals↗
PubMed1984

Genetic analysis of allergic disease in twins.

One hundred seven pairs of twins, sixty-one MZT and forty-six DZT, were investigated for allergic disease by a questionnaire, reaginic antibody levels, bronchial reactivity to inhaled methacholine, and skin test responses. Intrapair correlation coefficients (ri) of measured clinical markers of atopy were determined and a heritability analysis was performed. The intrapair correlation coefficient for serum IgE was 0.82 for MZT and 0.52 for DZT. The methacholine area demonstrated greater correlation in MZT with an ri of 0.67 compared to 0.34 for DZT. The total ISTS had an intrapair correlation coefficient of 0.82 in MZT and 0.46 in DZT. Our analysis demonstrates that methacholine sensitivity, total serum IgE levels, and total skin test scores to be heritable traits and suggests a genetic contribution to their expression.

Adolescent↗
PubMed1984

H2-uptake activity of the MoFe protein component of Azotobacter vinelandii nitrogenase.

The MoFe protein from Azotobacter vinelandii catalyzes the reduction of methylene blue and other oxidants by H2 under anaerobic conditions. H2 uptake followed manometrically or by 3H2 transfer from the gas to aqueous phase occurs concomitantly with methylene blue disappearance monitored optically or coulometrically. The stoichiometry was found to be 1:1 methylene blue/H2. MoFe protein oxidized by transfer of approximately 4 e- seems to be the redox state of the protein most active in the catalytic step, although both the S2O4(2-)-reduced and 6-e- oxidized state have been shown to react, but at a much lower rate. The presence of H2 in the atmosphere above the MoFe protein offers increased protection against O2 inactivation.

Azotobacter↗
PubMed1981

Stoichiometry and spectral properties of the MoFe cofactor and noncofactor redox centers in the MoFe protein of nitrogenase from Azotobacter vinelandii.

Reductive EPR and optical titrations of oxidized MoFe protein using reduced methyl viologen as reductant were used to quantitate the stoichiometry of the various spectroscopically and electrochemically distinct redox centers in the oxidized MoFe protein. Three centers were found to correlate with the EPR signal development (MoFe cofactor centers), and three centers were found to be independent of the EPR signal (P clusters) but to demonstrate distinct optical and kinetic properties. Oxidative EPR and optical titrations of reduced MoFe protein are reported which support the presence of three P-cluster centers. The optical titrations show a distinct change in kinetic behavior between the MoFe cofactor and P-cluster centers. Controlled potential coulometry demonstrates that incremental oxidation of reduced protein by methylene blue, thionine, or indigodisulfonate occurs specifically at three P-cluster sites. Subsequent oxidation by methylene blue and thionine (but not indigodisulfonate) causes the EPR signal to disappear. Three P-cluster sites, two EPR sites, and one presently uncharacterized site are suggested by the results of this study.

Anaerobiosis↗
PubMed1980

Redox and spectroscopic properties of oxidized MoFe protein from Azotobacter vinelandii.

The MoFe protein from Azotobacter vinelandii undergoes a six-electron oxidation by various organic dye oxidants with full retention of initial activity. Reduction of the oxidized protein by S2O42- and by controlled potential electrolysis indicates the presence of two reduction regions at -290 and -480 mV, each requiring three electrons for complete reaction. Control of the oxidation conditions provides a means for preparing two distinct MoFe protein species selectively oxidized by three electrons. Selective reduction of the redox region at -290 mV causes development of the EPR signal associated with fully reduced MoFe protein while reduction at -480 mV produces a change in the visible spectrum but has no effect on the EPR signal intensity. Kinetic differences for reduction of the two redox regions indicate that the cofactor region undergoes a more rapid reaction with reductant than the other metal redox sites.

Azotobacter↗
PubMed1980

Isolation and characterization of two rubredoxins from Clostridium thermoaceticum.

Two rubredoxins with similar molecular weights (about 6000) have been purified from Clostridium thermoaceticum, a thermophile and strict anaerobe. They exhibit minor differences in several properties like elution pattern from DEAE-cellulose column, isoelectric point, amino acid composition, absorption and EPR spectra and redox potential. Their chemical and physical properties are similar to those of other rubredoxins from anaerobic microorganisms.

Amino Acids↗
PubMed1980

Protective effect opf lodoxamide tromethamine on allergen inhalation challenge.

Lodoxamide tromethamine (U-42,585E) is a new drug intended for prophylaxis of mast cell-mediated allergic disease. It is a water-soluble, cromolyn-like agent with demonstrated activity in rat peritoneal mast cell assay, rat percutaneous anaphylaxis (rat PCA) and sensitized rhesus monkey airway system. Ten allergen-sensitive asthmatics were pretreated with lodoxamide (0.01, 0.1, or 1.0 mg) or placebo, then challenged with serial dilutions of allergen extract. Analysis of allergen dose-response curve parameters shows that pretreatment with lodoxamide offers significant protection against experimental allergen-induced bronchoconstriction. At 0.01 mg, lodoxamide was effective in over half the subjects tested. Administration of lodoxamide by inhalation at doses of 0.1 and 1.0 mg uniformly allowed subjects to tolerate significantly larger doses of inhaled allergen. Side effects observed at these doses were minimal.

Adolescent↗
PubMed1977

Kinetics of dithionite ion utilization and ATP hydrolysis for reactions catalyzed by the nitrogenase complex from Azotobacter vinelandii.

The kinetics of S2O42-utilization and ATP hydrolysis during the nitrogenase-catalyzed H2 evolution and acetylene and nitrogen-reducing reactions were studied using a polarographic technique to monitor-S2O42-concentration. Rate constants for both S2O42-utilization and ATP hydrolysis were determined as a function of temperature and corresponding activation energies determined. The activation energy for ATP hydrolysis differs from that for product formation or S2O42-utilization by 5 kcal/mol above 20 degrees C and by 25 kcal/mol below 20 degrees C. The rate law for S2O42-utilization was determined and describes the enzyme catalyzed rate over a 1000-fold variation in S2O42-concentration and at least a 100-fold change in ATP concentration. The rate law for S2O42-utilization under N2-reducing conditions at 25 degrees C is given by -d([S2O42-]/dt = (2.3 x 10(-3) ET[S2O42-]1/2-[ATP]2)/([ATP]2 + K1[ATP] + K2), where ET is total enzyme concentration in mg/ml and K1 and K2 are equilibrium constants for ATP binding to nitrogenase. The half-order dependence of the rate on S2O42-concentration in interpreted in terms of the equilibrium S2O42- = 2SO2-, in which SO2- is the actual electron donor to nitrogenase. A partial mechanism incorporating these results is presented.

Acetobacter↗
PubMed1975

Stoichiometry, ATP/2e values, and energy requirements for reactions catalyzed by nitrogenase from Azotobacter vinelandii.

The stoichiometry of the nitrogenase ATP-dependent H2 evolution and ecetylene reduction reactions using S2O4(2-) as an electron source was studied by various techniques. For each mole of S2O4(2-) oxidized to 2SO3(2-) by the enzyme-catalyzed reactions at 25 degrees and pH 8, 1 mol of H2 (1 mol of ethylene for acetylene reduction) and two protons are produced. Under these conditions, 4.5 mol of ATP was hydrolyzed to ADP and inorganic phosphate for each S2O4(2-) oxidized. ATP/S2O4(2-) (ATP/2e) values determined at 5 degree intervals from 10 to 35 degrees were found to go through a minimum at 20 degrees. This effect is explained in terms of possible enzyme structure modifications. Calorimetric measurements for the enzyme-catalyzed H2 evolution and acetylene reduction reactions gave deltaH values of -32.4 and -75.1 kcal/mol of S2O4(2-), respectively.

Acetylene↗