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Biomedical subjects

C H Williams

Publications and source records attributed to C H Williams.

At least 145 records · Page 8Linked to original sources
PubMed1983

Studies of the ferredoxin from Thermus thermophilus.

The soluble ferredoxin from Thermus thermophilus was examined by Mössbauer and EPR spectroscopies and by reductive titrations. These studies demonstrate the presence of one 3Fe center, responsible for the characteristic g = 2.02 EPR signal in the oxidized protein, and one [4Fe-4S] center which is responsible for the rhombic EPR spectrum of the fully reduced protein. These assignments should replace those made by Ohnishi et al. (Ohnishi, T., Blum, H., Sato, S., Nakazawa, K., Hon-nami, K., and Oshima, T. (1980) J. Biol. Chem. 255, 345-348) prior to the discovery of the 3Fe clusters. The amino acid composition was determined and is discussed with reference to recent structural studies of 7Fe ferredoxins.

Amino Acids↗
PubMed1983

Proton nuclear magnetic resonance investigation of the mechanism of flavin C-4a adduct formation induced by oxidized nicotinamide adenine dinucleotide binding to monoalkylated pig heart lipoamide dehydrogenase.

The active center thiol of monoalkylated pig heart lipoamide dehydrogenase, EHR, is induced to form an adduct to the enzyme-bound flavin adenine dinucleotide (FAD) at the C-4a position upon binding oxidized nicotinamide adenine dinucleotide (NAD+) [Thorpe, C., & Williams, C. H., Jr. (1976) J. Biol. Chem. 251, 7726-7728]. In light of hypotheses on covalent electron transfer between pyridine nucleotide and flavin, the induction of the thiol-flavin C-4a adduct by NAD+ is reasonably envisioned as involving a covalent bond between the modified flavin and the NAD+. The double-resonance proton nuclear magnetic resonance technique of cross saturation was used to probe the existence of covalent bond formation between the modified flavin of EHR and its inducer molecule, NAD+. Cross-saturation of the free NAD+ signals was not observed even though the spin-lattice relaxation time of NAD+ and the rate of exchange between free NAD+ and NAD+ bound to EHR were well within the limits required for cross-saturation. We conclude that a noncovalent interaction between NAD+ and FAD induces the formation of the thiol-flavin C-4a covalent adduct in EHR. A model by which NAD+ binding induces nucleophilic attack by the nascent thiolate of EHR is discussed.

Animals↗
PubMed1982

The primary structure of D-amino acid oxidase from pig kidney. I. Isolation and sequence of the tryptic peptides.

D-Amino acid oxidase from pig kidney cortex was digested with trypsin. Thirty-two tryptic peptides were isolated by ion exchange chromatography, high voltage paper electrophoresis, descending paper chromatography, and reverse-phase high performance liquid chromatography. The last method permitted the isolation of 29 tryptic peptides, many in a single step, in yields usually greater than 75%. The purified peptides were characterized by amino acid analysis and their sequences determined by the manual 5-dimethylaminonaphthalene-1-sulfonyl-Edman degradation procedure or by the automated Edman-Begg degradation method. These peptides accounted for all 12 lysine and 21 arginine residues observed by amino acid analysis of the intact protein and for 347 amino acid residues of the 345 predicted by the analysis.

Amino Acid Sequence↗
PubMed1982

A new specific inhibitor of monoamine oxidase A.

A new propynylamine has been tested as an inhibitor of MAO. Loosely based upon the structure of clorgyline, it is an irreversible inhibitor of MAO A but is apparently indifferent towards MAO B. This compound, N1-(2,4-dinitrophenyl)-N2-prop-2-ynyl 1,3-diaminopropane, may be useful as a model for the design of more potent but equally specific inhibitors of MAO A.

Animals↗
PubMed1982

Pre-column derivatization and high-performance liquid chromatography of biogenic amines in blood of normal and malignant hyperthermic pigs.

A sensitive, selective, pre-column derivatization method was used with high-performance liquid chromatography to measure norepinephrine, dopamine and serotonin in plasma from normal and malignant hyperthermic (MH) pigs. Samples were carefully collected from control and stressed animals under halothane anesthesia. Using a simple extraction method involving pre-column derivatization with o-phthaladehyde and ethyl acetate partitioning, the samples were chromatographed in less than 50 min. Norepinephrine was found to be elevated in MH pigs as the syndrome progressed, reaching levels eight-fold greater than control pigs under anesthesia. These experiments provide some evidence for our hypothesis that a failure to metabolize excess norepinephrine may be one of the key metabolic defects in causing the pathophysiology of the malignant hyperthermia-stress syndrome. The application of our chromatographic method in animal and human tests may provide a pattern of biogenic amine types and levels that could be diagnostic in identifying susceptible humans and carrier animals.

Animals↗
PubMed1982

Chemical modification of D-amino acid oxidase. Amino acid sequence of the tryptic peptides containing tyrosine and lysine residues modified by fluorodinitrobenzene.

D-Amino acid oxidase can be inactivated by covalent modification of predominantly tyrosine residue(s) at pH 7.4 by a low molar excess of fluorodinitrobenzene, which appears to act as an active site-directed reagent (Nishino, T., Massey, V., and Williams, C. H., Jr. (1980) J. Biol. Chem. 255, 3610-3616). Peptide mapping by high performance liquid chromatography of tryptic digests of protein modified with radiolabeled reagent revealed two major radioactive fractions with substantially different retention times which were not observed in protein modified in the presence of benzoate, a potent competitive inhibitor. Isolation and sequence analysis of the major radiolabeled peptides, as well as other direct chemical analyses, are presented which unambiguously demonstrate that these fractions represent modification of two different regions of the protein. The majority of the radiolabel was found within a 61-amino acid residue peptide containing an O-DNP-tyrosine residue exclusively at position 17. The substantial sequence surrounding this tyrosine residue indicates that it is different from that shown to react with N-chloro-D-leucine (Ronchi, S., Galliano, M., Minchiotti, L., Curti, B., Rudie, N. G., Porter, D. J. T., and Bright, H. J. (1980) J. Biol. Chem. 255, 6044-6046). The second fraction consisted of a 12-residue peptide containing an epsilon-DNP-lysine residue at position 5. Together, these two modified amino acids represented 0.89 mol of DNP incorporated/protein monomer. Both modifications must contribute to inactivation to account for the 90% decrease in enzymatic activity. Evidence is presented which suggests that both groups are within the active center of the enzyme and are modified in a mutually exclusive manner.

Amino Acid Sequence↗
PubMed1982

Amino acid sequence homology between pig heart lipoamide dehydrogenase and human erythrocyte glutathione reductase.

Extensive amino acid sequence homology has been found between nine tryptic peptides of pig heart lipoamide dehydrogenase (NADH:lipoamide oxidoreductase, EC 1.6.4.3] and the sequence of human erythrocyte glutathione reductase [NAD(P)H:glutathione oxidoreductase, EC 1.6.4.2]. The average homology is 40%. Six lipoamide dehydrogenase peptides are homologous with segments of the two parts of the FAD domain of glutathione reductase, one with the NADPH domain, and two with the interface domain. Thus, the homology extends throughout the molecule.

Amino Acid Sequence↗
PubMed1982

Bilingual districts as an instrument in Canadian language policy.

"Accommodation of a multi-ethnic population within the territory of a single state has been recognized as a world-wide problem. Within such a society language maintenance is frequently the avenue through which cultural groups seek to preserve their identity. This paper describes and evaluates the Canadian experience of language planning by concentrating on the bilingual district, the key spatial instrument in the federal Official Languages Act. The paper argues for a cautious and spatially varied application of the 1969 Act and seeks to suggest ways in which a geolinguistic analysis may further our understanding of plurilingual societies."

Americas↗
PubMed1981

Glutathione reductase from yeast. Differential reactivity of the nascent thiols in two-electron reduced enzyme and properties of a monoalkylated derivative.

Two-electron reduced glutathione reductase from yeast reacted with iodoacetamide is alkylated almost exclusively in the nascent thiol nearer the amino terminus of the protein. The charge-transfer absorbance, maximal at 530 nm, characteristic of the two-electron reduced enzyme is not lost as the alkylation proceeds, and the product has a spectrum virtually identical with that of the two-electron reduced enzyme. This observation demonstrates that the thiol alkylated is not the charge-transfer-donor thiolate which interacts with the FAD. The spectrum of the monoalkylated derivative is stable in the presence of oxidized glutathione, indicating that the charge-transfer-donor thiol is not involved in interchange with the substrate in the native enzyme. Thus, the nascent thiols produced upon two-electron reduction of glutathione reductase have distinct functions, interchange with the substrate and interaction with the FAD. Treatment of the monoalkylated derivative with the apolar phenylmercuric acetate eliminates the charge-transfer interaction. The spectrum of the resulting species is similar to that of the oxidized enzyme but less resolved and blue shifted by 10 nm. The dependence on pH of the absorbance associated with the thiolate to FAD charge-transfer interaction in native two-electron reduced glutathione reductase is biphasic, with pK values at approximately 4.8 and 7.4. By analogy with glyceraldehyde-3-phosphate dehydrogenase and papain, these data indicate that the thiolate is stabilized by an adjacent basic residue. The pK 7.4 is associated with the titration of the base to give the ion pair, and the pK of 4.8 is associated with the titration of the thiolate. Unlike lipoamide dehydrogenase, glutathione reductase is sufficiently stable to allow titration with dithionite at pH 3.7. The spectrum at this pH is essentially the same as that of the monoalkylated derivative treated with phenylmercuric acetate. The changes with pH are completely reversible.

Alkylation↗
PubMed1981

NADH inhibition and NAD activation of Escherichia coli lipoamide dehydrogenase catalyzing the NADH-lipoamide reaction.

A unique form of inhibition by NADH and partial reversal by NAD+ has been demonstrated with Escherichia coli lipoamide dehydrogenase. Substrate inhibition by NADH is consistent with its reduction of the active two-electron reduced enzyme intermediate to the inactive four-electron reduced form. NAD+ partially overcomes this inhibition by mass action reversal of this reduction. NAD+ activation is only partial since the presence of both NAD+ and NADH forces the accumulation of two binary enzyme-pyridine nucleotide complexes. These are intermediates in the two-electron to four-electron reduction of the enzyme and thus are not on the catalytic pathway. NAD+ is also shown to inhibit by binding to the oxidized enzyme to give a dead-end complex. From the steady state rate equations, it is apparent that the degree of inhibition will depend on the oxidation-reduction potential for two- to four-electron reduction of the enzyme. Thus, the wide variation in the severity of NADH inhibition between the E. coli and pig heart enzymes is explained by quantitative differences in the basic lipoamide dehydrogenase mechanism. A possible physiological role for this type of inhibition as a mechanism of control in E. coli is discussed.

Dihydrolipoamide Dehydrogenase↗
PubMed1981

Electromyographic evaluation of malignant hyperthermia-susceptible pigs.

Electromyography, with the aid of telemetry, was used to examine muscles of 4 young and 4 adult pigs from a genetic line highly susceptible to malignant hyperthermia (MH) and 4 young pigs (controls) from a line with no history of susceptibility to MH. In the young pigs, the intercostales, vastus lateralis, triceps brachii, biceps femoris, and gastrocnemius muscles were examined, whereas in the adult pigs only the latter 3 were examined. Spontaneous electrical activity, such as fibrillation potentials and abnormal insertion activity, was not observed in the electromyograms, indicating that neither denervation nor muscle membrane hyperirritability was associated with MH susceptibility in these pigs. Amplitude and duration of motor unit potentials (MUP) in the young MH-susceptible (MHS) pigs were significantly larger (P less than 0.01) and longer (P less than 0.05), respectively, than these values for MUP in the control pigs. These changes are opposite in direction to those expected in a myopathy. The mean values (+/- SD) for amplitude and duration for all muscles were 300 +/- 160 microV and 3.2 +/- 0.7 ms, respectively, in the control pigs and 555 +/- microV and 3.6 +/- 0.3 ms, respectively, in the young MHS pigs. Mean values for amplitude and duration of MUP for the adult MHS pigs were larger than were those for the young MHS pigs, but the differences were not significant (P greater than 0.05).

Animals↗
PubMed1980

Chemical modifications of D-amino acid oxidase. Evidence for active site histidine, tyrosine, and arginine residues.

1. D-amino acid oxidase is inactivated by reaction with a low molar excess of dansyl chloride at pH 6.6, with complete inactivation accompanied by incorporation of 1.7 dansyl residues per mol of enzyme-bound flavin. The presence of benzoate, a potent competitive inhibitor, protects substantially against inactivation. Evidence is presented that the inactivation is due to dansylation of an active site histidine residue. Reactivation may be obtained by incubation with hydroxylamine. Diethylpyrocarbonate also inactivates the enzyme and modifies the labeling pattern with dansyl chloride. 2. Butanedione in the presence of borate reacts rapidly to inactivate D-amino acid oxidase. Reactivation is obtained spontaneously on removal of borate, implicating reaction of butanedione with an active site arginine residue. 3. Fluorodinitrobenzene appears to behave as an active site-directed reagent when mixed with D-amino acid oxidase at pH 7.4. Complete inactivation is obtained with incorporation of 2.0 dinitrophenyl residues per mol of enzyme-bound flavin. Again benzoate protects against inactivation; only one dinitrophenyl residue is incorporated in the presence of benzoate. The active site residue attacked by fluorodinitrobenzene has been identified as tyrosine.

Animals↗