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A T Smith

Publications and source records attributed to A T Smith.

At least 37 records · Page 2Linked to original sources
PubMed2000

Global motion adaptation.

Image motion is initially detected locally. Local motion signals are then integrated across space in order to specify the global motion of objects or surfaces. It is well known that prolonged exposure to motion causes adaptation at the local motion level. We have investigated whether adaptation also occurs at the global motion level. We have devised a global motion stimulus (a random dot kinematogram) which has equal motion energy in opposite directions but nonetheless gives rise to global motion perception. At the local motion level, adaptation to this stimulus should cause equal adaptation in both directions and should not give rise to an aftereffect. Any aftereffect seen must therefore be attributable to adaptation at the global motion level. We find that following adaptation to this stimulus, judgements of the perceived direction of a test pattern are systematically biased towards the direction opposite to the adapting direction, suggesting that adaptation does occur at a level of visual processing at which global motion is represented.

Adaptation, Physiological↗
PubMed2000

No local cancellation between directionally opposed first-order and second-order motion signals.

Despite strong converging evidence that there are separate mechanisms for the processing of first-order and second-order motion, the issue remains controversial. Qian, Andersen and Adelson (J. Neurosci., 14 (1994), 7357-7366) have shown that first-order motion signals cancel if locally balanced. Here we show that this is also the case for second-order motion signals, but not for a mixture of first-order and second-order motion even when the visibility of the two types of stimulus is equated. Our motion sequence consisted of a dynamic binary noise carrier divided into horizontal strips of equal height, each of which was spatially modulated in either contrast or luminance by a 1.0 c/deg sinusoid. The modulation moved leftward or rightward (3.75 Hz) in alternate strips. The single-interval task was to identify the direction of motion of the central strip. Three conditions were tested: all second-order strips, all first-order strips, and spatially alternated first-order and second-order strips. In the first condition, a threshold strip height for the second-order strips was obtained at a contrast modulation depth of 100%. In the second condition, this height was used for the first-order strips, and a threshold was obtained in terms of luminance contrast. These two previously-obtained threshold values were used to equate visibility of the first-order and second-order components in the third condition. Direction identification, instead of being at threshold, was near-perfect for all observers. We argue that the first two conditions demonstrate local cancellation of motion signals, whereas in the third condition this does not occur. We attribute this non-cancellation to separate processing of first-order and second-order motion inputs.

Contrast Sensitivity↗
PubMed1999

The structures of the horseradish peroxidase C-ferulic acid complex and the ternary complex with cyanide suggest how peroxidases oxidize small phenolic substrates.

We have solved the x-ray structures of the binary horseradish peroxidase C-ferulic acid complex and the ternary horseradish peroxidase C-cyanide-ferulic acid complex to 2.0 and 1.45 A, respectively. Ferulic acid is a naturally occurring phenolic compound found in the plant cell wall and is an in vivo substrate for plant peroxidases. The x-ray structures demonstrate the flexibility and dynamic character of the aromatic donor binding site in horseradish peroxidase and emphasize the role of the distal arginine (Arg(38)) in both substrate oxidation and ligand binding. Arg(38) hydrogen bonds to bound cyanide, thereby contributing to the stabilization of the horseradish peroxidase-cyanide complex and suggesting that the distal arginine will be able to contribute with a similar interaction during stabilization of a bound peroxy transition state and subsequent O-O bond cleavage. The catalytic arginine is additionally engaged in an extensive hydrogen bonding network, which also includes the catalytic distal histidine, a water molecule and Pro(139), a proline residue conserved within the plant peroxidase superfamily. Based on the observed hydrogen bonding network and previous spectroscopic and kinetic work, a general mechanism of peroxidase substrate oxidation is proposed.

Binding Sites↗
PubMed1999

Kinetics and interactions of molybdenum and iron-sulfur centers in bacterial enzymes of the xanthine oxidase family: mechanistic implications.

For isoquinoline 1-oxidoreductase (IsoOr), the reaction mechanism under turnover conditions was studied by EPR spectroscopy using rapid-freeze methods. IsoOr displays several EPR-active Mo(V) species including the "very rapid" component found also in xanthine oxidase (XanOx). For IsoOr, unlike XanOx or quinoline 2-oxidoreductase (QuinOr), this species is stable for about 1 h in the absence of an oxidizing substrate [Canne, C., Stephan, I., Finsterbusch, J., Lingens, F., Kappl, R., Fetzner, S., and Hüttermann, J. (1997) Biochemistry 36, 9780-9790]. Under rapid-freeze conditions in the presence of ferricyanide the very rapid species behaves as a kinetically competent intermediate present only during steady-state turnover. To explain the persistence of the very rapid species in IsoOr in the absence of an added oxidant, extremely slow product dissociation is required. This new finding that oxidative conditions facilitate decay of the very rapid signal for IsoOr supports the mechanism of substrate turnover proposed by Lowe, Richards, and Bray [Lowe, D. J., Richards, R. L., and Bray, R. C. (1997) Biochem. Soc. Trans. 25, 774-778]. Additional stopped-flow data reveal that alternative catalytic cycles occur in IsoOr and show that the product dissociates after transfer of a single oxidizing equivalent from ferricyanide. In rapid-freeze measurements magnetic interactions of the very rapid Mo(V) species and the iron-sulfur center FeSI of IsoOr and QuinOr were observed, proving that FeSI is located close to the molybdopterin cofactor in the two proteins. This finding is used to relate the two different iron-sulfur centers of the aldehyde oxidoreductase structure with the EPR-detectable FeS species of the enzymes.

Anisotropy↗
PubMed1999

Evidence from spin-trapping for a transient radical on tryptophan residue 171 of lignin peroxidase.

The heme enzyme lignin peroxidase contains a unique Cbeta-hydroxylated tryptophan residue (Trp171) on the surface of the enzyme. Mutagenetic substitution of Trp171 abolishes completely the veratryl alcohol oxidation activity of the enzyme. This led us to surmise that Trp171 may be involved in electron transfer from natural substrates to the heme cofactor. Here we present evidence for the formation of a transient radical on Trp171 using spin-trapping in combination with peptide mapping. The spin-trap methyl nitroso propane forms a covalent adduct with Trp171 in the presence of hydrogen peroxide which can be detected by its characteristic visible absorbance spectrum. A very similar chromophore can be obtained in a small molecular model system from N-acetyl tryptophanamide, the spin-trap, and a single-electron abstracting system. The precise site the spin-trap is attached to could be identified in a crystal structure of spin-trap/hydrogen peroxide-treated enzyme as the C6 atom of the indole ring of Trp171. These results indicate that Trp171 is redox-active and that it forms an indole radical by transfer of an electron to the heme of compound I and/or II. Apart from cytochrome c peroxidase and DNA photolyase, lignin peroxidase appears to be the third enzyme only which utilizes a tryptophan residue as an integral part of its redox catalysis.

Amino Acid Substitution↗
PubMed1999

Reactions of dimethylsulfoxide reductase from Rhodobacter capsulatus with dimethyl sulfide and with dimethyl sulfoxide: complexities revealed by conventional and stopped-flow spectrophotometry.

Improved assays for the molybdenum enzyme dimethylsulfoxide reductase (DMSOR) with dimethyl sulfoxide (DMSO) and with dimethyl sulfide (DMS) as substrates are described. Maximum activity was observed at pH 6.5 and below and at 8.3, respectively. Rapid-scan stopped-flow spectrophotometry has been used to investigate the reduction of the enzyme by DMS to a species previously characterized by its UV-visible spectrum [McAlpine, A. S., McEwan, A. G., and Bailey, S. (1998) J. Mol. Biol. 275, 613-623], and its subsequent reoxidation by DMSO. Both these two-electron reactions were faster than enzyme turnover under steady-state conditions, indicating that one-electron reactions with artificial dyes were rate-limiting. Second-order rate constants for the two-electron reduction and reoxidation reactions at pH 5.5 were (1.9 +/- 0.1) x 10(5) and (4.3 +/- 0.3) x 10(2) M-1 s-1, respectively, while at pH 8.0, the catalytic step was rate-limiting (62 s-1). Kinetically, for the two-electron reactions, the enzyme is more effective in DMS oxidation than in DMSO reduction. Reduction of DMSOR by DMS was incomplete below approximately 1 mM DMS but complete at higher concentrations, implying that the enzyme's redox potential is slightly higher than that of the DMS-DMSO couple. In contrast, reoxidation of the DMS-reduced state by DMSO was always incomplete, regardless of the DMSO concentration. Evidence for the existence of a spectroscopically indistinguishable reduced state, which could not be reoxidized by DMSO, was obtained. Brief reaction (less than approximately 15 min) of DMS with DMSOR was fully reversible on removal of the DMS. However, in the presence of excess DMS, a further slow reaction occurred aerobically, but not anaerobically, to yield a stable enzyme form having a lambdamax at 660 mn. This state (DMSORmod) retained full activity in steady-state assays with DMSO, but was inactive toward DMS. It could however be reconverted to the original resting state by reduction with methyl viologen radical and reoxidation with DMSO. We suggest that in this enzyme form two of the dithiolene ligands of the molybdenum have dissociated and formed a disulfide. The implications of this new species are discussed in relation both to conflicting published information for DMSOR from X-ray crystallography and to previous spectroscopic data for its reduced forms.

Benzyl Viologen↗
PubMed1999

What motion distributions yield global transparency and spatial segmentation?

We have examined the ability of observers to parse bimodal local-motion distributions into two global motion surfaces, either overlapping (yielding transparent motion) or spatially segregated (yielding a motion boundary). The stimuli were random dot kinematograms in which the direction of motion of each dot was drawn from one of two rectangular probability distributions. A wide range of direction distribution widths and separations was tested. The ability to discriminate the direction of motion of one of the two motion surfaces from the direction of a comparison stimulus was used as an objective test of the perception of two discrete surfaces. Performance for both transparent and spatially segregated motion was remarkably good, being only slightly inferior to that achieved with a single global motion surface. Performance was consistently better for segregated motion than for transparency. Whereas transparent motion was only perceived with direction distributions which were separated by a significant gap, segregated motion could be seen with abutting or even partially overlapping direction distributions. For transparency, the critical gap increased with the range of directions in the distribution. This result does not support models in which transparency depends on detection of a minimum size of gap defining a bimodal direction distribution. We suggest, instead, that the operations which detect bimodality are scaled (in the direction domain) with the overall range of distribution. This yields a flexible, adaptive system that determines whether a gap in the direction distribution serves as a segmentation cue or is smoothed as part of a unitary computation of global motion.

Computer Graphics↗
PubMed1998

Two substrate interaction sites in lignin peroxidase revealed by site-directed mutagenesis.

It has been shown recently that Trp171 of lignin peroxidase (LiP) is hydroxylated at the Cbeta position [Blodig, W., Doyle, W. A., Smith, A. T., Winterhalter, K., Choinowski, T., and Piontek, K. (1998) Biochemistry 37, 8832-8838]. Comparative experiments, carried out on both wild-type fungal and recombinant LiP isoenzyme H8 (LiPH8), indicate that the process of hydroxylation is autocatalytic and that Trp171 may be implicated in catalysis. The role of this residue has therefore been examined using site-directed mutagenesis to obtain recombinant enzymes with Trp171 substituted by Phe or Ser (W171F and W171S LiPH8, respectively). The wild-type recombinant enzyme (LiPH8) was analyzed in solution using 1H NMR spectroscopy and its integrity confirmed prior to the kinetic and spectroscopic characterization of LiPH8 mutants. A charge neutralization mutation in the "classical heme edge" substrate access channel of LiP, in which Glu146 was substituted by Gly (E146G LiPH8), showed substantial activity with respect to veratryl alcohol (VA) oxidation and a marked (2.4 pH units) increase in pKa for the oxidation of a negatively charged difluoroazo dye. More surprisingly, the Trp171 LiPH8 mutants W171F and W171S LiPH8 were found to have lost all activity with VA as substrate, and compounds I and II were unable to react with VA. Both mutants, however, retained substantial activity with two dye substrates. These data provide the first direct evidence for the existence of two distinct substrate interaction sites in LiP, a heme-edge site typical of those encountered in other peroxidases and a second, novel site centered around Trp171 which is required for the oxidation of VA. Stopped-flow kinetic studies showed that all the mutants examined reacted normally with hydrogen peroxide to give a porphyrin cation radical (compound I). However, the rapid phase of spontaneous compound I reduction (2.3 s-1), typical of wild-type LiP, was absent in the Trp171 mutants, strongly suggesting that an electron-transfer pathway must exist within the protein leading from the heme to a surface site in close proximity to Trp171. The kinetic competence of such a pathway is dependent on interaction of the enzyme with VA, at or near Trp171.

Basidiomycota↗
PubMed1998

Stereoscopic and contrast-defined motion in human vision.

There is considerable evidence for the existence of a specialized mechanism in human vision for detecting moving contrast modulations and some evidence for a mechanism for detecting moving stereoscopic depth modulations. It is unclear whether a single second-order motion mechanism detects both types of stimulus or whether they are detected separately. We show that sensitivity to stereo-defined motion resembles that to contrast-defined motion in two important ways. First, when a missing-fundamental disparity waveform is moved in steps of 0.25 cycles, its perceived direction tends to reverse. This is a property of both luminance-defined and contrast-defined motion and is consistent with independent detection of motion at different spatial scales. Second, thresholds for detecting the direction of a smoothly drifting sinusoidal disparity modulation are much higher than those for detecting its orientation. This is a property of contrast-modulated gratings but not luminance-modulated gratings, for which the two thresholds are normally identical. The results suggest that stereo-defined and contrast-defined motion stimuli are detected either by a common mechanism or by separate mechanisms sharing a common principle of operation.

Depth Perception↗
PubMed1998

Autocatalytic formation of a hydroxy group at C beta of trp171 in lignin peroxidase.

In the high-resolution crystal structures of two lignin peroxidase isozymes from the white rot fungus Phanerochaete chrysosporium a significant electron density at single bond distance from the C beta of Trp171 was observed and interpreted as a hydroxy group. To further clarify the nature of this feature, we carried out tryptic digestion of the enzyme and isolated the Trp171 containing peptide. Under ambient conditions, this peptide shows an absorbance spectrum typical of tryptophan. At elevated temperature, however, the formation of an unusual absorbance spectrum with lambda max = 333 nm can be followed that is identical to that of N-acetyl-alpha, beta-didehydrotryptophanamide, resulting upon water elimination from beta-hydroxy tryptophan. The Trp171 containing tryptic peptide isolated from the recombinant and refolded lignin peroxidase produced from Escherichia coli does not contain the characteristic 333 nm absorbance band at any temperature. However, treatment with 3 equiv of H2O2 leads to complete hydroxylation of Trp171. Reducing substrates compete with this process, e.g., in the presence of 0.5 mM veratryl alcohol, about 7 equiv of H2O2 is necessary for complete modification. We conclude that the hydroxylation at the C beta of Trp171 is an autocatalytic reaction which occurs readily under conditions of natural turnover, e.g., in the ligninolytic cultures of P. chrysosporium, which are known to contain an oxidase-based H2O2-generating system. No dependence on dioxygen was found for this oxidative process. Chemical modification of fungal lignin peroxidase with the tryptophan-specific agent N-bromo succinimide leads to a drastically reduced activity with respect to the substrate veratryl alcohol. This suggests that Trp171 is involved in catalysis and that electron transfer from this surface residue to the oxidized heme cofactor is possible under steady-state conditions.

Basidiomycota↗
PubMed1998

Structural interactions between horseradish peroxidase C and the substrate benzhydroxamic acid determined by X-ray crystallography.

The three-dimensional structure of recombinant horseradish peroxidase in complex with BHA (benzhydroxamic acid) is the first structure of a peroxidase-substrate complex demonstrating the existence of an aromatic binding pocket. The crystal structure of the peroxidase-substrate complex has been determined to 2.0 A resolution with a crystallographic R-factor of 0.176 (R-free = 0. 192). A well-defined electron density for BHA is observed in the peroxidase active site, with a hydrophobic pocket surrounding the aromatic ring of the substrate. The hydrophobic pocket is provided by residues H42, F68, G69, A140, P141, and F179 and heme C18, C18-methyl, and C20, with the shortest distance (3.7 A) found between heme C18-methyl and BHA C63. Very little structural rearrangement is seen in the heme crevice in response to substrate binding. F68 moves to form a lid on the hydrophobic pocket, and the distal water molecule moves 0.6 A toward the heme iron. The bound BHA molecule forms an extensive hydrogen bonding network with H42, R38, P139, and the distal water molecule 2.6 A above the heme iron. This remarkably good match in hydrogen bond requirements between the catalytic residues of HRPC and BHA makes the extended interaction between BHA and the distal heme crevice of HRPC possible. Indeed, the ability of BHA to bind to peroxidases, which lack a peripheral hydrophobic pocket, suggests that BHA is a general counterpart for the conserved hydrogen bond donors and acceptors of the distal catalytic site. The closest aromatic residue to BHA is F179, which we predict provides an important hydrophobic interaction with more typical peroxidase substrates.

Binding Sites↗
PubMed1998

The processing of first- and second-order motion in human visual cortex assessed by functional magnetic resonance imaging (fMRI).

We have examined the activity levels produced in various areas of the human occipital cortex in response to various motion stimuli using functional magnetic resonance imaging (fMRI) methods. In addition to standard luminance-defined (first-order) motion, three types of second-order motion were used. The areas examined were the motion area V5 (MT) and the following areas that were delineated using retinotopic mapping procedures: V1, V2, V3, VP, V3A, and a new area that we refer to as V3B. Area V5 is strongly activated by second-order as well as by first-order motion. This activation is highly motion-specific. Areas V1 and V2 give good responses to all motion stimuli, but the activity seems to be related primarily to the local spatial and temporal structure in the image rather than to motion processing. Area V3 and its ventral counterpart VP also respond well to all our stimuli and show a slightly greater degree of motion specificity than do V1 and V2. Unlike V1 and V2, the response in V3 and VP is significantly greater for second-order motion than for first-order motion. This trend is evident, but less marked, in V3A and V3B and absent in V5. The results are consistent with the hypothesis that first-order motion sensitivity arises in V1, that second-order motion is first represented explicitly in V3 and VP, and that V5 (and perhaps also V3A and V3B) is involved in further processing of motion information, including the integration of motion signals of the two types.

Brain Mapping↗
PubMed1998

Redox- and anion-linked protonation sites in horseradish peroxidase: analysis of distal haem pocket mutants.

We have investigated the effects of mutations at residues His-42, Arg-38 and Phe-41 in the distal haem pocket of horseradish peroxidase on the changes in protonation state that accompany redox- and ligand-linked changes to the haem group. The mutations H42L and R38L result in the loss of a characteristic pH dependency in the visible spectrum of the ferrous form and a diminished dependency of the midpoint redox potential of the haem group on pH. The results support the view that His-42, with its pK probably modulated by Arg-38, provides the protonation site on the reduced enzyme that is responsible for these pH dependencies. The mutations H42L and R38L also have major effects on the binding of cyanide to the haem. We have already reported that binding of cyanide to the ferrous forms of these mutants becomes too weak to be measurable [Meunier, Rodriguez-Lopez, Smith, Thorneley and Rich (1995) Biochemistry 34, 14687-14692]. The pH dependency of the rate constants for binding of cyanide to the oxidized form of H42L suggests that CN- is the kinetically active species, in contrast with wild-type horseradish peroxidase, where HCN is the binding form. For the R38L variant, the pH dependency of cyanide binding suggests that the pK of His-42 in the absence of Arg-38 is raised to 7.5-8, in the oxidized form. In contrast with these changes, the mutant F41A exhibits cyanide-binding behaviour that is similar to that of the wild type, both in its oxidized and reduced forms. However, the rate constant for carbon monoxide recombination increases substantially, suggesting that the access route for carbon monoxide, but not for cyanide, is perturbed by this amino acid substitution.

Anions↗
PubMed1998

Effect of calcium, other ions, and pH on the reactions of barley peroxidase with hydrogen peroxide and fluoride. Control of activity through conformational change.

Transient-state kinetic analysis of compound I formation for barley grain peroxidase (BP 1) has revealed properties that are highly unusual for a heme peroxidase but which may be relevant to its biological function. The enzyme shows very little reaction with H2O2 at pH > 5 and exhibited saturation kinetics at higher H2O2 concentrations (kcatapp increases from 1.1 s-1 at pH 4.5 to 4.5 s-1 at pH 3.1 with an enzyme-linked pKa < 3.7 (Rasmussen, C.B., Bakovic, M., Welinder, K. G., and Dunford, H. B. (1993) FEBS Lett. 321, 102-105)). In the present paper, it is shown that the presence of Ca2+ gives rise to biphasic kinetics for compound I formation, with a slow phase as described above and a fast phase that exhibits a second order rate constant more typical of a classical peroxidase (K1app = 1.5 x 10(7) M-1 S-1, which is pH-independent between 3.3 and 5.0). The amount of enzyme reacting in the fast phase increases with Ca2+ concentration (Kd = 4 +/- 1 mM at pH 4.0), although it is also moderately inhibited by Cl-. The absorption spectrum of BP 1, which appears to be a five-coordinate high spin ferric in the resting state changes insignificantly in the presence of Ca2+. In the presence of Cl-, it becomes six-coordinate high spin (Kd approximately 60 mM at pH 4.0) but only if Ca2+ is also present. Fluoride binds to BP 1 with monophasic kinetics in the presence of 0-5 mM Ca2+. The activating effect of Ca2+ can be mimicked only by replacing it with Sr2+ and Ba2+ ions. Comparing these data with the crystal structure of the inactive neutral form of BP 1 (Henriksen, A., Welinder, K. G., and Gajhede, M. (1997) J. Biol. Chem. 273, 2241-2248) and similar data for wild-type and mutant peroxidases of plant and fungal origin suggests (i) a proton-induced conformational change from an inactive BP 1 at neutral pH to a low activity BP 1 form with a functional distal histidine and (ii) a Ca(2+)-induced slow conformational change (at least compared with compound I formation) of this low activity form to a high activity BP 1 with a typical peroxidase reactivity. BP 1 is the first example of a plant peroxidase whose activity can be reversibly controlled at the enzyme level by pH- and Ca(2+)-induced conformational changes.

Calcium↗
PubMed1998

Sensitivity to second-order motion as a function of temporal frequency and eccentricity.

There is considerable evidence that second-order motion, such as motion consisting of a drifting contrast modulation, is detected separately from first-order motion. Some previous studies have shown that the rate at which sensitivity declines as either drift speed or eccentricity increases is the same for both types of motion. However, these studies have used second-order motion stimuli based on static noise carriers, which we have shown (Smith & Ledgeway, 1997) may be inappropriate because they can give rise to local first-order artifacts. By using dynamic noise carriers, we isolate the second-order motion mechanism and show that its temporal response is much worse than that of the first-order system but that its rate of sensitivity loss with increasing stimulus eccentricity is indeed similar to that of the first-order motion system.

Humans↗
PubMed1998

Independent speed-tuned global-motion systems.

Several experiments were conducted to investigate the role of speed in global-motion processing; the extraction of the direction of motion of a small subset of coherently-moving (signal) dots in a stimulus in which the other (noise) dots move in random directions. The specific aim of the experiments was to determine whether multiple speed-tuned global-motion systems exist. The results of these experiments are: (1) when the signal dots were chosen from a group of dots moving at 1.2 degrees s-1, the speed of additional-noise dots had to be below 4.8 degrees s-1 for them to affect global-motion extraction; (2) the addition of static dots did not impair the extraction of a global-motion signal carried by dots moving at 1.2 degrees s-1; (3) noise dots moving at 1.2 degrees s-1 impaired the extraction of a global-motion signal from dots moving at 10.8 degrees s-1, though not to the same extent as dots moving at a higher speed; and (4) these results were dependent upon speed, not spatial-step size or luminance contrast. These results are interpreted as indicating that global-motion extraction occurs within at least two independent speed tuned systems. One of these systems is sensitive to high speeds and the other to low speeds.

Humans↗
PubMed1998

Substrate binding and catalysis in heme peroxidases.

Peroxidase-catalysed reactions are being analysed at an increasingly advanced level of structural and mechanistic sophistication. A significant development in this respect has been the long-anticipated solution of crystal structures for several plant peroxidases and a fungal peroxidase complexed to benzhydroxamic acid. New insights into peroxide binding and catalysis have been obtained through site-directed mutagenesis, a technique also crucial to recent progress in understanding the diversity of substrate interaction sites associated with peroxidases from different sources.

Binding Sites↗