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A T Smith

Publications and source records attributed to A T Smith.

At least 19 recordsLinked to original sources
PubMed1992

Characterisation of a haem active-site mutant of horseradish peroxidase, Phe41----Val, with altered reactivity towards hydrogen peroxide and reducing substrates.

A horseradish peroxidase variant ([F41V] HRP-C*), in which Val replaces the conserved Phe at position 41 adjacent to the distal His, has been constructed. Its composition and spectroscopic, catalytic and substrate-binding properties were compared with those of the wild-type recombinant (HRP-C*) and plant (HRP-C) enzymes. Presteady-state kinetic measurements of the rate constant for compound I formation (k1) revealed an eightfold decrease in the reactivity of the Phe41----Val variant towards H2O2, in comparison with HRP-C or HRP-C*. Measurement of the remaining rate constants, k2 and k3, for the two single-electron reduction reactions of [F41V] HRP-C with para-aminobenzoic acid as reducing substrate, showed that they were 2.5-fold and 1.3-fold faster, respectively. In contrast, analysis of data from steady-state assays with 2,2'-azinobis(3-ethylbenzthiazoline-6-sulphonate) as reducing substrate, showed decreased reactivity of the mutant enzyme to this compound, indicating a change in substrate specificity. Over the substrate range studied, the data for HRP-C* and for [F41V] HRP-C conformed to a simple modification of the accepted peroxidase mechanism in which a first-order step (ku), assumed to be product dissociation, becomes rate-limiting under our standard assay conditions. Calculations of rate constants from steady-state data yielded values of k1 for both enzyme forms in adequate agreement with those from pre-steady state measurements. They showed, furthermore, that both k3 for 2,2'-azinobis(3-ethylbenzthiazoline-6-sulphonate) and ku were substantially decreased, fivefold and tenfold, respectively, in the mutant. Analogous to the decrease in ku, we observed a twofold increase in the affinity of the mutant variant for the inhibitor benzhydroxamic acid. The coordination-state equilibrium of the haem iron also appeared shifted towards the hexacoordinate high-spin form. These observations indicate that in addition to affecting reactivity to H2O2, mutations in the distal region and close to the haem iron also affect reactivity towards different reducing substrates, inducing perturbations in the neighbourhood of the aromatic-substrate-binding site, known to be 0.8-1.2 nm from the haem iron.

Amino Acid Sequence↗
PubMed1992

Structural studies by proton-NMR spectroscopy of plant horseradish peroxidase C, the wild-type recombinant protein from Escherichia coli and two protein variants, Phe41----Val and Arg38----Lys.

Wild-type recombinant horseradish peroxidase isoenzyme C and two protein variants, Phe41----Val and Arg38----Lys, have been characterised using both one- and two-dimensional NMR spectroscopy. Proton NMR spectra recorded in both resting and cyanide-ligated states of the proteins were compared with those of the corresponding plant peroxidase. The latter contains 18% carbohydrate in eight N-linked oligosaccharide side chains whereas the recombinant proteins are expressed in nonglycosylated form. The spectra of the plant enzyme and refolded recombinant protein are essentially identical with the exception of carbohydrate-linked resonances in the former, indicating that their solution structures are highly similar. This comparison also identifies classes of carbohydrate resonances in the plant enzyme which provides new information on the local environment and mobility of the oligosaccharide side chains. Comparison of the spectra of the cyanide-ligated states of the two variants and those of plant horseradish peroxidase C indicated that there were significant differences with respect to haem and haem-linked resonances. These could not be rationalised simply on the basis of the local perturbation expected from a single-site substitution. The two substitutions made to residues on the distal side of the haem apparently influenced the degree of imidazolate character of the proximal His170 imidazole ring thus perturbing the magnetic environment of the haem group. Inspection of the spectra of the Phe41----Val variant also showed that the resonances of a phenylalanine residue in the haem pocket had been incorrectly assigned to Phe41 in a previous study. A new assignment, based on additional information from two-dimensional nuclear Overhauser enhancement spectroscopy, was made to Phe152. The assignments made for the Phe41----Val variant were also used as a basis to investigate the structure of the complex formed with the aromatic donor molecule, benzhydroxamic acid.

Arginine↗
PubMed1992

Two temporal channels or three? A re-evaluation.

The initial filtering of the image by the human visual system involves only a small number of temporal filters. Several studies suggest there are in fact only two, but some suggest that a third filter, sensitive to high frequencies, exists, at least at low spatial frequencies. This conclusion is derived in part from the observation that temporal frequency discrimination performance is better at very high (30-40 Hz) than at medium (20 Hz) temporal frequencies. We show that this apparent improvement at high frequencies is not real but is an artifact of differences in the rate of perceptual fading as a function of temporal frequency. Using suprathreshold counterphase gratings and a stimulus duration of 1.5 sec we replicated the finding of an improvement at high frequencies at a low (0.5 c/deg) spatial frequency. But when duration was reduced to 300 msec, to minimize fading cues, this improvement disappeared. Similarly, at 4 c/deg, the improvement was present at 3 sec duration but absent at 1.5 sec or less. Direct evidence that this effect of duration reflects differences in the ability to use fading cues was obtained in an experiment in which naive subjects were instructed to discriminate on the basis of fading: at high temporal frequencies and long durations performance was as good or better than for subjects instructed to use frequency; at short durations performance on this task was poor. Thus, the claim that a third temporal channel exists may need to be re-evaluated.

Discrimination, Psychological↗
PubMed1992

Coherence of plaids comprising components of disparate spatial frequencies.

The limits of plaid coherence were measured in terms of the maximum spatial frequency difference between the components at which coherence is sustained, for a variety of contrasts and speeds. In some circumstances the maximum spatial frequency difference is between three and four octaves. This suggests that one-dimensional components of a moving image are not combined within spatial frequency channels to give overall pattern direction but that there is a pooling of information across spatial frequencies prior to the stage at which pattern direction is computed. Coherence range declines as contrast is decreased or drift speed is increased.

Contrast Sensitivity↗
PubMed1992

Motion defined exclusively by second-order characteristics does not evoke optokinetic nystagmus.

We showed high-contrast, second-order motion stimuli to subjects whilst recording their horizontal eye movements. These stimuli were very poor at evoking optokinetic nystagmus. Smooth-pursuit eye movements and fixation were reduced by a masking band +/- 2.5 deg above and below an imaginary fixation point. First-order stimuli evoked vigorous optokinetic nystagmus (OKN) under identical conditions and also when matched for apparent contrast. These findings are discussed in terms of the site of detection of second-order motion.

Adult↗
PubMed1991

Use of plaid patterns to distinguish the corticofugal and direct retinal inputs to the brainstem optokinetic nystagmus generator.

We have recorded the direction of optokinetic nystagmus (OKN) elicited by moving plaid patterns in order to dissociate the pathways that mediate horizontal OKN. The plaids used comprised two drifting sinusoidal gratings arranged such that their individual directions of drift were very different from the direction of coherent motion of the overall pattern. The direction of OKN with binocular viewing was close to the mean of the component directions, suggesting a dominant influence of cortical visual neurons that respond to oriented one-dimensional components of the image. But the direction of OKN was consistently shifted slightly towards the direction of motion of the overall pattern, suggesting a secondary influence responsive to pattern direction. OKN recordings obtained during monocular viewing suggest that this secondary influence reflects the direct retinal pathway to the brainstem structures mediating OKN.

Animals↗
PubMed1991

The separability of temporal frequency and velocity.

McKee, Silverman and Nakayama (1986; Vision Research, 26, 609-619) have shown that velocity discrimination performance is little affected by quite large random changes in the spatial frequency, and hence temporal frequency, of the grating patterns to be discriminated. We show that the converse is also true: temporal frequency discrimination can be performed despite random changes in velocity. This casts doubt on McKee et al.'s conclusion that velocity is the fundamental dimension of the temporal mechanisms mediating discrimination and that temporal frequency has to be inferred indirectly from velocity and spatial frequency. We suggest instead that both velocity and temporal frequency are represented in the visual system but that the two dimensions are only partially separable at the perceptual level.

Discrimination, Psychological↗
PubMed1991

Perceived speed and direction of complex gratings and plaids.

Measurements of perceived speed were obtained for a variety of drifting simple and complex gratings, and measurements of perceived speed and direction were obtained for plaids. For sine gratings, perceived speed falls off at high spatial frequencies, the effect of spatial frequency being greatest at high speeds. Speed matches obtained from a variety of one-dimensional complex gratings are in some cases consistent with a simple averaging of the discrepant speeds signaled by their spatial Fourier components when seen alone. However, in other cases the results do not fit such an interpretation but suggest the involvement of a mechanism other than Fourier decomposition and recombination. Measurements of the perceived speed of plaids suggest that the observed spatial-frequency-dependent variations in encoded speed of gratings arise largely at a low level, before the aperture problem is solved. Measurements of the perceived direction of plaids whose components are of different spatial frequencies and hence have different perceived speeds show large deviations from the direction predicted by the intersection-of-velocity-constraints model [Nature 300, 523 (1982)] and are suggestive of a revised two-stage model in which the computation of pattern direction is based on the (sometimes disparate) perceived speeds of the components rather than their actual speeds.

Humans↗
PubMed1990

Expression of a synthetic gene for horseradish peroxidase C in Escherichia coli and folding and activation of the recombinant enzyme with Ca2+ and heme.

A synthetic gene encoding horseradish peroxidase isoenzyme C (HRP C) has been synthesized and expressed in Escherichia coli. The nonglycosylated recombinant enzyme (HRP C*) was produced in inclusion bodies in an insoluble inactive form containing only traces of heme. HRP C* was solubilized and conditions under which it folded to give active enzyme were determined. Folding was shown to be critically dependent upon the concentrations of urea, Ca2+, and heme and on oxidation by oxidized glutathione. Purification of active HRP C* from the folding mixture gave a peroxidase, with about half the activity of HRP C. Glycosylation is thus not essential for correct folding and activity. The C-terminal and N-terminal extensions to HRP identified previously in cloned cDNA sequences are also not required for correct folding. However, Ca2+ appears to play a key role in folding to give the active enzyme. The overall yield of purified active enzyme was 2-3%, but this could be increased by reprocessing material that precipitated during folding.

Amino Acid Sequence↗
PubMed1990

Comparison of the effects of Alzheimer's disease, normal aging and scopolamine on human transient visual evoked potentials.

Transient visual evoked potentials elicited by the onset of a patterned stimulus were recorded in patients suffering from Alzheimer's disease (AD), in healthy elderly controls and in healthy young individual. The latencies and amplitudes of both the components studied were adversely affected by normal aging and one of the components, CI, but not the other, CII, showed further deterioration in AD. These changes occurred over a range of stimulus contrast levels. The changes found in AD, but not those seen in normal aging, could be mimicked by administration of the cholinergic antagonist scopolamine to young volunteers.

Adult↗
PubMed1990

The influence of spatial frequency on perceived temporal frequency and perceived speed.

Speed matching experiments were conducted using drifting gratings of different spatial frequencies in order to assess the influence of spatial frequency on perceived speed. It was found that gratings of high spatial frequency appear to drift more slowly than low spatial frequency gratings of the same actual velocity. The perceived temporal frequency of a counterphase grating similarly declines as spatial frequency increases. The previously reported effect of temporal frequency on perceived spatial frequency probably does not contribute to these phenomena. Our results suggest that the motion sensors thought to operate within different spatial frequency ranges have different velocity transfer functions, a fact not incorporated in existing computational models of motion perception.

Contrast Sensitivity↗
PubMed1990

Flicker adaptation in the periphery at constant perceived modulation depth.

At constant physical flicker modulation depth, the time taken to adapt to flicker in the periphery varies inversely with temporal frequency. It has recently been suggested that this effect may indicate differential susceptibility to adaptation of the underlying temporal mechanisms. Using suprathreshold gratings, temporally modulated in contrast at constant perceived, rather than physical, modulation depth, we found the opposite result: the time required to adapt increased with temporal frequency. Given some uncertainty concerning the appropriateness of employing apparent or physically constant modulation depths, we conclude that rate of adaptation does not, at present, provide clear evidence as to the nature of the underlying temporal mechanisms.

Adaptation, Ocular↗
PubMed1990

Hemifield differences in perceived spatial frequency.

Measurements of the perceived spatial frequency of stationary sinewave gratings were made with the gratings presented at the same eccentricity in the left, right, upper, and lower visual hemifields. Ten subjects performed the task binocularly with spatial frequencies of 1, 2, and 4 cycles deg-1. Two of these subjects also performed the task monocularly at 2 cycles deg-1. In the majority of cases, the spatial frequency of stimuli presented in the left and lower visual hemifields was overestimated relative to stimuli presented in the right and upper visual hemifields. The results were similar for all spatial frequencies tested, and the direction of the asymmetry was the same whether viewing was with the left eye, right eye or binocular, suggesting that the differences in perceived spatial frequency are not retinal in origin.

Adult↗
PubMed1990

Roles for enteric d-type cytochrome oxidase in N2 fixation and microaerobiosis.

Escherichia coli strains that lacked the d-type cytochrome oxidase, the terminal oxidase with a high affinity for O2, grew anaerobically as well as the wild type did and were not impaired in the ability to evolve H2 from either glucose or formate. The anaerobic synthesis and activity of nitrogenase in transconjugants of these strains carrying Klebsiella pneumoniae nif genes were also normal. However, the behavior towards O2 of anaerobically grown bacteria lacking the d-type oxidase differed from that of the wild type in the following ways: the potential O2 uptake was lower, H2 evolution and nitrogenase activity supported by fermentation were more strongly inhibited by O2, and microaerobic O2-dependent nitrogenase activity in the absence of a fermentable carbon source did not occur. These results show that the d-type oxidase serves two functions in enteric bacteria--to conserve energy under microaerobic conditions and to protect anaerobic processes from inhibition by O2.

Aerobiosis↗
PubMed1988

Neural correlates of motion after-effects in cat striate cortical neurones: monocular adaptation.

Motion after-effects were elicited from striate cortical cells in lightly-anaesthetized cats, by adapting with square-wave gratings or randomly textured fields drifting steadily and continuously in preferred or null directions. The time-course and recovery of responsiveness following adaptation were assessed with moving bars, gratings or textured fields. Results were compared with controls in which the adapting stimulus was replaced by a uniform field of identical mean luminance, and also assessed in relation to the strength and time course of adaptation. Within 30-60 s adaptation, firing declined to a steady-state. Induced after-effects were direction-specific, and manifest as a transitory depression in response to the direction of prior adaptation, recovering to control levels in 30-60 s. Maximal after-effects were induced by gratings of optimal drift velocity and spatial frequency. With rare exceptions after-effects were restricted to driven activity; no consistent effects on resting discharge were observed. The onset of adaptation, and the recovery period, were more rapid in simple cells, although after-effects of comparable strength were elicited from simple and from standard complex cells. Special complex cells, including many of the more profoundly texture-sensitive neurones in the cortex, were more resistant to adaptation. The results support the conclusion that psychophysically measured adaptation and induced motion after-effect phenomena reflect the known properties of cortical neurones.

Animals↗