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R C Stewart

Publications and source records attributed to R C Stewart.

At least 37 records · Page 2Linked to original sources
PubMed1993

CheZ mutants with enhanced ability to dephosphorylate CheY, the response regulator in bacterial chemotaxis.

CheZ is a component of the chemotaxis signal-transduction pathway in Escherichia coli and Salmonella typhimurium. It is responsible for accelerating dephosphorylation of CheY and thereby antagonizing the tumble-promoting activity of CheY. In the absence of functional CheZ, cells are non-chemotactic and tumble constantly. We characterized the effects of two mutations in CheZ, R54C and V166G, that are unusual in that they cause cells to have a smooth swimming bias. These mutations were isolated as second-site suppressors of mutations in the switch complex responsible for regulating the direction of flagellar rotation (Yamaguchi, S., Aizawa, S.-I., Kihara, M. Isomura, M., Jones, C.J. and Macnab, R.M. (1986) J. Bacteriol. 168, 1172-1179). When produced at low levels in a delta cheZ host strain, CheZ R54C and CheZ V166G supported chemotaxis. However, when moderately overproduced they markedly inhibited chemotactic ability. In vitro studies revealed that these mutations enhanced the ability of CheZ to accelerate dephosphorylation of CheY. These results are discussed in relation to the possible roles and interactions of CheZ in the chemotaxis system.

Alleles↗
PubMed1993

The short form of the CheA protein restores kinase activity and chemotactic ability to kinase-deficient mutants.

Escherichia coli expresses two forms of the chemotaxis-associated CheA protein, CheAL and CheAS, as the result of translational initiation at two distinct, in-frame initiation sites in the gene cheA. The long form, CheAL, plays a crucial role in the chemotactic signal transduction mechanism by phosphorylating two other chemotaxis proteins: CheY and CheB. CheAL must first autophosphorylate at amino acid His-48 before transferring its phosphono group to these other signal transduction proteins. The short form, CheAS, lacks the N-terminal 97 amino acids of CheAL and, therefore, does not possess the site of autophosphorylation. Here we demonstrate that although it lacks the ability to autophosphorylate, CheAS can mediate phosphorylation of kinase-deficient variants of CheAL each of which retains a functional autophosphorylation site. This transphosphorylation enables these kinase-deficient CheAL variants to phosphorylate CheY. Because it mediates this activity, CheAS can restore to kinase-deficient E. coli cells the ability to tumble and, thus, to perform chemotaxis in swarm plate assays.

Bacterial Proteins↗
PubMed1993

Activating and inhibitory mutations in the regulatory domain of CheB, the methylesterase in bacterial chemotaxis.

In the chemotaxis system of Escherichia coli, CheB promotes sensory adaptation by interacting with the chemotaxis receptor-transducer proteins to catalyze removal of their gamma-glutamyl methyl ester groups. CheB is comprised of two functional domains; the C-terminal domain contains the methylesterase active site, and the N-terminal domain regulates the activity of this active site. The chemotaxis system controls CheB methylesterase activity via a mechanism involving phosphorylation of the CheB regulatory domain by the chemotaxis protein kinase CheA. To further explore the communication between the regulatory and methylesterase domains of CheB, I generated mutations in the CheB regulatory domain that affect methylesterase activity in vitro. Three of these mutations (D11K, E58K, and E91K) caused increased methylesterase activity in the absence of phosphorylation, and several other mutations (R42H, R73H, and K107R) caused decreased methylesterase activity in the purified proteins. Several of these mutations (D10N, D11K, R42H, E58K, and K107R) also affected the phosphorylation biochemistry of CheB by reducing the rate of CheA-mediated phosphorylation of CheB and/or by decreasing the autodephosphorylation rate of CheB. In addition, all of these mutations diminished the ability of excess CheA to inhibit CheB methylesterase activity. The locations of these mutations in the deduced three-dimensional structure of the CheB N-terminal domain indicate that the region of the protein surrounding the putative phosphorylation site plays important roles in its interaction with the CheB C-terminal domain as well as in its interactions with CheA.

Adenosine Triphosphatases↗
PubMed1990

Mutations that affect control of the methylesterase activity of CheB, a component of the chemotaxis adaptation system in Escherichia coli.

Sensory adaptation by the chemotaxis system of Escherichia coli requires adjustments of the extent of methyl esterification of the chemotaxis receptor proteins. One mechanism utilized by E. coli to make such adjustments is to control the activity of CheB, the enzyme responsible for removing receptor methyl ester groups. Previous work has established the existence of a multicomponent signal transduction pathway that enables the chemotaxis receptor proteins to control the methylesterase activity in response to chemotactic stimuli. We isolated and characterized CheB mutants that do not respond normally to this control mechanism. In intact cells these CheB variants could not be activated in response to negative chemotaxis stimuli. Further characterization indicated that these CheB variants could not be phosphorylated by the chemotaxis protein kinase CheA. Disruption of the mechanism responsible for regulating methylesterase activity was also observed in cells carrying chromosomal deletions of either cheA or cheW as well as in cells expressing mutant versions of CheA that lacked kinase activity. These results provide further support for recent proposals that activation of the methylesterase activity of CheB involves phosphorylation of CheB by CheA. Furthermore, our findings suggest that CheW plays an essential role in enabling the chemotaxis receptor proteins to control the methylesterase activity, possibly by controlling the CheA-CheB phosphotransfer reaction.

Adaptation, Physiological↗
PubMed1989

Control of transducer methylation levels in Escherichia coli: investigation of components essential for modulation of methylation and demethylation reactions.

During bacterial chemotaxis in Escherichia coli, adaptation is accomplished by reversible methylation of the transmembrane signal transducers. Methyl groups are added by the CheR protein in a slow response to attractants and removed by the CheB protein in response to repellents. The methylesterase activity of the CheB protein is modulated by a factor that is controlled in a global fashion throughout the cell. By controlling the level of expression of the cheR, cheB, and transducer genes with exogenous promoters on multicopy plasmids, we demonstrate that the modulating factor exists in stoichiometric concentrations relative to CheB protein and that the generation or efficacy of this factor requires the cheA and/or cheW gene products, suggesting that phosphorylation of the methylesterase by CheA may be involved in its global activation. We show that in the absence of any modulation of the CheB activity, the CheR methyltransferase activity is modulated in a local fashion at the transducers, most likely as a result of a conformational change in the transducer protein brought about by the binding of ligand, and does not require CheA or CheW.

Carboxylic Ester Hydrolases↗
PubMed1988

N-terminal half of CheB is involved in methylesterase response to negative chemotactic stimuli in Escherichia coli.

The chemotactic receptor-transducer proteins of Escherichia coli are responsible for directing the swimming behavior of cells by signaling for either straight swimming or tumbling in response to chemostimuli. The signaling states of these proteins are affected not only by the concentrations of various stimuli but also by the extent to which they have been methylated at specific glutamyl residues. The activities of a chemotaxis-specific methyltransferase (CheR) and a chemotaxis-specific methylesterase (CheB) are regulated in response to chemotactic stimuli to enable sensory adaptation to unchanging levels of stimuli by appropriately shifting the signaling states of the transducer proteins. For CheB this regulation involves a feedback loop that requires some of the components making up the chemotactic signal transduction machinery of the cell. This feedback loop causes the methylesterase activity of CheB to decrease transiently in response to attractant stimuli and to increase transiently in response to negative stimuli (repellent addition or attractant removal). In this report we demonstrate that the methylesterase response to negative stimuli involves the N-terminal half of the CheB protein, whereas the response to positive stimuli does not require this segment of the protein. Both aspects of the methylesterase response to positive stimuli does not require this segment of the protein. Both aspects of the methylesterase response require CheA. In addition, we demonstrate that mutant forms of CheB lacking methylesterase activity can adversely affect the swimming behavior and chemotactic ability of cells and can markedly diminish modulation of the wild-type methylesterase activity in response to negative stimuli. The significance of these results is discussed in relation to the recent demonstration of phosphoryl transfer from CheA to CheB (J. F. Hess, K. Oosawa, N. Kaplan, and M. I. Simon, Cell 53:79-87, 1988) and the discovery of sequence homology between the N-terminal half of CheB and CheY (A. Stock, D. E. Koshland, Jr., and J. Stock, Proc. Natl. Acad. Sci. USA 82:7989-7993, 1985).

Carboxylic Ester Hydrolases↗
PubMed1985

Potentiometric studies of native and flavin-substituted Old Yellow Enzyme.

We have measured the redox potentials for the flavin cofactor of native Old Yellow Enzyme and for a series of chemically modified flavin derivatives bound to the apoprotein. These flavin derivatives have midpoint potentials ranging from -120 to -300 mV in free solution. For the native enzyme, the midpoint potential of the first one-electron couple EFMNox + e- in equilibrium EFMN-. is E1 = -245 +/- 5 mV and that for the second one-electron couple EFMN-. + e- in equilibrium EFMN red is E2 = -215 +/- 5 mV in 0.1 M phosphate buffer at pH 7.0 and 25 degrees C. Thus, the apoprotein lowers the two-electron midpoint potential of FMN below its value in free solution (Emid,bound = -230 mV, Emid,free = -210 mV). A similar effect on the two-electron midpoint potentials of most of the chemically modified flavins is observed upon binding to the apoprotein. Therefore, the relative order of the respective midpoint potentials of this series of flavins is maintained upon binding to the apoenzyme. However, the effect of the apoprotein on the separation between E1 and E2 varies considerably over this series of flavin derivatives, resulting in quite different levels of thermodynamic stability for the one-electron-reduced (semiquinone) forms of the flavin-substituted enzymes. The optical absorption spectra of these flavin-substituted enzymes were determined in the presence of several phenolic compounds which are known to bind to the native enzyme and to give characteristic long wavelength transitions. We have found that the positions of the wavelength maxima of these transitions are shifted to longer wavelengths as the measured redox potentials of the enzyme-bound flavins are increased. The relationship between the energy of these long wavelength transitions (expressed as vCT = 1/lambda max) and the redox potential of the enzyme-bound flavin is interpreted as further evidence that these transitions arise from charge-transfer complexes between the phenolic compounds and the flavin.

Dithionite↗
PubMed1985

The reaction of arsenite-complexed xanthine oxidase with oxygen. Evidence for an oxygen-reactive molybdenum center.

The effects of arsenite on the reaction of reduced xanthine oxidase with oxygen are determined. The kinetics of the reaction monitoring the return of enzyme absorbance are investigated as are the kinetics and stoichiometries of peroxide and superoxide formation. Although some of the effects of arsenite are qualitatively consistent with expectations based on the known perturbation of the molybdenum midpoint potentials by arsenite, several results cannot be so easily explained. Specifically, arsenite introduces a very rapid phase (kobs = 110 s-1 at 125 microM oxygen) to the oxidative half-reaction which is not observed with the native enzyme. Arsenite also diminishes the amount of superoxide produced and eliminates one-electron reduced enzyme as a detectable kinetic intermediate in the reoxidation pathway. These differences appear to result from the ability of arsenite to greatly enhance the oxygen- and/or superoxide-reactivity of the reduced molybdenum center. This is reflected in the observation that reduced forms of arsenite-complexed xanthine oxidase lacking functional FAD (iodoacetamide-alkylated enzyme and deflavo enzyme) react relatively rapidly with oxygen whereas these reactions are quite slow in the absence of arsenite.

Arsenic↗
PubMed1984

Characterization of arsenite-complexed xanthine oxidase at room temperature. Spectral properties and pH-dependent redox behavior of the molybdenum-arsenite center.

Several aspects of the interaction of xanthine oxidase with arsenite are investigated. Room temperature potentiometric titrations using EPR to monitor Molybdenum reduction reveal midpoint potentials of -225 mV for the Mo(VI)-arsenite/Mo(V)-arsenite couple and -440 mV for the Mo(V)-arsenite/Mo(IV)-arsenite couple at pH 8.3. Under the same conditions, the values for native enzyme are -395 mV and -420 mV, respectively. The predicted effects of the altered Mo(VI)/Mo(V) potential on the distributions of reducing equivalents in partially reduced enzyme are compared with the experimentally observed effects in optical experiments. The bleaching that occurs on reduction of the chromophore that is generated when arsenite binds to oxidized enzyme is characterized and found to be associated with reduction of Mo(V)-arsenite to Mo(V)-arsenite. This probe enables determination of the midpoint potential for this conversion using optical data. From such data at a series of pH values ranging from 6.15 to 9.9, a pH dependence of -60 mV/pH unit increase is determined for this couple above pH 7. The ability of arsenite to bind to reduced xanthine oxidase and to desulfo enzyme are also investigated. Reduced active enzyme binds arsenite much more tightly (Kd less than 0.1 microM) and more rapidly than does oxidized active enzyme (Kd = 8 microM); oxidized desulfo enzyme binds arsenite almost as tightly (Kd = 20 microM) as does the oxidized active enzyme.

Animals↗
PubMed1984

The inhibition of xanthine oxidase by 8-bromoxanthine.

The interaction of xanthine oxidase with the substrate analog 8-bromoxanthine has been examined in an effort to determine the nature of interaction of purines with the active site of the enzyme. It is found that 8-bromoxanthine is an inhibitor of xanthine oxidase with a Ki of approximately 400 microM; inhibition is uncompetitive with respect to xanthine and noncompetitive with respect to molecular oxygen. While 8-bromoxanthine has only a slight effect on the reaction of reduced enzyme with oxygen, it dramatically slows the rate of enzyme reduction by xanthine, suggesting that inhibition does involve the interaction of 8-bromoxanthine with the molybdenum center of the enzyme. KD determinations for binding of 8-bromoxanthine to oxidized and reduced xanthine oxidase indicate that the inhibitor binds preferentially to the fully reduced form of the molybdenum center (MoIV), with dissociation constants of 1.5 mM and 18 microM for oxidized and reduced enzyme, respectively. This preferential binding to the reduced form of the enzyme is manifested in a significant increase in the oxidation-reduction potentials of the molybdenum center as determined by potentiometric titrations with 8-bromoxanthine complexed with xanthine oxidase. The shape of the Mov EPR signal observed in the course of these titrations as well as a comparison with results of reductive titrations and KD determinations with uric acid and xanthine indicate that 8-bromoxanthine interacts with the molybdenum center of xanthine oxidase in a way that is typical of purine substrates and products, despite the presence of the bulky Br group. The inhibitor thus has a potential as a probe of enzyme-substrate interactions, particularly using the technique of x-ray absorption spectroscopy.

Animals↗
PubMed1983

The interaction of arsenite with xanthine oxidase.

The binding of arsenite to the molybdenum center of milk xanthine oxidase is re-examined. The Kd for the arsenite complex has been determined to be 24 microM from equilibrium binding studies and this value has been confirmed by determination of the association and dissociation rate constants for the interaction of arsenite with xanthine oxidase. Formation of the complex is not prevented by prior reaction of the enzyme with thiol reagents such as 5,5'-dithiobis-(2-nitrobenzoic acid) or methyl methanethiosulfonate. Binding of arsenite to the enzyme perturbs both the oxidation-reduction potentials and the electron paramagnetic resonance signal of the molybdenum center observed after partial reduction of the enzyme with sodium dithionite. The EPR signal of the partially reduced arsenite-complexed enzyme is further modified in two different ways by the addition of xanthine or salicylate. Other purine and pteridine substrates and products for the enzyme yield EPR signals indistinguishable from that generated by xanthine, whereas aromatic aldehydes and carboxylic acids give signals similar to that observed in the presence of salicylate. It is thus clear that while arsenite prevents enzyme turnover, it does not preclude binding of substrate and product molecules. Binding of arsenite at the molybdenum center of xanthine oxidase does not disturb the oxidation-reduction potentials of the iron-sulfur centers of the enzyme, but evidence is presented to suggest that the midpoint potential of the FAD site is decreased by approximately 15 mV. A structure for the arsenite complex is proposed to provide a framework in which to interpret the EPR signals in a quantitative fashion.

Animals↗
PubMed1979

Chondritis of the ear: a method of treatment.

Chrondritis may develop as a secondary complication of trauma to the ear. Its onset is often insidious and may be delayed until after apparent healing has occurred. Treatment is difficult, but if postponed, the result may be complete destruction of the external ear. A method of aggressive surgical therapy combined with antibiotics is presented which, when utilized early, results in good healing with minimal residual otic deformity.

Anti-Bacterial Agents↗
PubMed1975

Secondary orgasmic dysfunction. I. Analysis and strategies for treatment.

Following treatment of six cases of primary orgasmic dysfunction and six cases of secondary orgasmic dysfunction, an analysis of assessment and outcome data indicated that (1)clients with secondary orgasmic dysfunction and those with primary orgasmic dysfunction did not differ significantly in most aspects of sexual behavior before treatment; (2) before treatment, secondary orgasmic dysfunction tended to be associated with a disturbed marital relationship, while primary orgasmic dysfunction did not; (3) following a behavioral treatment program focused on anxiety reduction and sexual skill training, organism in coitus was attained by clients with primary orgasmic dysfunction but not by clients with secondary orgasmic dysfunction. Based on these findings, suggestions for effective treatment of secondary orgasmic dysfunction are made.

Adult↗
PubMed1975

Inactivation of myxoviruses by calcium elenolate.

Calcium elenolate inactivates all myxoviruses so far tested. The pH of the reaction mixture is less critical for myxovirus inactivation than that required for coxsackie A-21 virus; the myxoviruses are inactivated at a broad spectrum of pH with the maximum activity occurring at a pH below 7.0. The infectivity of the virus is more susceptible to the action of calcium elenolate than is either the neuraminidase activity or the hemagglutinin. The inactivation of Newcastle disease virus by calcium elenolate also destroys the ability of the virus to induce interferon formation in cell culture and in mice.

Animals↗