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The steady-state kinetics of Pseudomonas aeruginosa cytochrome oxidase were studied. Reduced cytochrome c551 and azurin from the same bacteria were used as the electron-donating substrates, while dioxygen served as the electron acceptor. Oxidized cytochrome c551 and azurin exhibited product inhibition of the reaction. However, apo-azurin and azurin derivatives in which the copper was substituted by the redox-inert ions Ni2+, Co2+, Cd2+ and Zn2+, did not show any effect on the kinetics. These observations implied that complex formation between the substrates or the products and the enzyme is not a rate-limiting step and is not the cause for product inhibition. The integrated rate law for a reaction scheme in which we assumed that complex formation was not rate limiting was fitted to the complete reaction traces. The results suggested that it is the low thermodynamic driving force, expressed in the small differences in redox potential between the substrates and heme c of the enzyme, which cause the observed product inhibition. 相似文献
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Mike A. Steer Jayson M. Semmens 《Journal of experimental marine biology and ecology》2003,290(2):165-178
The influence of both predator and prey size on the shift from a pulling to a drilling predatory response was examined in the intertidal octopus Octopus dierythraeus, using an experimental program. Additionally, selective drilling, where particular regions of the prey are targeted, was examined for a variety of bivalve and gastropod prey. O. dierythraeus always initially attempted to pull bivalves apart. Shells that were eventually drilled were always subjected to significantly more pulling attempts than those that could be pulled apart, indicating that octopus are willing to expend more energy to access the flesh quickly. There was no defined threshold where bivalve size caused an octopus to switch from a pulling to a drilling response. Instead, there was a broad size range where the octopus could adopt either handling method and it varied for each individual. Octopus may only able to pull open bivalves before the molecular ratchet or ‘catch’ mechanism that many bivalves possess is engaged. This might explain the lack of a relationship between either octopus or bivalve size and the success of pulling, as it is likely that when the bivalves were presented to individual octopus they were either setting the ‘catch’ mechanism, or had already engaged it. O. dierythraeus demonstrated selective drilling on a variety of molluscan prey, with penetration sites differing between prey species. O. dierythraeus targeted the valve periphery, which was the thinnest part of the shell, therefore minimizing handling time. O. dierythraeus always drilled gastropods, but did not target the thinnest regions of the shells, with drill site varying according to the morphology of the prey. Elongate species with pronounced aperture lips were drilled in the apical region, close to the columella on the side of the opercula whereas nonelongate species were drilled immediately above the aperture. The location of drilling sites may represent a trade-off between targeting the most effective places to inject paralyzing secretions and the mechanically simplest places to drill. 相似文献
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Triplet-state detection of labeled proteins using fluorescence recovery spectroscopy 总被引:1,自引:0,他引:1
The experimental procedures for detecting the triplet states of chromophores in solutions (cuvettes) by fluorescence recovery spectroscopy (FRS) are described in detail, together with applications in studies of protein structure and protein-cell interactions in the microsecond to millisecond time domain. The experimental configuration has been characterized by measuring the emission intensities and anisotropies of eosin and erythrosin immobilized in poly(methyl methacrylate). The fluorescence data are compared with those from phosphorescence emission measurements and with theoretical predictions. Triplet-state lifetimes were obtained in 5 mM phosphate buffer, pH 7.0, of concanavalin A labeled with eosin, tetramethylrhodamine, and fluorescein and of alpha 2-macroglobulin labeled with the first two probes. In the case of labeled concanavalin A, iodide quenching measurements gave bimolecular rate constants of approximately 10(9) M-1 s-1. The usefulness of FRS for studying protein-cell interactions is exemplified with eosin-labeled concanavalin A bound to living A-431 human epidermoid carcinoma cells. Finally, the advantages and disadvantages of the technique are compared to those of the alternative phosphorescence emission method. 相似文献
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Extracellular production of a heat-stable somatic antigen (HSSA) by Bacillus thuringiensis subsp. dendrolimus strain T84A1-A [flagellar (H) serotype 4a: 4b] was serologically detected. In Ouchterlony tests, the HSSA antiserum gave single precipitin lines against both untreated and heat-treated culture supernatants. These two precipitin lines fused completely. When colonies of strain T84A1-A were grown on nutrient agar plates containing the homologous HSSA antiserum, precipitin halos were formed around the colonies. Of 27 type strains of B. thuringiensis subspecies tested, only the type strains of B. thuringiensis subsp. sotto (H serotype 4a: 4b) and B. thuringiensis subsp. israelensis (H serotype 14) formed [precipitin halos on nutrient agar plates containing antiserum against the HSSA of strain T84A1-A. 相似文献
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Purification and some properties of the methyl-CoM reductase of Methanothrix soehngenii 总被引:1,自引:0,他引:1
Mike S.M. Jetten Alfons J.M. Stams Alexander J.B. Zehnder 《FEMS microbiology letters》1990,66(1-3):183-186
Abstract The methyl-CoM reductase from Methanothrix soehngenii was purified 18-fold to apparent homogeneity with 50% recovery in three steps. The native molecular mass of the enzyme estimated by gel-fitration was 280 kDa. SDS-polyacrylamide gel electrophoresis revealed three protein bands corresponding to M r 63 900, 41 700 and 30 400 Da. The methyl-coenzyme M reductase constitutes up to 10% of the soluble cell protein. The enzyme has K m apparent values of 23 μM and 2 mM for N -7-mercaptoheptanoylthreonine phosphate (HS- HTP = component B ) and methyl-coenzyme M (CH3 CoM) respectively. At the optimum pH of 7.0 60 nmol of methane were formed per min per mg protein. 相似文献