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Biomedical subjects

C H Williams

Publications and source records attributed to C H Williams.

At least 127 records · Page 7Linked to original sources
PubMed1987

An improved spectrophotometric assay for leucine aminopeptidase.

A sensitive assay to determine the activity of leucine aminopeptidase (EC 3.4.11.1), using L-leucine thiobenzyl ester as substrate, was developed. Hydrolysis of the ester by leucine aminopeptidase can be monitored in the presence of 5,5-dithiobis-(2-nitrobenzoic acid) by continuous spectrophotometric measurement at 412 nm. Comparison with some amide substrates showed that the thiol ester provides a much more sensitive assay, its specificity constant (Vmax./Km) being some 3000-fold higher than that of leucine p-nitroanilide.

Animals↗
PubMed1987

Influence of steroids on hemorrhagic and traumatic shock.

A hemorrhagic-traumatic shock model in 20 domestic pigs (Sus scrofa) was used to evaluate the pharmacologic effect of methylprednisolone in preventing disseminated intravascular coagulation (DIC). Pairs of animals were anesthetized with thiopental and both femoral arteries cannulated. Four ml/kg-1 of blood were withdrawn, frozen, thawed, and returned to the animal. One femoral artery cannula was connected to a blood pressure monitor for arterial systolic, diastolic, and mean pressures along with heart rate and ECG. The other catheter was used to bleed the animal down to and maintain a mean arterial pressure of 50 mm Hg for 2 hours. Blood samples were taken during the control period before bleeding, and at the end of the 2-hour shock period. Blood samples were analyzed for prothrombin time, partial thromboplastin time, fibrinogen level, and platelet count. The combination of hemorrhagic shock and hemolysis produced DIC and a fatal shock. Pharmacologic doses (30 mg/kg-1) of methylprednisolone significantly prevent the DIC.

Adrenal Cortex Hormones↗
PubMed1987

[Atracurium and vecuronium: interaction with three orally administered calcium antagonists in animal experiments].

Interactions between calcium antagonists (CA) and muscle relaxants have been reported however due to the extremely high dosages of CA used in these studies, the clinical relevance of the observed interactions has been questioned. In order to simulate clinical practice--which entails long-term oral treatment and significant reductions in systolic blood pressure--CA were administered to rats orally (by gavage) for 8 days at a dosage that induced a systolic blood pressure decrease of not more than 25 +/- 5 mmHg. Ten groups of six male Sprague-Dawley rats each, body weight (BW) 313 +/- 23 g, were subjected to oral administration of three different CA: nitrendipine (1 mg/kg BW), nisoldipine (1 mg/kg BW), and diltiazem (100 mg/kg BW) or their solvents (control) three times daily by gavage for a period of 8 days. One hour after the last dose of drug or solvent, the rats were anesthetized (pentobarbital 60 mg/kg BW) and mechanically ventilated via tracheostomy. Monitoring included rectal temperature, carotid artery pressure, central venous pressure, heart rate, blood gases, and end-tidal CO2. Evoked train-of-four (T4) twitch tension of the right tibialis anterior muscle was recorded continuously. After equilibration of vital signs and muscle twitch tension, vecuronium (150 micrograms/kg BW) or atracurium (500 micrograms/kg BW) was injected into the internal jugular vein four times at 5-min intervals after twitch tension had totally recovered and T4 fade had disappeared. Twitch depression, duration 90, and onset time of neuromuscular block in CA-pretreated animals were compared to control animals.(ABSTRACT TRUNCATED AT 250 WORDS)

Administration, Oral↗
PubMed1986

Electrophilic amination of a single methionine residue located at the active site of D-amino acid oxidase by O-(2,4-dinitrophenyl)hydroxylamine.

O-(2,4-Dinitrophenyl)hydroxylamine is a rapid active-site-directed inhibitor of D-amino acid oxidase: modification results in specific incorporation of an amine group into an accessible nucleophilic residue with concomitant release of 2,4-dinitrophenol. The reaction is prevented by the competitive inhibitor benzoate, indicating an active-site-directed reaction. A stoichiometry of 1-1.5 mol of amine residues per enzyme bound flavin adenine dinucleotide monomer was observed at pH 7.0. Amino acid and sequence analyses show that His-217 is not the target of the modification reaction. Dependence of the modification on pH, model studies on functional groups present on amino acids, and thiolysis studies on aminated enzyme collectively indicate that the modification is located on a methionine residue at or near the active site of the enzyme. Aminated enzyme, although spectrally similar to native enzyme, exhibits a 7-9-nm blue shift in the 455-nm flavin absorption. Benzoate perturbs the spectrum of aminated enzyme, but binding relative to native enzyme is much weaker (Kd ca. 300 times greater at pH 8.0).

Animals↗
PubMed1986

Two-electron reduced mercuric reductase binds Hg(II) to the active site dithiol but does not catalyze Hg(II) reduction.

Mercuric reductase contains FAD and a redox-active disulfide which is reduced to a thiol/thiolate pair in two-electron reduced enzyme (EH2) (Fox, B. and Walsh, C.T. (1982) J. Biol. Chem. 257, 2498-2503). A charge transfer interaction between the thiolate and oxidized FAD gives EH2 a characteristic absorption spectrum, very similar to that found with other flavoprotein disulfide oxidoreductases. We have examined the reaction of EH2 with HgCl2 (+/- mercaptoethanol) in stopped-flow kinetic and static titration experiments. In the absence of mercaptoethanol, reaction of EH2 with HgCl2 yields a final spectrum which is indistinguishable from that of oxidized enzyme. The nature of the final species was examined by titration of enzyme thiols with 5,5'-dithiobis-2,2'-nitrobenzoic acid under denaturing conditions in the presence of NaI to displace any Hg(II) bound to enzyme thiols. These studies demonstrate that EH2 tightly complexes Hg(II) with its active site thiols, but is incapable of reducing Hg(II) to Hg0. For the latter reaction to occur, additional reducing equivalents are required. In catalysis, the enzyme must first be reduced to EH2 after which it cycles between EH2 and EH2 X NADPH forms. This is in contrast to other flavoprotein disulfide oxidoreductases which cycle between Eox and EH2 forms in catalysis (Williams, C. H., Jr. (1976) in The Enzymes (Boyer, P. D., ed) 3rd Ed., Vol. 13, pp. 89-173, Academic Press, New York). With mercuric reductase, exogenous thiols are required for catalytic reduction of Hg(II) to Hg0. We have shown that this is due to prevention or reversal of formation of an abortive complex of Hg(II) with the thiol/thiolate pair of EH2.

Binding Sites↗
PubMed1985

Reaction of both active site thiols of reduced thioredoxin reductase with N-ethylmaleimide.

Thioredoxin reductase from Escherichia coli, only in its reduced state, reacts rapidly with 2 mol of N-ethylmaleimide, which specifically alkylates both active site cysteine residues. This dual modification supports previous studies indicating that a base lowers the pK of both active site cysteine residues. The dual modification also indicates that the region around the active site dithiol is more open than is the case with the related enzymes lipoamide dehydrogenase and glutathione reductase, both of which can be alkylated only on one nascent thiol. Enhanced nucleophilicity of the active site thiols is consistent with the proposed chemical mechanism of thioredoxin reductase. The sequence of the amino-terminal 16 residues is presented.

Amino Acid Sequence↗
PubMed1985

Plasma levels of norepinephrine and epinephrine during malignant hyperthermia in susceptible pigs.

Malignant hyperthermia (MH) is a genetic disease of man, swine, dogs, cats, and horses. The syndrome is normally triggered by inhalational anesthetics or the administration of depolarizing muscle relaxants such as succinylcholine or various environmental stress factors. We have used the MH-susceptible pig as an animal model to study the hormonal changes developing during this highly lethal syndrome. High-performance liquid chromatography with electrochemical detection was used for the quantitation of the plasma levels of norepinephrine and epinephrine during MH. This research presents evidence that the rapid release of massive quantities of norepinephrine (up to 108 ng/ml) into the blood stream occurs simultaneously with the initiation of tachycardia which is the herald signal of the onset of MH. Norepinephrine levels exceed epinephrine by a 4:1 ratio early in the syndrome. Even pigs with MH which do not develop the muscle rigor phase have high levels of circulating norepinephrine. Tachycardia, pulmonary hypertension, increased venous oxygen desaturation, and increasing core temperature develop as the syndrome progresses.

Animals↗
PubMed1985

Diltiazem inhibits halothane-induced contractions in malignant hyperthermia-susceptible muscles in vitro.

The ability of diltiazem to suppress halothane and halothane-caffeine induced contractures in malignant hyperthermia (MH) susceptible pig muscle, was tested in vitro. Muscle specimens were divided into two groups and tested with a modified halothane-caffeine contracture test. One group acted as the control; the other group was pretreated with diltiazem 20 mumol litre-1. The control muscles developed contractures attributable to halothane and halothane-caffeine, whereas the diltiazem-treated specimens did not. Increases in muscle twitch tension as a result of halothane or halothane-caffeine exposure occurred in treated and untreated specimens, but were significantly delayed in the presence of diltiazem. Muscle exhaustion observed after halothane and halothane-caffeine exposure in the control specimens did not occur in the diltiazem treated muscles.

Animals↗
PubMed1985

Vecuronium and porcine malignant hyperthermia.

Vecuronium was studied in eight malignant hyperthermia (MH) susceptible pigs for its potential to either trigger or prevent MH. Two sets of experiments were performed in the same animals: 1-hr total neuromuscular blockade by vecuronium infusion with thiopental anesthesia in the absence of invasive monitoring and halothane; and 1-hr infusion of vecuronium with thiopental anesthesia with invasive monitoring in the absence of and then, followed by 30-min infusion in the presence of halothane, followed in turn by exposure to halothane alone. One-hour infusion of vecuronium in the absence of halothane and invasive monitoring did not trigger MH in any animal. During the second set of experiments, MH, evidenced by rising rectal temperature, elevated end-tidal PCO2, mixed venous oxygen desaturation and muscle rigor, occurred in one animal during vecuronium alone, in four animals during vecuronium infusion and simultaneous exposure to halothane, and in three animals during exposure to halothane alone after recovery from vecuronium neuromuscular blockade. In view of the results of control experiments, the development of MH during vecuronium neuromuscular blockade before exposure to halothane was attributed to surgical stress rather than to vecuronium itself. It is concluded that vecuronium is not a trigger to MH in susceptible pigs.

Anesthesia, General↗
PubMed1984

Methylation of the active center histidine 217 in D-amino acid oxidase by methyl-p-nitrobenzenesulfonate.

Incubation of D-amino acid oxidase with excess methyl-p-nitrobenzenesulfonate results in a pseudo-first order, irreversible loss of 95% of the assayable activity using D-phenylglycine as substrate. The rate of inactivation reaches a limiting value of 0.021 min-1 (pH 7.7, 22 degrees C) as the concentration of inhibitor is increased, consistent with a two-step mechanism with complex formation prior to covalent inactivation. This rate decreases with decreasing pH in a manner that suggests that the basic form of a group within the enzyme, with an apparent pKa of 6.7, is required for inactivation. Approximately three [14C]methyl residues are covalently incorporated at maximal inactivation. The competitive inhibitor benzoate prevents inactivation and the incorporation of approximately 1 methyl residue. Histidine 217, the residue modified by 5-dimethylaminonaphthalene-1-sulfonyl chloride (Swenson, R. P., Williams, C. H., Jr., and Massey, V. (1983) J. Biol. Chem. 258, 497-502), was identified as the major residue modified in the absence of benzoate. Both the 1-methyl and 3-methyl derivatives of histidine 217 were observed in a mole ratio of 0.64:0.36.

Amino Acids↗
PubMed1984

Reconstitution of Escherichia coli thioredoxin reductase with 1-deazaFAD. Evidence for 1-deazaFAD C-4a adduct formation linked to the ionization of an active site base.

The flavin prosthetic group (FAD) of thioredoxin reductase has been replaced by 1-deazaFAD (carbon substituted for nitrogen at position 1). Reduction of 1-deazaFAD-thioredoxin reductase by four electrons proceeds in two stages having midpoint potentials that are separated by 0.063 V. Two-electron reduced 1-deazaFAD-thioredoxin reductase (EH2) has spectral characteristics that are different from both the fully oxidized and fully reduced enzyme. The fluorescence of the 2-electron reduced enzyme shows a mixture of two EH2 species. The spectrum of one EH2 species has a single absorption peak (lambda max, 414 nm; epsilon 414, 8750 M-1 cm-1) which is similar to the spectrum of 1-deazaFAD-C-4a adducts (referred to as the 414-nm absorbing species). In the other EH2 species the electrons are in the dithiol, and it has an oxidized 1-deazaFAD spectrum (referred to as the 550-nm EH2 species). The equilibrium between the two EH2 species of 1-deazaFAD-thioredoxin reductase is pH dependent, forming more of the 414-nm absorbing species as the pH is lowered. The pH dependence suggests the presence of an active center base having a pK of 7.41 on the 414-nm EH2 species and a thiol of pK 6.73 on the 550-nm EH2 species. These pK values are similar to the pK values determined for native enzyme having a disulfide or a dithiol (7.59 and 6.98, respectively). Thus, the pH dependence of the equilibrium between the two EH2 species of 1-deazaFAD-thioredoxin reductase is further evidence for an active site base with an ionization behavior that is linked to the chemical state of the active site disulfide moiety. The nature of the linked ionization is consistent with a thiol base ion pair formed upon disulfide reduction.

Binding Sites↗
PubMed1984

Graphical analysis of interactions between oxidation-reduction sites in two site oxidation-reduction proteins.

Many enzymes that catalyze electron-transfer reaction contain multiple oxidation-reduction centers (sites). The oxidation-reduction potential of one site as well as the kinetics of electron transfer through this site may be altered by the state of reduction of a neighboring site. Oxidation-reduction site interactions may be mechanistically important and quantitation of site interactions would aid the interpretation of thermodynamic data and possibly kinetic data. A graphical means to detect and quantitate interactions between oxidation-reduction sites from oxidation-reduction equilibrium data (type A + B in equilibrium C + D) is described and has its roots in the Scatchard analysis of ligand binding equilibria (type A + B in equilibrium C). Oxidation-reduction sites often have distinct physical properties allowing the titration behavior of specific sites to be monitored. Equilibrium measurements on specific sites of a two site protein allow a further analysis of the data which can be combined with the oxidation-reduction Scatchard analysis to solve for all four specific site equilibrium constants. Ligand binding systems can usually measure only total site binding and simplifying assumptions of identical sites or noninteracting sites are required to solve for the site specific equilibrium constants. Thus, specific site equilibrium measurements offer a distinct advantage over total site measurements. The principles of the method are illustrated by applying the graphical analysis to the two site protein, thioredoxin reductase, which contains an oxidation-reduction active site disulfide in addition to FAD. The specific site oxidation-reduction midpoint potentials (Em) of the FAD and disulfide couples of thioredoxin reductase at pH 6.0, 12 degrees C, were found to be FAD/FADH2-enzyme-(S)2 = -0.183 V, FAD/FADH2-enzyme-(SH)2 = -0.199 V, (FAD)-enzyme-(S)2/(SH)2 = -0.202 V, and (FADH2)-enzyme-(S)2/(SH)2 = -0.218 V. Hence, at pH 6.0, the FAD and disulfide sites of thioredoxin reductase have Em values that differ by approximately 0.019 V and have a negative interaction of about 0.016 V.

Binding Sites↗
PubMed1983

Proton stoichiometry in the reduction of the FAD and disulfide of Escherichia coli thioredoxin reductase. Evidence for a base at the active site.

The oxidation-reduction midpoint potentials, Em, of the FAD and active site disulfide couples of Escherichia coli thioredoxin reductase have been determined from pH 5.5 to 8.5. The FAD and disulfide couples have similar Em values and thus a linked equilibrium of four microscopic enzyme oxidation-reduction states exists. The binding of phenylmercuric acetate to one enzyme form could be monitored which allowed solving the four microscopic Em values. The Em values at pH 7.0 and 12 degrees C of the four couples of thioredoxin reductase are: (S)2-enzyme-FAD/FADH2 = -0.243 V, (SH)2-enzyme-FAD/FADH2 = -0.260 V, (FAD)-enzyme-(S)2/(SH)2 = -0.254 V, and (FADH2)-enzyme-(S)2/(SH)2 = -0.271 V. Thus, at pH 7.0, the FAD and disulfide moieties have a 0.017-V negative interaction and Em values which are different by 0.011 V. The delta Em/delta pH of the FAD couples E2m and E3m are about 0.060 V/pH throughout the pH range studied, showing an approximately 2-proton stoichiometry of reduction of the enzyme FAD. The delta Em/delta pH of the disulfide couples E1m and E4m are about 0.052 V/pH from pH 5.5 to 8.5, showing an apparently nonintegral proton stoichiometry of reduction of 1.8 in this pH range. This proton stoichiometry suggests the presence of a base with an ionization behavior that is linked to the oxidation-reduction state of the disulfide. A novel method is presented for determining the pK values on oxidized and reduced enzyme which agrees with the less accurate classical method. The proton stoichiometry results are consistent with the presence of a thiol-base ion pair in which the pK of the base is elevated from 7.6 in disulfide containing enzyme to greater than 8.5 upon forming an ion pair with a thiol anion of pK 7.0 generated upon reduction of the disulfide. The fluorescence of the FAD in thioredoxin reductase decreases as the pH is lowered with a pK of 7.0, direct evidence for a base near the FAD probably distinct from the base interacting with the dithiol.

Binding Sites↗