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P B Garland

Publications and source records attributed to P B Garland.

At least 19 recordsLinked to original sources
PubMed1991

Chemiosmotic systems in medicine.

The concept of chemiosmotic systems arises from the pioneering work of Peter Mitchell on two fronts. One is concerned with the mechanisms by which molecules are transported across membranes which are generally barriers to such transport. These mechanisms are inevitably molecular, and are now yielding their secrets to a combination of structural protein chemistry and molecular biology. The other front is more physiological, and explores the functional relationships between metabolism and transport. Nevertheless, the two fronts form a continuum of mutually related structure and function. Chemiosmotic systems provide a hierarchy of complexity, starting from say a uniporter reconstituted in a chemically defined bilayer, and proceeding to greater complexity in mitochondria, chloroplasts, eukaryotic and prokaryotic cell membranes, and multicellular systems. Their relationship to medicine is profound, because they provide many opportunities for therapeutic intervention. In this paper I present an overview of chemiosmotic systems at different levels of complexity, both molecular and biological, of their involvements in pathology, and of possible pharmacological treatment or prevention of disease.

Biological Transport, Active↗
PubMed1979

Phosphorescence of protein-bound eosin and erythrosin. A possible probe for measurements of slow rotational mobility.

We used a pulsed dye laser working at 540 nm to excite triplet-state formation of eosin and erythrosin, either bound or unbound to bovine serum albumin, in aqueous solution anaerobically at pH 8 and 20-22 degrees C. Delayed emission from radiative transitions of the triplet state was readily detectable, both as delayed fluorescence and as red phosphorescence. Detection of the triplet state by measurement of phosphorescence at 645 nm upwards was at least 100-fold more sensitive than by absorbance measurements of ground-state depletion at 500 nm. When immobilized in poly(methyl methacrylate), the phosphorescence of eosin and erythrosin was polarized with an anisotropy parameter [Jablonski (1961) Z. Naturforsch. A16, 1-4] of about 0.25. The phosphorescence of erythrosin is sufficiently intense to be distinguishable from the long-wavelength end of fluorescence under conditions of continuous rather than pulsed excitation. Our observations suggest that phosphorescence depolarization of eosin or erythrosin probes could be used as a highly sensitive method of measuring rotational relaxation times in region from 10(-5) to 10(-3) s, such as those of the uniaxial rotation of membrane proteins.

Eosine Yellowish-(YS)↗
PubMed1979

Synthesis of cytoplasmic membrane during growth and division of Escherichia coli. Dispersive behaviour of respiratory nitrate reductase.

We have used the penicillin selection method of Autissier & Kepes [(1972) Biochimie 54, 93--101] to study the segregation of membrane-bound respiratory nitrate reductase (EC 1.9.6.1) in Escherichia coli for the three generations after cessation of nitrate reductase synthesis caused by withdrawal of nitrate from the growth medium. We also included a physical separation procedure that permitted direct assay for nitrate reductase activity among all fractions produced by the penicillin selection method. We conclude that the segregation of nitrate reductase after cell division is dispersive, and not semi-conservative as proposed by Autissier & Kepes (1972).

Cell Division↗
PubMed1977

Sites and specificity of the reaction of bipyridylium compounds with anaerobic respiratory enzymes of Escherichia coli. Effects of permeability barriers imposed by the cytoplasmic membrane.

The ability of the oxidized and singly reduced species of several bipyridylium cations to cross the cytoplasmic membrane of Escherichia coli was studied to locate the sites of reaction of the dyes with anaerobic respiratory enzymes. Benzyl Viologen radical crossed the membrane rapidly, whereas the oxidized species did not. The oxidized or radical species of Methyl Viologen, Morfamquat or Diquat did not rapidly cross the membrane. It was also shown that the dithionite anion does not cross the cytoplasmic membrane of E. coli. Diquat radical donates electrons to the nitrate reductase pathway at the periplasmic aspect of the membrane, whereas Benzyl Viologen radical reacted directly with nitrate reductase itself (EC 1.7.99.4) at the cytoplasmic aspect of the membrane. Thus the pathway of electron transfer in the nitrate reductase pathway is transmembranous. Formate hydrogenlyase (EC 1.2.1.2) and an uncharacterized nitrite reductase activity react with bipyridylium dyes at the periplasmic aspect of the membrane. Fumarate reductase (succinate dehydrogenase; EC 1.3.99.1) reacts with bipyridylium radicals, and formate dehydrogenase (cytochrome) (EC 1.2.2.1) with ferricyanide, at the cytoplasmic aspect of the membrane. The differing charge and membrane permeation of oxidized and radical species of bipyridylium dyes greatly complicate their use as potentiometric mediators in suspensions of cells or membrane vesicles.

Aldehyde Oxidoreductases↗
PubMed1977

Structure of mitochondria and vacuoles of Candida utilis and Schizosaccharomyces pombe studied by electron microscopy of serial thin sections and model building.

The structure of mitochondria and of vacuoles in Candida utilis and Schizosaccharomyces pombe has been studied by electron microscopy of serial thin sections and subsequent model building. The models of the two cells of C. utilis which were studied confirmed our earlier findings, made by high voltage electron microscopy of thick sections, that there is a single, branched and continuous mitochondrial network in the cell (Davison & Garland, 1975). A model of a S. pombe cell showed that the mitochondrial structure was far more continuous than expected from inspection of thin sections, there being but two large and two small mitochondria. The models demonstrated that the few large vacuoles in C. utilis were interconnected into a single cluster, whereas in S. pombe there were two separate complexes of interconnected vacuoles towards each pole of the cell.

Ascomycota↗
PubMed1976

Electron-paramagnetic-resonance studies on the molybdenum of nitrate reductase from Escherichia coli K12.

Studies on the respiratory nitrate reductase (EC 1.7.99.4) from Escherichia coli K12 by electron-paramagnetic-resonance spectroscopy indicate that its molybdenum centre is comparable with that in other molybdenum-containing enzymes. Two Mo(V) signals may be observed; one shows interaction of Mo(V) with a proton exchangeable with the solvent and has: A (1H) 0.9-1.2mT; g1 = 1.999; g2=1.985; g3 = 1.964; gav. = 1.983. Molybdenum of both signal-giving species may be reduced with dithionite and reoxidized with nitrate.

Electron Spin Resonance Spectroscopy↗
PubMed1976

A stopped-flow dual-wavelength spectrophotometer suitable for the study of respiratory chains.

The requirements for a dual-wavelength stopped-flow spectrophotometer to be suitable for studying limited quantities of respiratory-chain preparations are described. They can be met by a design using mainly commercially available components. The constructed apparatus has a dead-time of approx. 2.6 ms, a mixing ratio of 17:1, and a minimal requirement for 0.5 ml of mixed reactants per flow.

Cytochromes↗
PubMed1976

Kinetic characterization of the membrane-bound cytochromes of Escherichia coli grown under a variety of conditions by using a stopped-flow dual-wavelength spectrophotometer.

A study was made of the rapid oxidation kinetics of the cytochromes of Escherichia coli. The b-type cytochromes were kinetically heterogeneous, with one species (presumably cytochrome o) oxidized so rapidly that it could fully support observed oxidation rates. Cytochrome d but not cytochrome a1 was also kinetically competent to support respiration. However, in cells grown anaerobically in the presence of NO3-, cytochrome d exhibited slow oxidation kinetics and a red-shift in its reduced-minus-oxidized difference spectrum.

Anaerobiosis↗
PubMed1975

Electron-paramagnetic-resonance spectroscopy studies of iron-sulphur centres of submitochondrial particles from iron- and sulphur-deficient. Candida utilis.

1. Measurements were made at 12 degrees K of the electron-paramagnetic-resonance (e.p.r.) spectra of submitochondrial particles from Candida utilis cells grown under conditions that alter the amount of the mitochondrial NADH dehydrogenase (EC 1.6.99.3). 2. Iron-limited growth decreases the extent of iron-sulphur e.p.r. signals to undetectable values that are less than 1 percent of those normally found with glycerol-limited growth. 3. Small but significant signals attributable to the NADH dehydrogenase were detected in submitochondrial particles from sulphate-limited cells. 4. Measurements made on submitochondrial particles prepared from these and other phenotypically modified cells lead us to conclude that the presence of low-temperature e.p.r.-detectable iron-sulphur centres attributable to the NADH dehydrogenase are necessary but not sufficient for the coupling of ATP synthesis to the NADH dehydrogenase reaction in the mitochondrial membrane of C. utilis. 6. The amplitude of the g=2.01 signal observed in non-reduced submitochondrial particles is approximately tenfold diminished by iron limitation but not significantly altered by sulphate limitation.

Adenosine Triphosphate↗
PubMed1975

Proton translocation and the respiratory nitrate reductase of Escherichia coli.

Stoicheometries and rates of proton translocation associated with respiratory reduction of NO3- have been measured for spheroplasts of Escherichia coli grown anaerobically in the presence of NO3-. Observed stoicheiometries [leads to H+/NO3- ratio; P. Mitchell (1966) Chemiosmotic Coupling in Oxidative and Photosynthetic Phosphorylation, Glynn Research, Bodmin] were approx. 4 for L-malate oxidation and approx. 2 for succinate, D-lactate and glycerol oxidation. Measurements of the leads to H+/2e- ratio with formate as the reductant and oxygen or NO3- as the oxidant were complicated by pH changes associated with formate uptake and CO2 formation. Nevertheless, it was possible to conclude that the site of formate oxidation is on the inner aspect of the cytoplasmic membrane, that the leads to H+/O ratio for formate oxidation is approx. 4, and that the leads to H+/NO3- ratio is greater than 2. Measurements of the rate of NO3- penetration into osmotically sensitive spheroplasts demonstrated an electrogenic entry of NO3- anion. The permeability coefficient for nitrate entry at 30 degrees C was between 10(-9) and 10(-10) cm- s(-1). The calculated rate of nitrate entry at the concentration typically used for the assay of nitrate reductase (EC 1.7.99.4) activity was about 0.1% of that required to support the observed rate of nitrate reduction by reduced Benzyl Viologen. Measurements of the distribution of nitrate between the intracellular and extracellular spaces of a haem-less mutant, de-repressed for nitrate reductase but unable to reduce nitrate by the respiratory chain, showed that, irrespective of the presence or the absence of added glucose, nitrate was not concentrated intracellularly. Osmotic-swelling experiments showed that the rate of diffusion of azid anion across the cytoplasmic membrane is relatively low in comparison with the fast diffusion of hydrazoic acid. The inhibitory effect of azide on nitrate reductase was not altered by treatments that modify pH gradients across the cytoplasmic membrane. It is concluded that the nitrate-reducing azide-sensitive site of nitrate reductase is located on the outer aspect of the cytoplasmic membrane. The consequences of this location for mechanisms of proton translocation driven by nitrate reduction are discussed, and lead to the proposal that the nitrate reductase of the cytoplasmic membrane is vectorial, reducing nitrate on the outer aspect of the membrane with 2H+ and 2e- that have crossed from the inner aspect of the membrane.

Anaerobiosis↗