Biomedical subjects

M Revington

Publications and source records attributed to M Revington.

11 recordsLinked to original sources

PubMed2000

A re-examination of the structural and functional consequences of mutation of alanine-128 of the b subunit of Escherichia coli ATP synthase to aspartic acid.

The effects of mutation of residue Ala-128 of the b subunit of Escherichia coli ATP synthase to aspartate on the structure of the subunit and its interaction with the F(1) sector were analyzed. Determination of solution molecular weights by sedimentation equilibrium ultracentrifugation revealed that the A128D mutation had little effect on dimerization in the soluble b construct, b(34-156). However, the mutation caused a structural perturbation detected through both a 12% reduction in the sedimentation coefficient and also a reduced tendency to form intersubunit disulfide bonds between cysteine residues inserted at position 132. Unlike the wild-type sequence, the A128D mutant was unable to interact with F(1)-ATPase. These results indicate that the A128D mutation caused a structural change in the C-terminal region of the protein, preventing the binding to F(1) but having little or no effect on the dimeric nature of b.

Alanine↗

PubMed2000

The second stalk of Escherichia coli ATP synthase.

Two stalks link the F(1) and F(0) sectors of ATP synthase. The central stalk contains the gamma and epsilon subunits and is thought to function in rotational catalysis as a rotor driving conformational changes in the catalytic alpha(3)beta(3) complex. The two b subunits and the delta subunit associate to form b(2)delta, a second, peripheral stalk extending from the membrane up the side of alpha(3)beta(3) and binding to the N-terminal regions of the alpha subunits, which are approx. 125 A from the membrane. This second stalk is essential for binding F(1) to F(0) and is believed to function as a stator during rotational catalysis. In vitro, b(2)delta is a highly extended complex held together by weak interactions. Recent work has identified the domains of b which are essential for dimerization and for interaction with delta. Disulphide cross-linking studies imply that the second stalk is a permanent structure which remains associated with one alpha subunit or alphabeta pair. However, the weak interactions between the polypeptides in b(2)delta pose a challenge for the proposed stator function.

ATP Synthetase Complexes↗

PubMed2000

The b subunit of Escherichia coli ATP synthase.

The b subunit of ATP synthase is a major component of the second stalk connecting the F1 and F0 sectors of the enzyme and is essential for normal assembly and function. The 156-residue b subunit of the Escherichia coli ATP synthase has been investigated extensively through mutagenesis, deletion analysis, and biophysical characterization. The two copies of b exist as a highly extended, helical dimer extending from the membrane to near the top of F1, where they interact with the delta subunit. The sequence has been divided into four domains: the N-terminal membrane-spanning domain, the tether domain, the dimerization domain, and the C-terminal delta-binding domain. The dimerization domain, contained within residues 60-122, has many properties of a coiled-coil, while the delta-binding domain is more globular. Sites of crosslinking between b and the a, alpha, beta, and delta subunits of ATP synthase have been identified, and the functional significance of these interactions is under investigation. The b dimer may serve as an elastic element during rotational catalysis in the enzyme, but also directly influences the catalytic sites, suggesting a more active role in coupling.

Amino Acid Sequence↗

PubMed1999

The dimerization domain of the b subunit of the Escherichia coli F(1)F(0)-ATPase.

In this study a series of N- and/or C-terminal truncations of the cytoplasmic domain of the b subunit of the Escherichia coli F(1)F(0) ATP synthase were tested for their ability to form dimers using sedimentation equilibrium ultracentrifugation. The deletion of residues between positions 53 and 122 resulted in a strongly decreased tendency to form dimers, whereas all the polypeptides that included that sequence exhibited high levels of dimer formation. b dimers existed in a reversible monomer-dimer equilibrium and when mixed with other b truncations formed heterodimers efficiently, provided both constructs included the 53-122 sequence. Sedimentation velocity and (15)N NMR relaxation measurements indicated that the dimerization region is highly extended in solution, consistent with an elongated second stalk structure. A cysteine introduced at position 105 was found to readily form intersubunit disulfides, whereas other single cysteines at positions 103-110 failed to form disulfides either with the identical mutant or when mixed with the other 103-110 cysteine mutants. These studies establish that the b subunit dimer depends on interactions that occur between residues in the 53-122 sequence and that the two subunits are oriented in a highly specific manner at the dimer interface.

Amino Acid Sequence↗

PubMed1997

Sympathetic and parasympathetic interaction in vascular and secretory control of the nasal mucosa in anaesthetized dogs.

1. In dogs anaesthetized with pentobarbitone, electrical stimulation of the parasympathetic nerve fibres to the nasal mucosa evoked frequency dependent increases in both nasal arterial blood flow and nasal secretion. Blood flow was measured using a transonic flow probe placed around the artery. 2. Sympathetic nerve stimulation for 3 min at 10 Hz evoked significant and prolonged (> 30 min) attenuation of the vasodilator and secretory responses to subsequent parasympathetic stimulation. 3. Intravenous and intranasal administration of the neuropeptide Y (NPY) analogue N-acetyl [Leu28,Leu31] NPY 24-36, a selective NPY Y2 receptor agonist (20 nmol kg-1), significantly attenuated both vasodilator and secretory effects of subsequent parasympathetic nerve stimulation. When given intravenously, the inhibitory effect of this Y2 receptor agonist on vascular and secretory effects of parasympathetic nerve stimulation was rapid in onset (5 min) and lasted for more than 60 min. The modulatory effect of the Y2 receptor agonist was also seen with intranasal administration, but was slower in onset (15 min), and lasted less than 45 min. The effects of the intranasal pretreatment with the Y2 receptor agonist were significantly prolonged in the presence of the endopeptidase inhibitor phosphoramidon (10 nM). 4. Atropine pretreatment did not significantly reduce the change in vascular conductance evoked by parasympathetic nerve stimulation. Subsequent pretreatment with the NPY Y2 receptor agonist N-acetyl [Leu28,Leu31] NPY 24-36 reduced the stimulation induced increase in conductance by 30%. Nasal secretion was reduced by 70% following pretreatment with atropine and a further 30% by pretreatment with the NPY Y2 receptor agonist. Dose dependent vasodilator and secretory effects of local intra-arterial infusion of acetylcholine and vasoactive intestinal peptide were not modified by the NPY Y2 agonist. 5. Total protein and albumin concentration were measured in nasal lavage fluid collected after nerve stimulation. Atropine pretreatment increased the percentage of the total protein that was albumin in nasal lavage fluid. Neither sympathetic nerve stimulation nor Y2 receptor agonist pretreatment further modified the albumin exudation (a marker of vascular permeability) in nasal fluid lavage collected after parasympathetic nerve stimulation. 6. We propose that sympathetic nerve stimulation releases NPY, which acts on Y2 receptors, probably located on parasympathetic nerve endings, to attenuate both vasodilatation and nasal secretion evoked by subsequent parasympathetic nerve stimulation. This effect is also observed after pretreatment with the Y2-selective NPY analogue N-acetyl [Leu28,Leu31] NPY 24-36.

Administration, Intranasal↗

PubMed1995

Rapid corepressor exchange from the trp-repressor/operator complex: an NMR study of [ul-13C/15N]-L-tryptophan.

[ul-13C/15N]-L-tryptophan was prepared biosynthetically and its dynamic properties and intermolecular interaction with a complex of Escherichia coli trp-repressor and a 20 base-pair operator DNA were studied by heteronuclear isotope-edited NMR experiments. The resonances of the free and bound corepressor (L-Trp) were unambiguously identified from gradient-enhanced 15N-1H HSQC, 13C-1H HSQC, 13C- and 15N-edited 2D NOESY spectra. The exchange off-rate of the corepressor between the bound and free states was determined to be 3.4 +/- 0.52 s-1 at 45 degrees C, almost three orders of magnitude faster than the dissociation of the protein-DNA complex. Examination of the experimental NOE buildup curves indicates that it may be desirable to use longer mixing times than would normally be used for a large molecule, in order to detect weak intermolecular NOEs in the presence of exchange. Intermolecular NOEs from bound corepressor to trp-repressor and DNA were analyzed with respect to the mechanism of ligand exchange. This analysis suggests that, in order for the ligand to diffuse out of the complex, there must be significant movement or 'breathing' of the protein and/or DNA.

Bacterial Proteins↗

PubMed1994

The solution structures of the trp repressor-operator DNA complex.

The solution structures of the complex between Escherichia coli trp holorepressor and a 20 base-pair consensus operator DNA were determined. The majority of proton chemical shifts of the trp holorepressor and operator DNA were assigned from homonuclear 2D NOESY spectra of selectively deuterated analog-operator DNA complexes and the 3D NOESY-HMQC spectrum of a uniformly 15N-labeled repressor-operator DNA complex. The structures were calculated using restrained molecular dynamics and sequential simulated annealing with 4086 NOE and other experimental constraints. The root-mean-squared deviation (RMSD) among the calculated structures and their mean is 0.9(+/- 0.3)A for the repressor backbone, 1.1(+/- 0.5)A for the DNA backbone, and 1.3(+/- 0.3)A for all heavy atoms. The DNA is deformed to a significant extent from the standard B DNA structure to fit the helix-turn-helix (HTH) segment of the repressor (helices D and E) into its major grooves. Little change is found in the ABCF core of the repressor on complexation in comparison to the free repressor, but changes in the cofactor L-tryptophan binding pocket and the HTH segment are observed. The N-terminal residues (2 to 17) are found to be disordered and do not form stable interactions with DNA. Direct H-bonding to the bases of the operator DNA is consistent with all of our observed NOE constraints. Hydrogen bonds from NH eta 1 and NH eta 2 of Arg69 to O-6 and N-7 of G2 are compatible with the solution structure, as they are with the crystal structure. Other direct H-bonds from Lys72, Ala80, Ile79, Thr83 and Arg84 to base-pair functional groups can also be formed in our solution structures.

Amino Acid Sequence↗

PubMed1990

Prolonged inhibition of cardiac vagal action following sympathetic stimulation and galanin in anaesthetized cats.

1. Stimulation of the right cardiac sympathetic nerve for 3 min at 16 Hz in the presence of effective beta-adrenoceptor blockade evoked prolonged attention of cardiac vagal action in the cat: 40.8 +/- 5.4% maximum inhibition of cardiac vagal action on prolonging pulse interval, with half-time to recovery of 8.3 +/- 1.4 min. 2. Intravenous injection of galanin (1.6-3.1 nmol/kg) evoked prolonged attenuation of cardiac vagal action: 40.9 +/- 8.2% maximum inhibition with a half-time to recovery of 13.6 +/- 2.6 min. This effect of galanin was not significantly different from the action of sympathetic nerve stimulation. A slight depressor response (-14.4 +/- 1.9 mmHg) was seen in nine of sixteen cats. 3. Intravenous injection of neuropeptide Y (NPY) (2.8-6.3 nmol/kg) evoked slight attenuation of cardiac vagal action: 11.9 +/- 4.5% maximum inhibition of cardiac vagal action on pulse interval, with a half-time to recovery of 4.1 +/- 1.7 min. Blood pressure increased by 68.6 +/- 5.7 mmHg. 4. Following administration of guanethidine (1 mg/kg I.V.) the inhibitory effect of sympathetic nerve stimulation on cardiac vagal action was significantly reduced (P less than 0.001). The responses to exogenous NPY and galanin on vagal action were unchanged after guanethidine. 5. The prolonged attenuation of cardiac vagal action can be mimicked by exogenous galanin in the cat but not by exogenous NPY.

Anesthesia, Intravenous↗

PubMed1988

Neuropeptide Y and control of vascular resistance in skeletal muscle.

The effect of neuropeptide Y (NPY) on the increase in skeletal muscle vascular resistance caused by exogenous noradrenaline and by sympathetic stimulation was examined in gracilis muscles of anaesthetised dogs. NPY potentiated the increases in resistance caused by both of these to similar degrees. Although NPY itself often caused an elevation in the basal resistance, correlation coefficients for the percentage increase in basal resistance due to NPY and the percentage increase in the evoked sympathetic and noradrenergic responses in the presence of NPY indicated that it was the NPY itself (rather than the increase in basal resistance per se) which was responsible for the potentiation. The potentiation was apparently biphasic, with an initial peak in response during the first 20 min following administration of NPY followed by a secondary peak between 30 and 60 min. Radioimmunoassay for plasma levels of NPY indicated that the secondary increase of vascular resistance was not associated with a secondary peak in the plasma level of NPY.

Animals↗

PubMed1983

Haematology of the racing Thoroughbred in Australia 1: reference values and the effect of excitement.

Eight hundred and sixteen blood samples were collected from horses at Sydney race tracks, 1 to 3 h before racing, and subjected to haematological analysis. Haemograms were also performed on 65 blood samples taken from horses at rest in their stalls. These were used as reference values of prerace and resting haemograms, respectively. The haemograms of 29 of the resting horses were compared with the haemograms of the same 29 horses the following day at the race track. Both samples were considered to be representative of their reference populations. In general, there was a significant increase from the resting to prerace packed cell volume, haemoglobin, red cell count, white cell count and total plasma protein, although the extent of the increases varied between horses and, in a minority, levels were unchanged or decreased. The degree of variability in the prerace samples was not greater than that of the resting samples.

Animals↗

PubMed1983

Haematology of the racing Thoroughbred in Australia 2: haematological values compared to performance.

Eight hundred and sixteen blood samples were collected from Thoroughbred racehorses at the race track, 1 to 3 h before racing, and subjected to routine haematological examination. Attempts were made to correlate the haemogram with subsequent performance. Races were classified according to age, class and distance, and performances were grouped according to distance from the winner. Intra- and interclass comparisons were made but no relationship emerged between racing performance and the haemogram. The haemograms of individual horses on different occasions were compared with subsequent performance, but no consistent or significant relationships were apparent. The extent of the rise in red and white cell parameters between horses at rest and immediately before racing were examined as indicators of performance, but no correlations found. It was concluded that under the conditions of this survey no relationship existed between the haemogram of the Thoroughbred racehorse and its racing performance.

Animals↗