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R K Poole

Publications and source records attributed to R K Poole.

At least 109 records · Page 6Linked to original sources
PubMed1983

Nitrate, but not silver, ions induce spectral changes in Escherichia coli cytochrome d.

The absorbance maximum (630 nm) of reduced cytochrome d in Escherichia coli membrane particles was diminished by 160 microM AgNO3 or NaNO3 and accompanied by the formation of a species with an absorption maximum at 640-645 nm. Nitrite, trioxodinitrate and nitric oxide elicited qualitatively similar, but faster, changes in the spectrum of cytochrome d, suggesting that formation of a nitrosyl complex may be involved in all cases. In direct contrast to an earlier report, silver ions (160 microM) were without effect on the alpha-bands of reduced cytochromes d, b or a 1.

Cytochrome d Group↗
PubMed1983

The 650 and chromophore in Escherichia coli is an 'oxy-' or oxygenated compound, not the oxidized form of cytochrome oxidase d: an hypothesis.

The form of cytochrome d in Escherichia coli and Azotobacter vinelandii that shows an absorption maximum at 648 to 652 nm ('cytochrome d650') is generally regarded as the oxidized form of this terminal oxidase. Membranes from E. coli grown under oxygen-limited conditions, when treated with ferricyanide, do not reveal cytochrome d650, whereas a sharp symmetrical band at 652 nm results from the reaction of the reduced enzyme with O2 at either room temperature or after flash photolysis of the CO-liganded form at -130 degrees C. Electron paramagnetic resonance spectroscopy of cytochrome d650 trapped at -130 degrees C shows that its spectrum is indistinguishable from the CO-liganded form and does not reveal resonances of high spin ferric haem previously attributed to cytochrome d. An hypothesis is proposed in which cytochrome d650 is an early intermediate in the reaction of reduced cytochrome d650 and oxyhaemoglobin is presented and the hypothesis discussed in relation to earlier work, in which the indirect interconversions of reduced cytochrome d and d650 have been explained by proposing the existence of an 'invisible' form. It is suggested that this form could be the oxidized enzyme.

Cytochrome d Group↗
PubMed1983

The reaction with oxygen of cytochrome oxidase (cytochrome d) in Escherichia coli K12: optical studies of intermediate species and cytochrome b oxidation at sub-zero temperatures.

Optical changes in d- and b-type cytochromes, following initiation of the reaction of cytochrome oxidase d with O2, have been studied in cells and derived membrane particles from oxygen-limited cultures of Escherichia coli K12. At successively higher temperatures between -132 and -88 degrees C, the first scan after photolysis of the Co-liganded, reduced oxidase in the presence of O2 and a slow increase in absorbance at 675 to 680 nm due to an unidentified chromophore. A similar sequence occurs when a single sample is scanned repetitively at -91 degrees C. At higher temperatures, oxidation of at least two spectrally distinct cytochromes b occurs. Selective photolysis of the cytochrome d-CO complex with a He-Ne laser shows that neither of these cytochromes is the CO-binding cytochrome o436. In all oxidation states examined, no absorbance in the 720 to 860 nm region was observed; it is concluded that both cytochromes d and o436 lack redox-active copper that has an environment similar to the copper(s) in mitochondrial cytochrome c oxidase. The amount of cytochrome d650 (but not the amount of reduced cytochrome o436) formed after photolysis is directly proportional to the oxygen concentration in the sample at the time of freeze trapping. The results are discussed in relation to the composition and mechanism of action of cytochrome d.

Carbon Monoxide↗
PubMed1983

The cell cycle of the budding yeast Sterigmatomyces halophilus: culture fractionation by zonal centrifugation and the accumulation of DNA, RNA and protein.

Sterigmatomyces halophilus is an unusual budding yeast in which daughter cells are formed, remote from the mother cell, on fine projections called sterigmata. Some fundamental properties of the cell cycle have been explored by separating cells from an exponentially growing culture into size, and thus age, classes by density-gradient centrifugation. Rate separations on high capacity, high resolution, equivolumetric gradients of sucrose, or, alternatively, isopycnic separations on gradients of Urografin revealed consistent and reproducible patterns of accumulation of DNA, RNA and protein through the cell cycle. Total DNA accumulation was stepwise, synthesis occurring late in the cycle, whilst protein accumulated continuously with no evidence for the discontinuities reported in some other lower eukaryotes. Total RNA accumulation, measured either colorimetrically or by long-term incorporation of radioactively-labelled uracil was transiently elevated early in the cycle and then accumulated continuously. A mathematical analysis of the volume distributions of the cells in fractions from the gradients showed that there is a hyperbolic relationship between cell age and size but that, to a first approximation, measurements of cell size (and density) are direct measures of age. The results are discussed with reference to (1) the unusually high buoyant density of this yeast, (2) the resolution of zonal cell separation methods and (3) macromolecular accumulation in the cell cycles of other eukaryotic micro-organisms.

Cell Cycle↗
PubMed1983

Mitochondria of Tetrahymena pyriformis: enumeration and sizing of isolated organelles using a Coulter Counter and pulse-height analyser.

An electronic particle counter (Coulter Counter ZBI) and pulse-height analyser (Channelyzer C1000) have been used to measure numbers and sizes of mitochondria isolated from the ciliated protozoon Tetrahymena pyriformis. Differential centrifugation of disrupted organisms, followed by single-step sub-fractionation of the mitochondrial fraction on sucrose gradients yielded a population of organelles extensively enriched in the activities of mitochondrial marker enzymes. Gradient-purified mitochondria (approx. 3 X 10(9) particles (mg protein)-1) were stable in electrolyte, exhibited unimodal volume distributions and were somewhat larger (0.93 +/- 0.13 (S.D.) microns 3; 19 preparations) than organelles in a crude mitochondrial fraction. Glutaraldehyde fixation of mitochondria in sucrose gradients decreased the apparent volume to 0.6 +/- 0.06 micron 3 (6 preparations). Based on the recovery in the mitochondrial fraction of mitochondrial membrane-bound cytochromes from a suspension of intact cells, the number of mitochondria per cell was estimated to be approximately 1440, representing 15% of the total cell volume. Isolated mitochondria were osmotically sensitive and exhibited an apparent marked contraction on adding Ca2+ (10 microM-10mM). Addition of chloramphenicol (500 micrograms ml-1) to exponentially growing Tetrahymena cultures resulted in an almost immediate cessation of cell division and a dramatic decrease in cell volume. Mitochondria purified from such cells were much smaller than control mitochondria (0.21 +/- 0.02 microns 3; 7 measurements); their population density was approximately 900 per cell, equivalent to 11% of total cell volume. The measurements of mitochondrial populations using the Coulter Counter and electron microscopy are in good agreement.

Animals↗
PubMed1982

A re-examination of the cytochromes of Escherichia coli using fourth-order finite difference analysis: their characterization under different growth conditions and accumulation during the cell cycle.

The cytochromes of Escherichia coli K12 have been studied using low-temperature (77 K) difference spectrophotometry. Numerical (i.e. fourth-order finite differences) analysis resolved the alpha band of reduced minus oxidized spectra of whole cells from aerobically growth cultures into five components, with absorption maxima at 548, 551.5, 555.5, 560 and 563 nm. Using the same differencing intervals, numerical analysis of cells grown under oxygen-limited conditions revealed only two components, at 555.5 and 559 nm. Similar analysis of cells grown anaerobically with fumarate as electron acceptor revealed four absorption maxima, at 548, 550.5, 555.5 and 559 nm. Membrane particles from aerobically grown cells showed the same absorption bands as intact cells; preliminary evidence was obtained for a component of the 555.5 nm band that could be relatively easily washed from the membrane. The contribution of cytochrome o or its CO-liganded form to the alpha region could not be determined by numerical analysis. We conclude, in contrast to a previous application of numerical analysis to the cytochromes of E. coli, that growth under anaerobic or oxygen-limited conditions results in the appearance of cytochromes spectrally distinct from those in aerobically grown cells. An attempt has been made to reconcile the presence of multiple components detected in the alpha region by numerical analysis of aerobically grown cells with the diverse components described by others. Quantification of cytochromes revealed by numerical analysis in aerobically grown cells separated into size (and thus age) classes by zonal centrifugation showed that the major components accumulated continuously, probably exponentially, throughout the cell cycle.

Aerobiosis↗
PubMed1982

A dimeric complex of ruthenium: a new inhibitor of respiration-driven calcium transport in Escherichia coli K12.

Succinate-driven calcium uptake by everted membrane vesicles from Escherichia coli was sensitive to 2 mM-KCN and a dimeric, mixed valence complex of ruthenium, Ru2 (NH3)6Br5(H2O) (100 micro M), but relatively insensitive to 100 micro M-Ruthenium Red. The sensitivity of uptake to KCN and the slight inhibition caused by Ruthenium Red could be attributed to the different potencies of these compounds as respiratory inhibitors. Respiration was completely insensitive to the dimeric ruthenium complex, however, suggesting that this compound is an inhibitor of respiration-driven calcium transport.

Biological Transport, Active↗
PubMed1981

Respiratory biogenesis during the cell cycle of aerobically grown Escherichia coli K 12. The accumulation and ligand binding of cytochrome o.

A quantitative assay is described for the measurement of cytochrome o in intact cells of E. coli. The procedure involves flash photolysis of the CO-liganded, reduced enzyme in the absence of O2 at temperatures (approx. -100 degrees C) at which the rate of recombination of CO is immeasurably slow. Other CO-binding pigments known to be present, particularly cytochrome d, are excluded from the photodissociation spectrum under these conditions. Measurement of the content of cytochrome o in bacteria separated into size (and thus age) classes by zonal centrifugation shows that the cytochrome accumulates continuously, probably exponentially, throughout the cell cycle and thus constitutes a constant proportion of cell protein during the cycle. The velocity of recombination of CO with cytochrome o at -65 degrees C is invariant over the cell cycle.

Carbon Monoxide↗
PubMed1981

Respiratory biogenesis during the cell cycle of aerobically grown Escherichia coli K12. The accumulation of iron-sulphur clusters and their orientation in the membrane.

The magnitudes of four signals detectable in intact cells by electron paramagnetic resonance spectroscopy, and assigned to membrane-associated protein, increase continuously during the cell cycle of Escherichia coli K12. Studies on membrane multilayers prepared from cells separated according to their age in the cycle suggest that the orientation within the membrane of the ferredoxin-type signal is invariant throughout the cycle.

Cell Cycle↗
PubMed1981

The light-reversible binding of carbon monoxide to cytochrome a1 in Escherichia coli K12.

Difference spectra at 77 K of intact Escherichia coli K 12 grown under oxygen-limited conditions revealed the presence of cytochrome a1. In the presence of CO, the band of the reduced form, observed in both the alpha and gamma regions of the spectrum, was decreased. Dual-wavelength scanning spectrophotometry at sub-zero temperatures revealed a flash-dissociable CO-binding pigment with a broad band around 595 nm, identified as cytochrome alpha 1. Photolysis in the presence of O2 revealed no such band in difference spectra where the reference spectrum was that of the CO-liganded form, a result consistent with the binding of O2 to cytochrome a1. Repeated cycles of photolysis and recombination of the cytochrome with CO were demonstrated at --46 degrees C. The apparent energy of activation for the reaction with CO was 10.9 kcal mol-1 (45.6 kJ mol-1). The results are discussed in relation to previous assumptions and results regarding ligand binding to cytochrome a1 and the function of this cytochrome in bacterial respiration.

Carbon Monoxide↗
PubMed1981

The reaction of cytochrome o in Escherichia coli K12 with oxygen. Evidence for a spectrally and kinetically distinct cytochrome o in cells from oxygen-limited cultures.

Intact cells harvested from O2-limited batch cultures of Escherichi coli K12 contained high levels of the CO-binding cytochromes d, o and a1. In photodissociation difference spectra (i.e. photolysed minus reduced + CO), a peak at 436 nm and a trough at 415 nm have been assigned to an 0-type cytochrome, and not cytochrome d, by photolysis with white light and an He-Ne laser. The reaction of reduced cytochrome o436 with O2 at sub-zero temperatures involved O2 binding to give intermediate(s) with spectral characteristics similar to those of the reduced oxidase-CO complex. The reaction with O2 at successively higher temperatures (range -98 to -59 degrees C) was accompanied by the formation of a trough (with reference to the CO-liganded state) at 436 nm which eventually shifted to 432 nm, indicative of the oxidized form. The apparent energy of activation at low temperatures was 44.6 kJ mol-1 (10.7 kcal mol-1). There was a linear relationship between the rate of formation of the oxygen compound and the O2 concentration up to about 0.5 mM. The second-order constant for this reaction was 10.9 M-1 s-1 at 100 degrees C, at least 10-fold greater than for the reaction of cytochrome o432 with O2 in cells from vigorously aerated cultures. The reaction of both types of cytochrome o with O2 was not readily reversible in the light or in the dark and was further distinguished from the reaction with CO by the markedly lower velocity of the CO reaction. Comparisons are drawn between the reactions with O2 of cytochrome(s) o in E. coli from O2-sufficient and O2-limited cultures and of mitochondrial cytochrome a3. It is proposed that, like the synthesis of cytochrome d, the formation of cytochrome o436 represents an adaptation of the organism to reduced O2 availability.

Carbon Monoxide↗
PubMed1980

The orientation of iron-sulphur clusters in membrane multilayers prepared from aerobically-grown Escherichia coli K12 and a cytochrome-deficient mutant.

1. Membrane particles prepared from ultrasonically-disrupted, aerobically-grown Escherichia coli were centrifuged on to a plastic film that was supported perpendicular to the centrifugal field to yield oriented membrane multilayers. In such preparations, there is a high degree of orientation of the planes of the membranes such that they lie parallel to each other and to the supporting film. 2. When dithionite- or succinate-reduced multilayers are rotated in the magnetic field of an e.p.r. spectrometer, about an axis lying in the membrane plane, angular-dependent signals from an iron-sulphur cluster at g(x)=1.92, g(y)=1.93 and g(z)=2.02 are seen. The g=1.93 signal has maximal amplitude when the plane of the multilayer is perpendicular to the magnetic field. Conversely, the g=2.02 signal is maximal when the plane of the multilayer is parallel with the magnetic field. 3. Computer simulations of the experimental data show that the cluster lies in the cytoplasmic membrane with the g(y) axis perpendicular to the membrane plane and with the g(x) and g(z) axes lying in the membrane plane. 4. In partially-oxidized multilayers, a signal resembling the mitochondrial high-potential iron-sulphur protein (Hipip) is seen whose g(z)=2.02 axis may be deduced as lying perpendicular to the membrane plane. 5. Appropriate choice of sample temperature and receiver gain reveals two further signals in partially-reduced multilayers: a g=2.09 signal arises from a cluster with its g(z) axis in the membrane plane, whereas a g=2.04 signal is from a cluster with the g(z) axis lying along the membrane normal. 6. Membrane particles from a glucose-grown, haem-deficient mutant contain dramatically-lowered levels of cytochromes and exhibit, in addition to the iron-sulphur clusters seen in the parental strain, a major signal at g=1.90. 7. Only the latter may be demonstrated to be oriented in multilayer preparations from the mutant. 8. Comparisons are drawn between the orientations of the iron-sulphur proteins in the cytoplasmic membrane of E. coli and those in mitochondrial membranes. The effects of diminished cytochrome content on the properties of the iron-sulphur proteins are discussed.

Cell Membrane↗
PubMed1980

Low-temperature spectral and kinetic properties of cytochromes in Escherichia coli K-12 grown at lowered oxygen tension.

Escherichia coli K-12 was grown in batch culture in a medium containing succinate as carbon source, supplemented with casein hydrolysate, and with a rate of oxygen supply that resulted in dissolved O2 tension falling to 10% of saturation in the latter stages of growth. Cytochromes in such cells were qualitatively indistinguishable from those present in cells grown under conditions of vigorous aeration where dissolved O2 tensin remained greater than 80% saturation. Spectra recorded at 77 K and their fourth-order finite difference analyses revealed the absence of cytochrome b-558 and only low concentrations of cytochromes a1 and d(a2). At low temperatures, the reaction of cytochrome o with O2 in intact cells, grown under lowered O2 tension, proceeds through the same stage as observed previously in cells grown with vigorous aeration (Poole, R.K., Waring, A.J. and Chance, B. (1979) Biochem. J. 184, 379-389). However, much higher temperatures are required for comparable progress of the reaction in cells grown at lowered O2 tensions. AT 91 degrees C, the reaction with O2 involves ligand binding to give intermediate(s) with spectral characteristics similar to those of the reduced oxidase-CO complex. Temperatures of approx. -79 degrees C are required for the observation of biphasic kinetics and the attainment of an 'end point' in the reaction, features that are seen at temperatures below -98 degrees C in cells from vigorously-aerated cultures. At -32.5 degrees C, oxidation of cytochrome o is observed. The energy of activation for this reaction at low temperatures is 29.9 kJ x mol-1. Binding with CO, in contrast to binding with O2, is characterized by high photolytic reversibility and appears to be less affected by the degree of aeration of cells during growth.

Cold Temperature↗