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A Watts

Publications and source records attributed to A Watts.

At least 19 recordsLinked to original sources
PubMed1995

The conformations of a functional spin-labeled derivative of gastric H/K-ATPase investigated by EPR spectroscopy.

A spin-labeled derivative of porcine gastric H/K-ATPase with high ATP hydrolyzing activity (77 mumol of Pi/(mg.h)) has been prepared. Over 65% of initial ATPase activity (115 mumol of Pi/(mg.h)) was preserved after complete reaction of the enzyme with the lysine reactive nitroxide spin-labeled TEMPO isothiocyanate (TITC). In contrast, rapid and complete loss of ATPase activity occurred after reaction of the enzyme with the lysine directed fluorescent probe FITC. Conventional EPR spectra of TITC labeled H/K-ATPase reflected mainly the slow rotational diffusion of the enzyme in the membrane. An upper limit enzyme intramembranous radius of 108 A was calculated on the basis of rotational correlation times estimated from saturation transfer (ST) EPR spectral lineshapes. Conventional EPR spectra exhibited two major components corresponding to at least two populations of strongly constrained spin-labels. Difference spectroscopy revealed that the proportion of these two components changed markedly with temperature. Moreover, the proportion of the components was sensitive to the presence of the activating ionic ligands Mg2+ and ATP, which induce enzyme conformational transitions, and to the reversible inhibitor SCH 28080, which binds to the K+ sensitive form of the enzyme. These findings show that EPR spectroscopy is able to report functionally coupled conformational changes of gastric H/K-ATPase and imply that the spin-labels are attached to lysines within functionally important regions of the enzyme.

Animals↗
PubMed1995

Effect of the C-terminal proline repeats on ordered packing of squid rhodopsin and its mobility in membranes.

Negative stain electron microscopy and saturation transfer electron spin resonance spectroscopy have been used to compare the lattice ordering and in-plane membrane mobility of full-length and C-terminally cleaved squid rhodopsin. The C-terminus of squid rhodopsin contains a negatively charged region followed by 9-10 repeats of a proline-rich sequence, not found in rhodopsins other than those of cephalopod invertebrates, but similar proline repeats are found in other, unrelated membrane proteins. We find that the proline repeats cluster the rhodopsins into small groups, interfering with two-dimensional crystallization and maintaining their mobility in the membrane.

Animals↗
PubMed1995

Initial stages in the development of valid criteria for the replacement of amalgam restorations.

OBJECTIVES: Replacement amalgam restorations account for the largest single cost in the General Dental Services in Scotland. There is little practical advice available to practitioners on the maintenance and replacement of amalgam restorations. A distance learning package is being prepared for distribution to general dental practitioners in Scotland and this paper describes the initial stages in the development of the policy statements for the proposed package. METHODS: In the first stage proposals are being developed by a panel of dental academics using a modified Delphi technique. This technique is designed to produce a consensus view. Seventeen statements have been set out by the expert group. RESULTS AND CONCLUSIONS: While agreement was rapidly reached that 'ditching' of amalgam restoration margins did not justify their replacement and that repair was preferable to total replacement, some difficulty was experienced in arriving at a consensus view on the correct management of 'white spot' lesions at restoration margins and dentinal staining adjacent to amalgam restorations. The next stage in the development will be to present these statements to a selected panel of general dental practitioners and to develop their consensus view of the proposals, again using the Delphi technique.

Delphi Technique↗
PubMed1994

Incorporation of fatty acids by concanavalin A-stimulated lymphocytes and the effect on fatty acid composition and membrane fluidity.

The fatty acid compositions of the neutral lipid and phospholipid fractions of rat lymph node lymphocytes were characterized. Stimulation of rat lymphocytes with the T-cell mitogen concanavalin A resulted in significant changes in the fatty acid composition of both neutral lipids and phospholipids (a decrease in the proportions of stearic, linoleic and arachidonic acids and an increase in the proportion of oleic acid). Membrane fluidity was measured using nitroxide spin-label e.s.r., and increased during culture with concanavalin A. Culturing the lymphocytes in the absence of mitogen did not affect fatty acid composition or membrane fluidity. The uptake and fate of palmitic, oleic, linoleic and arachidonic acids were studied in detail; there was a time-dependent incorporation of each fatty acid into all lipid classes but each fatty acid had a characteristic fate. Palmitic and arachidonic acids were incorporated principally into phospholipids whereas oleic and linoleic acids were incorporated in similar proportions into phospholipids and triacylglycerols. Oleic acid was incorporated mainly into phosphatidylcholine, palmitic and linoleic acids were incorporated equally into phosphatidylcholine and phosphatidylethanolamine, and arachidonic acid was incorporated mainly into phosphatidylethanolamine. Supplementation of the culture medium with particular fatty acids (myristic, palmitic, stearic, oleic, linoleic, alpha-linolenic, arachidonic, eicosapentaenoic or docosahexaenoic acid) led to enrichment of that fatty acid in both neutral lipids and phospholipids. This generated lymphocytes with phospholipids differing in saturated/unsaturated fatty acid ratio, degree of polyunsaturation, index of unsaturation and n - 6/n - 3 ratio. This method allowed the introduction into lymphocyte phospholipids of fatty acids not normally present (e.g. alpha-linolenic) or usually present in low proportions (eicosapentaenoic and docosahexaenoic). These three n - 3 polyunsaturated fatty acids replaced arachidonic acid in lymphocyte phospholipids. Fatty acid incorporation led to an alteration in lymphocyte membrane fluidity: palmitic and stearic acids decreased fluidity whereas the unsaturated fatty acids increased fluidity. It is proposed that the changes in lymphocyte phospholipid fatty acid composition and membrane fluidity brought about by culture in the presence of polyunsaturated fatty acids are responsible for the inhibition of lymphocyte functions caused by these fatty acids.

Animals↗
PubMed1994

Distorted structure of the retinal chromophore in bacteriorhodopsin resolved by 2H-NMR.

Structural details about the geometry of the retinal chromophore in the binding pocket of bacteriorhodopsin are revealed by measuring the orientations of its individual methyl groups. Solid-state 2H-NMR measurements were performed on macroscopically oriented samples of purple membrane patches, containing retinal specifically deuterium-labeled at one of the three methyl groups along the polyene chain (C18, C19, C20). The deuterium quadrupole splitting of each "zero-tilt" spectrum is used to calculate the orientation of the corresponding C-CD3 bond vector with respect to the membrane normal; however, two possible solutions may arise. These ambiguities in angle could be resolved by recording a tilt series of spectra at different sample inclinations to the magnetic field and analyzing the resulting complex line shapes with the aid of computer simulations. The angles for the C18, C19, and C20 group are found to be 37 +/- 1 degree, 40 +/- 1 degree, and 32 +/- 1 degree, respectively. These highly accurate values imply that the polyene chain of the retinal chromophore is not straight but rather has an in-plane curvature and possibly an out-of-plane twist. Together with the angles of the remaining methyl groups on the cyclohexene ring that have been measured previously, an overall picture has thus emerged of the intramolecular conformation and the three-dimensional orientation of retinal within bacteriorhodopsin. The deduced geometry confirms and refines the known structural information on the chromophore, suggesting that this 2H-NMR strategy may serve as a valuable tool for other membrane proteins.

Bacteriorhodopsins↗
PubMed1994

Phospholipid headgroup-headgroup electrostatic interactions in mixed bilayers of cardiolipin with phosphatidylcholines studied by 2H NMR.

The headgroup-headgroup interactions in binary mixed bilayers of diacylphosphatidylcholines (PC) and cardiolipin were analyzed by 2H NMR. Specific changes in the quadrupole splittings of the choline headgroup deuterated PC at alpha,beta-methylenes, and gamma-methyls are observed upon the insertion of the negatively charged tetraacylphospholipid, cardiolipin. The effects are consistent with an electrostatic interaction between PC and cardiolipin headgroups, in which a concerted conformational reorientation of the entire phosphocholine moiety toward the membrane surface is involved. On the basis of the "choline-tilt" model by Macdonald and co-workers (1991) the variations in the quadrupole splittings are consistent with a change in orientation of the choline P-N vector up to 23 degrees for the highest cardiolipin concentrations. Additional information on headgroup conformational changes was obtained through the analysis of the dependence on temperature of the quadrupole splittings for the various deuterium-labeled segments. Evaluation of the deuterium spin-lattice (T1) relaxation times for the deuterons in the various positions of the choline headgroup in mixed bilayers of PC and cardiolipin showed that the internal fast segmental motions were not affected on addition of cardiolipin to PC membranes.

Cardiolipins↗
PubMed1994

NMR observation of substrate in the binding site of an active sugar-H+ symport protein in native membranes.

NMR methods have been adopted to observe directly the characteristics of substrate binding to the galactose-H+ symport protein GalP, in its native environment, the inner membranes of Escherichia coli. Sedimented inner-membrane vesicles containing the GalP protein, overexpressed to levels above 50% of total protein, were analyzed by 13C magic-angle spinning NMR, when in their normal "fluid" state and with incorporated D-[1-13C]glucose. Using conditions of cross-polarization intended to discriminate bound substrate alone, it was possible to detect as little as 250 nmol of substrate added to the membranes containing about 0.5 mumol (approximately 26 mg) of GalP protein. Such high measuring sensitivity was possible from the fluid membranes by virtue of their motional contributions to rapid relaxation recovery of the observed nuclei and due to a high-resolution response that approached the static field inhomogeneity in these experiments. This good spectral resolution showed that the native state of the membranes presents a substrate binding environment with high structural homogeneity. Inhibitors of the GalP protein, cytochalasin B and forskolin, which are specific, and D-galactose, but not L-galactose, prevent or suppress detection of the 13C-labeled glucose substrate, confirming that the observed signal was due to specific interactions with the GalP protein. This specific substrate binding exhibits a preference for the beta-anomer of D-glucose and substrate translocation is determined to be slow, on the 10(-2) s time scale. The work describes a straightforward NMR approach, which achieves high sensitivity, selectivity, and resolution for nuclei associated with complex membrane proteins and which may be combined with other NMR methodologies to yield additional structural information on the binding site for the current transport system without isolating it from its native membrane environment.

Bacterial Proteins↗
PubMed1994

Hydration of DOPC bilayers by differential scanning calorimetry.

The phase diagram of the unsaturated lipid dioleoylphosphatidylcholine (DOPC) in aqueous multibilayer dispersions has been constructed from a series of differential scanning calorimetry (DSC) thermograms over the temperature range from -40 to +10 degrees C, covering a range of hydration levels from the monohydrate to excess free water. Both the lipid chain melting transition and the ice melting point are found to be hydration dependent. From their respective variations it is found that the bilayer in the gel phase binds approximately 9 H2O per lipid, while the liquid-crystalline state has a saturation limit near 20 H2O. The water transition exhibits a hydration-dependent melting point depression, which can be explained in terms of newly incorporated water between the bilayer surfaces upon melting of the acyl chains, and which is reminiscent of the events that occur at the pre-transition for saturated lipids. From the melting point depression, the thermodynamic activity of the interbilayer water can be calculated and thus the repulsive hydration force characterized quantitatively. We evaluate a (non-isothermal) hydration force decay constant around 2.8 H20, which demonstrates that this DSC approach is well-suited for quantitatively characterizing the hydration properties of unsaturated lipid dispersions at low temperature.

Calorimetry, Differential Scanning↗
PubMed1994

Lipid specificity in the interaction of cytochrome c with anionic phospholipid bilayers revealed by solid-state 31P NMR.

Phosphorus-31 NMR has been used to investigate the interaction of cytochrome c with bilayers of the anionic lipids dioleoylphosphatidylglycerol (DOPG), dioleoylphosphatidylserine (DOPS), and diacylphosphatidylinositol (diacylPI). All 31P NMR spectra revealed the typical line shapes characteristics of phospholipids in liquid-crystalline bilayers. The effects on the 31P chemical shift anisotropy (CSA) for each system reflect particular modes of phospholipid headgroup interaction with cytochrome c. A distinct increase in the CSA for DOPS bilayers was observed upon binding of cytochrome c, which is likely to arise from a partial restriction of the amplitude of motion on this phospholipid headgroup. 31P NMR spin-lattice (T1) relaxation times of the various phospholipid-cytochrome c complexes show that conformational changes occur in the protein on binding to anionic phospholipids. These protein conformational changes are observed through paramagnetic enhancement of the measured 31P spin-lattice relaxation times for lipid phosphates. However, the 31P T1 values for the various complexes with cytochrome c show a different temperature dependence for each lipid, revealing different modes of protein interaction for each of the different lipid headgroups. The phosphate of DOPS was most efficiently relaxed by cytochrome c, while the relaxation of the phosphate in the PI headgroup was not affected. The relaxation profile for DOPG-bound cytochrome c shows a more complex behavior, where the lipid phosphorus relaxation is strongly enhanced above 15 degrees C, but not significantly affected at lower temperatures. It was found that the enhancement of lipid phosphorus relaxation is a result of the conformational changes in the protein, in which the heme becomes accessible to lipid phosphate upon binding to charged bilayer surfaces.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1994

Resolution of individual lipids in mixed phospholipid membranes and specific lipid-cytochrome c interactions by magic-angle spinning solid-state phosphorus-31 NMR.

A model of the inner mitochondrial membrane was constructed with dioleoyphosphatidylcholine (PC), dioleoylphosphatidylethanolamine (PE), and cardiolipin (CL) at a PC:PE:CL molar ratio of 2:2:1, and the interaction of the peripheral membrane protein cytochrome c with this mixed membrane has been investigated by static and magic-angle spinning (MAS) solid-state 31P NMR. The static 31P NMR spectrum of the three-component membrane is a typical broad powder pattern for phospholipids in a bilayer structure, and is a result of three overlapping spectra of each individual phospholipid component in the mixed membrane, with an average effective chemical shift anisotropy of approximately 41 ppm. Using magic-angle spinning NMR methods, three resolved resonances are observed in the narrowed MAS 31P NMR spectrum, each of which has been assigned to each lipid component in the mixed membrane. This allows the investigation of individual phospholipid-protein interactions in multicomponent lipid bilayers. The interaction of cytochrome c with each lipid in a model mitochondrial membrane could now be evaluated. Phosphorus-31 spin-lattice (T1) relaxation times for each lipid phosphate were measured as a function of temperature, in the absence and presence of bound cytochrome c. T1 was not affected for any lipid upon binding of cytochrome c over the temperature range analyzed. However, averaging of the phosphorus-31 chemical shift anisotropy for the cardiolipin component in mixed PC/PE/CL bilayers at lower temperatures ceases to be axially symmetric on binding of cytochrome c, while for PC and PE components the axial symmetry is retained over the temperature interval studied here.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1994

Morphological and biochemical characterization of influenza vaccines commercially available in the United Kingdom.

Four vaccines are available in the United Kingdom against influenza virus. All are subunit vaccines, defined as either split-virion or purified surface antigen vaccine; there are two of each distinct type available. Both vaccine types are less reactogenic than whole inactivated virus, with antigenicity induced by viral surface glycoproteins. Here, each of the four vaccines has been characterized by electron microscopy and SDS-PAGE analysis, giving a unique vaccine profile. Three vaccines contain internal viral nucleoprotein which, in the presence of residual haemagglutinin, may induce an influenza A virus cross-reactive cytotoxic T-cell response and thus be of value to vaccine efficacy. Residual lipid was present in three vaccines and recent evidence suggests that pyrogenicity is correlated with the presence of viral lipid with clusters of surface glycoproteins. By a combination of electron microscopic evidence and biochemical characterization, it has been possible to resolve compositional differences, not only between vaccine type, but also between each individual currently available vaccine. Hence, there is the possibility that the morphological differences characterized here may be contributory to potential reactogenic effects subsequent to vaccination.

Blotting, Western↗
PubMed1994

Report on the Annual Conference of the British Association of Teachers of Conservative Dentistry.

The Annual Conference of the British Association of Teachers of Conservative Dentistry (BATCD) was held in Sheffield on 16 and 17 September 1993. The main programme addressed educational issues in three areas: the undergraduate/vocational-trainee interface, the teaching of implants in the undergraduate curriculum, and the changing role of dental instructors in restorative dentistry. The programme also included a number of research papers and poster demonstrations, which were submitted for the Association's new QED/Maillefer prizes in Restorative Dentistry. This paper presents reports of the four principal lectures, and abstracts of the research papers and poster demonstrations, with details of the prize winners.

Curriculum↗
PubMed1994

Molecular response of the lipid headgroup to bilayer hydration monitored by 2H-NMR.

The effect of hydration on the conformation and dynamics of the phosphatidylcholine headgroup has been investigated by 2H-NMR measurements of liquid crystalline dioleoylphosphatidylcholine in multilamellar liposomes. Deuterium quadrupole splittings (delta nu Q) and spin-lattice relaxation rates (1/T1) were recorded for three selectively labeled headgroup segments (alpha, beta, and gamma) over the range of water/lipid mole ratios from 4 to 100. The smooth changes in delta nu Q and 1/T1 are found to essentially parallel each other and can be described by a single exponential decay function. Progressive hydration thus induces a concerted change in headgroup conformation together with an increase in its rate of motion (detected by delta nu Q and 1/T1, respectively). The enhanced mobility is partially due to a shift in the lipid phase transition temperature (as monitored by differential scanning calorimetry) and is furthermore attributed to an entropic contribution. It is concluded that the choline dipole becomes slightly raised in its average orientation into the aqueous layer and that the rate is increased at which the headgroup is fluctuating and protruding. The observed molecular changes can thus be accommodated within a model where the effective accessible headgroup volume expands with increasing hydration.

Biophysical Phenomena↗
PubMed1994

High-resolution, non-crystallographic structural studies of large integral membrane proteins.

The work described here clearly demonstrates that n.m.r. is a viable method with which to resolve molecular details about membrane proteins and can give information at a resolution comparable with that gained from crystallographic studies, where, in the limited number of cases studied so far, such information is available. Sensitivity is not a major problem although other difficulties may prevent particular kinds of information being resolved. However, because bond orientational details are obtained ab initio, the method is quite model independent and interpretationally unique. The requirements for solid-state n.m.r. methods to be applied to a large membrane protein are that: a sufficient amount of functionally active protein is available; the protein should be located in bilayers; and suitable specific isotopic enrichment is achieved, in ideal cases, either by chemical or biosynthetic means. Achieving these requirements will need to draw on a range of skills, possibly genetic or chemical or both, and a full understanding of the biochemistry is essential to ensure that any structural information gained is functionally relevant. The structural information potentially available includes: (a) conformation details about specific parts of a protein or its binding sites; (b) distance measurements between specific labels introduced into the protein; (c) dynamics of particular parts of the protein; (d) chain folding and residue orientation; and (e) the mechanistic changes that can occur during a functional cycle. Much developmental work still needs to be done both on the sample handling instrumental side and with the theoretical aspect of the method.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1993

The antiviral peptide carbobenzoxy-D-phenylalanyl-L-phenylalanylglycine changes the average conformation of phospholipids in membranes.

The influence of the antiviral peptide, carbobenzoxy-D-phenylalanyl-L-phenylalanylglycine (ZfFG), on the average conformation of phosphatidylcholine in hydrated bilayers was investigated with multinuclear solid state magnetic resonance (NMR). Phosphatidylcholine was specifically deuterated (separately) in the choline N-methyls, the alpha and beta positions of the choline, the C2 carbon of the acyl chains, and at all the carbons of the acyl chains of the phosphatidylcholine. Phosphatidylcholine was also synthesized with the carbonyl carbons of the ester bonds between the glycerol and the hydrocarbon chains enriched in 13C. 2H NMR of the phosphatidylcholine perdeuterated in the acyl chains showed a loss of intensity from the deuteriums with the largest quadrupole splitting in the presence of ZfFG, while the remainder of the powder pattern was largely unaffected. The phosphatidylcholine specifically deuterated at the C2 carbon (representative of the C-D bonds giving rise to the largest quadrupole splittings) showed the same loss of intensity suggesting changes in the phospholipid conformation and conformational dynamics near the glycerol. Analysis of the powder patterns in the 13C NMR spectrum of phosphatidylcholine labeled with 13C in the carbonyl carbons revealed a significant change in the average orientation of the sn-1 carbonyl due to the presence of the ZfFG and no change in the sn-2 carbonyl orientation. Changes in the headgroup conformation, as detected by 2H NMR of the deuteriums in the alpha and beta methylenes of the choline headgroup and 31P NMR of the phosphate segment, reflected the electrostatic nature of the interaction of the carboxyl of ZfFG with phosphatidylcholine bilayers.(ABSTRACT TRUNCATED AT 250 WORDS)

1,2-Dipalmitoylphosphatidylcholine↗
PubMed1993

A role for band 4.2 in human erythrocyte band 3 mediated anion transport.

Human erythrocyte band 3 was purified essentially free of peripheral proteins, in particular band 4.2, using affinity chromatography. Band 3 protein was then reconstituted into liposomes of lipid type and ratio approximating that of erythrocyte membranes. Stilbenedisulfonate inhibition of band 3 mediated efflux of radiolabeled sulfate from preloaded liposomes was used to test the functionality and correct orientation of the protein. When sulfate efflux, mediated by purified band 3, was compared with partially purified band 3, which contained detectable amounts of bands 4.1 and 4.2, a clear difference in efflux was measured. Sulfate efflux was approximately 30% faster from liposomes containing purified band 3 compared with those containing partially purified protein. In order to investigate further any specific effect of band 4.2 protein on band 3 mediated anion transport, band 4.2 was purified. Increasing amounts of band 4.2 were complexed with purified band 3 and then reconstituted into liposomes. Increasing amounts of band 4.2 complexed with band 3 caused a decrease in band 3 mediated anion transport. The effect of band 4.2 on band 3 mediated anion transport appears to be specific since increasing concentrations of band 4.2 added exogenously to band 3 in reconstituted vesicles (rather than complexed with band 3 before reconstitution) produced no significant changes in sulfate efflux. Further, when increasing amounts of band 4.2 were added to the functionally active transmembrane domain of band 3 and then reconstituted into vesicles, there was also no significant change in sulfate efflux.(ABSTRACT TRUNCATED AT 250 WORDS)

4,4'-Diisothiocyanostilbene-2,2'-Disulfonic Acid↗