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Biomedical subjects

M R Bendall

Publications and source records attributed to M R Bendall.

25 records · Page 2Linked to original sources
PubMed1984

Estimation of H+ to adenosine 5'-triphosphate stoichiometry of Escherichia coli ATP synthase using 31P NMR.

High-field 31P NMR techniques have been used to measure transmembrane delta pH in wild-type, unc A, and hem A mutants of Escherichia coli. delta psi was measured by distribution methods with radioactive tetraphenylphosphonium bromide and 86Rb+ ions as the probes, while intracellular ATP, ADP, and inorganic phosphate concentrations were determined from the 31P NMR spectra. delta G'p and the stoichiometry for ATP synthesis [delta G'p/(F delta p)] were then calculated. The stoichiometry of the ATP synthase was found to vary as a function of the cellular metabolic state. In nongrowing, wild-type cells delta p was 192 +/- 16 mV with succinate as the substrate and saturating oxygen tension. With limiting oxygen (congruent to microM oxygen), delta p was 125 +/- 14 mV. Nucleoside triphosphate synthesis was observed in both cases. The H+/ATP stoichiometry varied from 2.15 +/- 0.35 under aerobic conditions to 3.6 +/- 0.8 at low oxygen tension. delta p for unc A cells was 140 +/- 14 mV with glucose as the substrate (greater than 2.5 microM oxygen) and for hem A mutants was 115 +/- 10 mV. The bulk phase potentials in oxygen-limited, wild-type cells and in respiratory deficient (hem A) cells are comparable, but in the former the ATPase is poised for synthesis while in the latter it generates delta p. The data support a role for localized interactions between the redox and the ATPase sites.

ATP Synthetase Complexes↗
PubMed1984

Depth pulse sequences for surface coils: spatial localization and T1 measurements.

The depth pulse sequences theta;[2 theta(+/- x, +/- y)]2 and 2 theta;theta(+/- x);[2 theta(+/- x, +/- y)]2 have been implemented with a 20 mm diameter two-turn surface coil operating at 31P resonance (89.96 MHz). In these sequences theta refers to the pi/2 rf pulse at the center of the sensitive region of the coil, +/- x and +/- y denote the four orthogonal phases of the rf pulses, and ";" represents an optional brief delay (e.g., 4 microseconds) between pulses to facilitate switching between different phases. Localization of the sensitive region was demonstrated with phantom samples by in vivo monitoring of rat livers and detection of necrotic regions of subcutaneously implanted tumors. The inversion-recovery pulse sequence, 2 theta-tau-theta(+/- x);[2 theta(+/- x, +/- y)]2, where tau is a variable delay, was employed to measure the spin-lattice relaxation time of a selected region, which could be varied by changing the pulse width and the size of the surface coil.

Animals↗
PubMed1983

High-field phosphorus NMR studies of the stoichiometry of the lactate/proton carrier in Streptococcus faecalis.

High-field 31P-NMR studies of whole cells of Streptococcus faecalis have shown that delta pH can be formed by ATP hydrolysis and also by lactate transport. We have used 31P-NMR to measure the pH dependence of the variable stoichiometry of the proton/lactate carrier. At low external pH (pH approximately equal to 6.5) the influx stoichiometry was 1.1 H+/lactate, while at high pH (7.5) the ratio was almost 2; the apparent midpoint pH of this variable stoichiometry is 7. delta psi measurements support the electrogenic nature of lactate transport at high pH; the variable rate of membrane depolarization caused by lactate transport also had a midpoint near pH 7.0. The data is consistent with a symmetrical carrier operating with variable stoichiometry as proposed by Michels et al.

Biological Transport↗
PubMed1977

Inhibition of papain by N-acyl-aminoacetaldehydes and N-acyl-aminopropanones. Evidence for hemithioacetal formation by a cross-saturation technique in nuclear-magnetic resonance spectroscopy.

N-Acyl-aminoacetaldehydes are potent inhibitors of the proteolytic enzyme, papain. Although they exist predominantly in their hydrated form in aqueous solution only the aldehyde is an effective inhibitor. The binding constants for related amides and methyl ketones confirm that it is principally the lower steric requirement of the aldehyde rather than its increased electrophilicity which is responsible for its powerful inhibitor properties. Using nuclear magnetic resonance spectroscopy, evidence is provided for an N-acetyl-aminoacetaldehyde-papain complex. Using a cross-saturation technique evidence is also provided for a hemithioacetal, formed from the aldehyde and the active-site thiol group. Hemithioacetal formation has also been detected between N-benzoyl-aminoacetaldehyde and papain. This provides the first direct evidence for a tetrahedral adduct with papain and supports the proposed involvement of such intermediates in papain-catalysed hydrolyses.

Acetaldehyde↗
PubMed1976

Co-operative ionisation of aspartic-acid-158 and histidine-159 in papain. Evidence from 19F nuclear-magnetic-resonance and fluorescence spectroscopy.

The chemical shift of the single resonance in the 19F nuclear magnetic resonance spectrum of papain which has been irreversibly inhibited by 3-bromo-1,1,1-trifluoropropanone, exhibits pH-dependence. The fluorescence intensity of this papain derivative shows pH-dependence on two groups which exhibit co-operative ionisation. This co-operative behaviour is probably a function of the probe since the fluorescence intensity of S-ethane-thio-papain is dependent on a single ionisation constant, whereas that of S-(2-hydroxyethane)-thio-papain is dependent on two ionisable groups again acting co-operatively. The 1,1,1-trifluoroketone probe will be hydrated in aqueous solution and would be capable of hydrogen bonding with the protein. The two groups detected are considered to be aspartic-acid-158 and histidine-159. The co-operative ionisation of these groups in substrate hydrolysis is discussed.

Aspartic Acid↗
PubMed1976

A spectroscopic investigation of S-trifluoroethylthiopapain. An investigation of the active site of papain.

The pH dependence of the 19F chemical shift and the fluorescence spectrum of S-2,2,2-trifluoro-1,1-dideuteroethyl-thio-paapain are analysed in terms of dependence on the ionisation of aspartic-acid-158 and histidine-159. The 19F probe causes negative cooperativity between these groups, and does not detected any ionisation at high pH. The intermediate chemical exchange rates for the ionisation of aspartic-acid-158 and histidine-159 allow the approxmate rate constants for proton transfer to be calculated. The rather low rate constants are explained in terms of the hydrophobicity of the active-site region and the net positive charge on the enzyme resulting from its high isoelectric point.

Binding Sites↗