Biomedical subjects

D Marsh

Publications and source records attributed to D Marsh.

At least 199 records · Page 11Linked to original sources

PubMed1990

Phase transition from a gel to a fluid phase of cubic symmetry in dimyristoylphosphatidylcholine/myristic acid (1:2, mol/mol) bilayers.

Aqueous dispersions (pH 4.0) of a 2:1 (mol/mol) mixture of myristic acid with dimyristoylphosphatidylcholine undergo a sharp transition at 45-47 degrees C from a lamellar gel phase to a fluid phase which is optically isotropic. This fluid phase gives rise to 31P-NMR spectra, and 2H-NMR spectra of the chain-deuterated components, which are also isotropic. X-ray diffraction studies of the fluid phase at 49 degrees C, reveal reflections with spacings in the ratio square root of 2: (square root of 3): square root of 4: square root of 6: square root of 8, accompanied by a strong diffuse scatter. These reflections index on a cubic lattice of primitive space group Pn3 or Pn3m, or possibly the body-centered group Im3m, with a lattice constant of 21.2 nm. The dimensions of the phase are consistent with a structure composed of two systems of tetrahedrally (octahedrally) oriented inverted lipid cylinders, found for other cubic lipid phases with Pn3m (Im3m) symmetry. At higher temperatures the cubic phase gradually converts, with increasing temperature, to a coexisting inverted hexagonal phase.

Dimyristoylphosphatidylcholine↗

PubMed1990

Incorporation of cytochrome oxidase into cardiolipin bilayers and induction of nonlamellar phases.

Cytochrome oxidase from beef heart has been lipid-substituted with beef heart cardiolipin. The lipid phase behavior and protein aggregation state of the reconstituted complexes have been studied with 31P NMR, freeze-fracture electron microscopy, and saturation-transfer ESR of the spin-labeled protein. In the absence of salt, the lipid has a lamellar arrangement, and the protein is integrated and uniformly distributed in the membrane vesicles and undergoes rapid rotational diffusion. The presence of the protein stabilizes the cardiolipin lamellar phase against salt-induced transitions to the inverted hexagonal phase. The threshold salt concentration becomes higher and the extent of conversion becomes lower with decreasing lipid:protein ratio. In high salt, lamellar-phase lipid with integrated protein coexists with hexagonal-phase lipid free of protein, and the rotational diffusion of the protein is drastically reduced as a result of the high packing density.

Animals↗

PubMed1990

Lipid-protein interactions in stacked and destacked thylakoid membranes and the influence of phosphorylation and illumination. Spin label ESR studies.

The effects of membrane destacking, protein phosphorylation, and continuous illumination have been studied in pea thylakoid membranes using ESR spectroscopy of an incorporated spin-labelled phosphatidylglycerol. This spin-labelled analogue of an endogenous thylakoid lipid has previously been shown to exhibit a selectivity of interaction with thylakoid proteins. Neither destacking, phosphorylation nor illumination was found to change the ESR spectra appreciably, suggesting that for phosphatidylglycerol at least, neither the number of protein-associated membrane lipids nor their pattern of selectivity was altered. The redistribution of the thylakoid protein complexes in the membrane, under these various conditions, therefore takes place with conservation of the properties of the lipid/protein interface.

Cell Membrane↗

PubMed1990

ESR of spin-labeled bacteriophage M13 coat protein in mixed phospholipid bilayers.

Bacteriophage M13 major coat protein was spin-labeled with a nitroxide derivative of iodoacetamide, preferentially at the single methionine that is located in the hydrophobic region of the protein. The spin-labeled protein was incorporated at different lipid-to-protein ratios in phospholipid bilayers composed of dimyristoylphosphatidylglycerol (DMPG), dimyristoylphosphatidylcholine (DMPC), or the 1:1 molar mixture of these lipids. Both conventional and saturation transfer (ST) ESR studies were performed to investigate the rotational motions of the protein over a large dynamic range. The conventional ESR spectra indicate that the mobility of the spin labelled protein in the lipid gel phase decreases in the order: DMPG greater than DMPC/DMPG (1:1) greater than DMPC. In the liquid crystalline phase, the largest mobility is found in DMPC/DMPG (1:1, mol/mol) mixtures, but the mobility is still greater in DMPG than in DMPC. The results are interpreted in terms of different degrees of protein aggregation in the different lipids. Segmental motion with rotational correlation times on the order of tens of nanoseconds, motional anisotropy, and spectral overlap complicate the analysis of the STESR spectra. An estimate of the size of the protein aggregates is found to be in the region of 85 monomer units. Removing the polar tails from the protein by proteolytic digestion results in an enhanced aggregation in the gel phase. In the liquid crystalline phase, the segmental wobbling mobility of the protein is increased relative to the native protein, whereas the overall rotational diffusion is not changed greatly.

Capsid↗

PubMed1990

Spin label ESR and 31P-NMR studies of the cubic and inverted hexagonal phases of dimyristoylphosphatidylcholine/myristic acid (1:2, mol/mol) mixtures.

The thermotropic phase behaviour and chain dynamics of the dimyristoylphosphatidylcholine/myristic acid (1:2, mol/mol) mixture at pH 4.0 have been studied with 31P-NMR and spin-label ESR. Broadline proton-dipolar decoupled 31P-NMR spectra show that the system undergoes a transition from a lamellar phase to a non-lamellar phase at 47 degrees C. The 31P-NMR spectrum collapses, from a lamellar powder pattern with apparent chemical shift anisotropy delta sigma = -38 ppm below 47 degrees C, to an isotropic spectrum with superimposed hexagonal powder pattern of delta sigma = +13 ppm above 47 degrees C. With increasing temperature, the hexagonal component grows at the expense of the isotropic component, finally transforming to a single hexagonal phase at approximately 70 degrees C. The ESR spectra from both fatty acid and phosphatidylcholine probes spin-labelled in the hydrocarbon chain have been used to study the ordering and mobility of the lipid chains through the lamellar-non lamellar transition. An abrupt increase in the chain mobility observed at approx. 47 degrees C demonstrates that the lamellar-non lamellar phase transition coincides with chain melting. The apparent order parameter and polarity profiles, deduced from the ESR spectra of 11 different chain-labelled positional isomers, demonstrate flexibility and polarity gradients characteristic of a lyotropic liquid crystalline phase. The temperature dependence of the order parameters does not show any changes in the fluid phase, indicating that the conversion from the isotropic (cubic) phase to the inverted hexagonal phase is not accompanied by large changes in chain order. A comparison of the apparent order parameters and polarity profiles of the phosphatidylcholine and fatty acid spin labels indicates that the fatty acid spin labels are intercalated approximately one CH2 unit more deeply in the hydrocarbon region than are the positionally isomeric phosphatidylcholine spin labels.

Chemical Phenomena↗

PubMed1990

Influence of polar residue deletions on lipid-protein interactions with the myelin proteolipid protein. Spin-label ESR studies with DM-20/lipid recombinants.

The lipid specificities of two related integral membrane proteins of central nervous system myelin, the proteolipid (PLP) and DM-20 proteins, which differ only by the deletion of a polar stretch of 35 contiguous amino acid residues, were studied with spin-labeled lipids after reconstitution into dimyristoyl-phosphatidylcholine. The selectivity in populating lipid association sites at the protein interface and in modulating the lipid exchange between protein and bulk lipid sites was quantitated by the relative association constants and the off-rate constants for exchange, respectively, for both proteins. The sequence deleted in DM-20 (residues 116-150 of PLP) is found to play a major role in determining the lipid selectivity for the parent PLP protein.

Animals↗

PubMed1990

Conformational changes in cytochrome c and cytochrome oxidase upon complex formation: a resonance Raman study.

The fully oxidized complex of cytochrome c and cytochrome oxidase formed at low ionic strength was studied by resonance Raman spectroscopy. The spectra of the complex and of the individual components were compared over a wide frequency range using Soret band excitation. In both partners of the complex, structural changes occur in the heme groups and in their immediate protein environment. The spectra of the complex in the 1600-1700 cm-1 frequency range were dominated by bands from the cytochrome oxidase component, whereas those in the 300-500 cm-1 range were dominated by bands from the cytochrome c component, hence allowing separation of the contributions from the two individual species. For cytochrome c, spectral changes were observed which correspond to the induction of the conformational state I and the six-coordinated low-spin configuration of state II on binding to cytochrome oxidase. While in state I the structure of cytochrome c is essentially the same as in solution, state II is characterized by a structural rearrangement of the heme pocket, leading to a weakening of the axial iron-methionine bond and an opening of the heme crevice which is situated in the center of the binding domain for cytochrome oxidase. The relative contributions of the two cytochrome c states were estimated to be approximately in the ratio 1:1 in the complex.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗

PubMed1990

Quantitative conformational analysis of cytochrome c bound to phospholipid vesicles studied by resonance Raman spectroscopy.

Resonance Raman spectra have been recorded from ferri-cytochrome c bound to phospholipid vesicles composed of dimyristoyl phosphatidylglycerol (DMPG), dioleoyl phosphatidylglycerol (DOPG) or dioleoyl phosphatidylglycerol-dioleoyl phosphatidylcholine (DOPG-DOPC) (70:30 mole/mole). Lipid binding induces very significant conformational changes in the protein molecule. The resonance Raman spectra differ in their content of bands originating from two different conformational species, I and II, of the protein, and from two different spin and coordination states of the heme in conformation II. Data of sufficiently high precision were obtained that the spectra of the individual species could be quantitated by a constraint interactive fitting routine using single Lorentzian profiles. In the high frequency, or marker band region (1200 to 1700 cm-1), the frequencies, half widths and relative intensities of the individual bands could be estimated from previous surface enhanced resonance Raman measurements on cytochrome c adsorbed on a silver electrode. These were then further optimized to yield both the spectral parameters and relative contents of the different species. In the low frequency, or fingerprint, region (200 to 800 cm-1), the spectral parameters of the individual species were obtained from difference spectra derived by sequential subtraction between the spectra of ferri-cytochrome c in the three different lipid systems, using the relative proportions of the species derived from the marker band region. These parameters were then subsequently refined by iterative optimization. The optimized spectral parameters in both frequency regions for the six-coordinated low spin states I and II, and for the five-coordinated high spin state II are presented.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗

PubMed1990

Vinyl ketone reagents for covalent protein modification. Nitroxide derivatives suited to rotational diffusion studies by saturation transfer electron spin resonance, using membrane-bound Na,K-ATPase as an example.

The reactivity of a series of substituted vinyl ketone nitroxides with an integral membrane protein, the Na,K-ATPase, is described. Increasing the electrophilicity of the conjugated double bond enhances reactivity markedly, with some spin labels showing higher reactivity than the conventionally used maleimide derivatives. The spectroscopic characteristics of the spin-labeled protein are also better suited for motional analysis by the saturation transfer electron spin resonance (STESR) method than with previous labeling procedures. The rotational correlation time, deduced from STESR experiments, is in the same range (100-300 microseconds) irrespective of the vinyl ketone derivative used, and the rotational mobility corresponds to an (alpha beta)2 or higher oligomer of the membrane-bound Na,K-ATPase.

Adenosine Triphosphate↗

PubMed1990

The validation of measures of outcome following suture of divided peripheral nerves supplying the hand.

Erik Moberg pioneered the idea of validating measures of sensory function following peripheral nerve suture by correlating their results with those of functional tests. However it is important that powerful prior variables (age at suture, time elapsed since suture and delay between injury and suture) be controlled. Failure to do this may result in spurious correlations, as illustrated by analysis of two sets of data, one collected by the author and the other given in the classic paper of Onne (1962).

Adolescent↗

PubMed1990

Saturation transfer electron spin resonance of Ca2(+)-ATPase covalently spin-labeled with beta-substituted vinyl ketone- and maleimide-nitroxide derivatives. Effects of segmental motion and labeling levels.

The Ca2(+)-ATPase in native sarcoplasmic reticulum membranes was selectively spin-labeled for saturation transfer electron spin resonance (ESR) studies by prelabeling with N-ethylmaleimide and by using low label/protein ratios. Results with the nitroxide derivative of the standard sulphydryl-modifying reagent, maleimide, were compared with a series of six novel nitroxide beta-substituted vinyl aryl ketone derivatives which differed (with two exceptions) in the substituent at the ketone position. The two exceptions had a different electron withdrawing group at the alpha-carbon, to enhance further the electrophilic character of the beta-carbon. Although differing in their reactivity, all the conjugated unsaturated ketone nitroxide derivatives displayed saturation transfer ESR spectra indicative of much slower motion than did the maleimide derivative. The saturation transfer ESR spectra of maleimide-labeled Ca2(+)-ATPase therefore most likely contain substantial contributions from segmental motion of the labeled group. The effects of the level of spin labeling were also investigated. With increasing degree of spin label incorporation, the linewidths of the conventional ESR spectrum progressively increased and the intensity of the saturation transfer spectrum dropped dramatically, as a result of increasing spin-spin interactions. The hyperfine splittings of the conventional spectrum and the outer lineheight ratios of the saturation transfer spectrum remained relatively unchanged. Extrapolation back to zero labeling level yielded comparable values for the effective rotational correlation times deduced from the saturation transfer spectrum intensities and from the lineheight ratios, for the vinyl ketone label. For the maleimide label the extrapolated values from the integral are significantly lower than those from the lineheight ratios, probably because of the segmental motion. Comparison is made of the effective rotational correlation time for the vinyl ketone label with the predictions of hydrodynamic models for the protein diffusion, in a discussion of the aggregation state of the Ca2(+)-ATPase in the native sarcoplasmic reticulum membrane. The implications for the study of protein rotational diffusion and segmental motion, and of the proximity relationships between labeled groups, using saturation transfer ESR spectroscopy are discussed.

Animals↗

PubMed1990

Serum levels of IgE-binding factor (soluble CD23) in diseases associated with elevated IgE.

Several in-vitro experiments suggest that the low affinity receptor for IgE (Fc epsilon RII) and its soluble fragment (IgE-binding factor, IgE-BF) are multi-functional molecules and more particularly that they are capable of regulating the synthesis of human IgE. In an attempt to examine the in-vivo significance of these in-vitro observations, the serum level of IgE-BF was measured in individuals with allergic or parasitic diseases, both associated with an increased production of IgE. IgE-BF was measured by a radioimmunoassay employing two mAbs against Fc epsilon RII (mAbER). We first compared 257 allergic subjects to 172 non-allergic controls matched for age and sex. Statistical analysis of the data, after logarithmic transformation of IgE-BF and IgE values, revealed that despite a great overlap, the allergic subjects had significantly higher levels of IgE-BF. The correlation between IgE and IgE-BF was very weak but significant. Allergic or non-allergic children had significantly higher IgE-BF levels than the corresponding groups of adults; moreover, the inverse correlation between age and IgE-BF levels was significant only in the children and not in the adults. The IgE-BF levels were not influenced by gender, by hyposensitization therapy or by treatment with local steroids. Subjects receiving systemic steroids had lower IgE-BF levels than untreated subjects.(ABSTRACT TRUNCATED AT 250 WORDS)

Adult↗

PubMed1989

Spin-label ESR of bacteriophage M13 coat protein in mixed lipid bilayers. Characterization of molecular selectivity of charged phospholipids for the bacteriophage M13 coat protein in lipid bilayers.

Bacteriophage M13 major coat protein has been incorporated at different lipid/protein ratios in lipid bilayers consisting of various ratios of dimyristoylphosphatidylcholine (DMPC) to dimyristoylphosphatidylglycerol (DMPG). Spin-label ESR experiments were performed with phospholipids labeled at the C-14 position of the sn-2 chain. For M13 coat protein recombinants with DMPC alone, the relative association constants were determined for the phosphatidylcholine, phosphatidylglycerol, and phosphatidic acid spin-labels and found to be 1.0, 1.0, and 2.1 relative to the background DMPC, respectively. The number of association sites for each phospholipid on the protein was found to be 4 per protein monomer. The intrinsic off-rates for lipid exchange at the intramembranous surface of the protein in DMPC alone at 30 degrees C were found to be 5 X 10(6), 6 X 10(6), and 2 X 10(6) s-1 for the phosphatidylcholine, phosphatidylglycerol, and phosphatidic acid spin-labels, respectively. Adding DMPG to the DMPC lipid system increased the exchange rates of the lipids on and off the protein. By gel filtration chromatography, it is found that protein aggregation is reduced after addition of DMPG to the lipid system. This is in agreement with measurements of tryptophan fluorescence, which show a decrease in quenching efficiency after introduction of DMPG in the lipid system. The results are interpreted in terms of a model relating the ESR data to the size of the protein-lipid aggregates.

Bacteriophages↗

PubMed1989

Spin-label ESR studies on the interaction of bovine spinal cord myelin basic protein with dimyristoylphosphatidylglycerol dispersions.

Electron spin resonance (ESR) spectroscopy and chemical binding assays were used to study the interaction of bovine spinal cord myelin basic protein (MBP) with dimyristoylphosphatidylglycerol (DMPG) membranes. Increasing binding of MBP to DMPG bilayers resulted in an increasing motional restriction of PG spin-labeled at the C-5 atom position in the acyl chain, up to a maximum degree of association of 1 MBP molecule per 36 lipid molecules. ESR spectra of PG spin-labels labeled at other positions in the sn-2 chain showed a similar motional restriction, while still preserving the chain flexibility gradient characteristic of fluid lipid bilayers. In addition, labels at the C-12 and C-14 atom positions gave two-component spectra, suggesting a partial hydrophobic penetration of the MBP into the bilayer. Spectral subtractions were used to quantitate the membrane penetration in terms of the stoichiometry of the lipid-protein complexes. Approximately 50% of the spin-labeled lipid chains were directly affected at saturation protein binding. The salt and pH dependence of the ESR spectra and of the protein binding demonstrated that electrostatic interaction of the basic residues of the MBP with the PG headgroups is necessary for an effective association of the MBP with phospholipid bilayers. Binding of the protein, and concomitant perturbation of the lipid chain mobility, was reduced as the ionic strength increased, until at salt concentrations above 1 M NaCl the protein was no longer bound. The binding and ESR spectral perturbation also decreased as the protein charge was reduced by pH titration to above the pI of the protein at approximately pH 10.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗

PubMed1989

Interaction of two complementary fragments of the bovine spinal cord myelin basic protein with phospholipid bilayers. An ESR spin-label study.

The myelin basic protein (MBP) from bovine spinal cord was cleaved at the single tryptophan residue to produce an N-terminal fragment (F1) of molecular weight 12.6K and a C-terminal fragment (F2) of molecular weight 5.8K. The interactions of the two fragments with bilayers of the acidic lipid dimyristoylphosphatidylglycerol (DMPG) were compared with those of the intact protein, by using both chemical binding assays and spin-label electron spin resonance spectroscopy. The saturation binding stoichiometries of the two fragments were found to sum to that of the MBP, having values of 11, 24, and 36 mol of DMPG/mol of protein for F2, F1, and the MBP, respectively. The strength of binding was found to increase in the order F2 less than F1 less than MBP, which follows that of the net charges on the different fragments. The ionic strength dependence of the protein binding indicated that the interaction is primarily of electrostatic origin. The efficiency of displacement of the proteins by salt was in the order F2 greater than F1 greater than MBP, which correlates with both the strength of binding and the net charge on the different protein fragments. Nitroxide derivatives of phosphatidylglycerol (PG) labeled on the sn-2 chain were used to probe the protein-induced changes in the acyl chain dynamics. Both the fragments and the MBP decreased the lipid chain mobility as recorded by the C-5 atom and C-12 atom position nitroxide-PG spin-labels, in a manner which followed the protein binding curves.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗

PubMed1989

Selectivity of interaction of phospholipids with bovine spinal cord myelin basic protein studied by spin-label electron spin resonance.

The selectivity of interaction between bovine spinal cord myelin basic protein (MBP) and eight different spin-labeled lipid species in complexes with dimyristoylphosphatidylglycerol (DMPG) and between spin-labeled phosphatidylglycerol and spin-labeled phosphatidylcholine in complexes of MBP with various mixtures of DMPG and dimyristoylphosphatidylcholine (DMPC) has been studied by electron spin resonance (ESR) spectroscopy. In DMPC/DMPG mixtures, the protein binding gradually decreased with increasing mole fraction of DMPC in a nonlinear fashion. The lipid-protein binding assays indicated a preferential binding of the protein to phosphatidylglycerol relative to phosphatidylcholine without complete phase separation of the two lipids. The outer hyperfine splittings (2Amax) of both phosphatidylglycerol and phosphatidylcholine labeled at C-5 of the sn-2 chain (5-PGSL and 5-PCSL, respectively) were monitored in the lipid-protein complexes as a function of the mole fraction of DMPC. The increases in the value of Amax induced on binding of the protein were larger for 5-PGSL than for 5-PCSL, up to 0.25 mole fraction of DMPC. Beyond this mole fraction the spectral perturbations induced by the protein were similar for both lipid labels. The ESR spectra of phosphatidylglycerol and phosphatidylcholine labeled at C-12 of the sn-2 chain were two component in nature, indicating indicating a direct interaction of the protein with the lipid chains, at mole fractions of DMPC up to 0.25. Quantitation of the motionally restricted spin-label population by spectral subtraction again indicated a preferential interaction of the protein with phosphatidylglycerol relative to phosphatidylcholine. Up to DMPC mode fractions of 0.25, the microenvironment of the protein was enriched in DMPG.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗

PubMed1989

A spin-label electron spin resonance study of the binding of mitochondrial creatine kinase to cardiolipin.

The binding of the mitochondrial creatine kinase to aqueous dispersions of beef heart cardiolipin has been studied via the perturbation of the mobility of spin-labelled cardiolipin, using electron spin resonance (ESR) spectroscopy. In the presence of creatine kinase (1:1 protein/lipid ratio, by mass), the ESR spectra of cardiolipin labelled in a single acyl chain [n-(4,4-dimethyl-oxazolidinyl-N- oxy)stearoylcardiolipin] indicate a restriction of motion both at the C-5 and C-14 positions (n = 5, 14) of the lipid chains. The restriction in mobility was reversed by addition of phosphate or adriamycin, which are thought to inhibit the binding of creatine kinase to the mitochondrial membrane or to displace it from its binding site on the membrane. The effect of the protein on the chain mobility is consistent with surface binding of the protein; no positive evidence was obtained for penetration of the protein into the hydrophobic region of the membrane.

Animals↗

PubMed1989

Specificity of the interaction of amino- and carboxy-terminal fragments of the mitochondrial precursor protein apocytochrome c with negatively charged phospholipids. A spin-label electron spin resonance study.

The contribution of the various regions of the mitochondrial precursor protein apocytochrome c to the interaction of the protein with phosphatidylserine dispersions has been studied with chemically and enzymatically prepared fragments of horse heart apocytochrome c and phospholipids spin-labeled at different positions of the sn-2 chain. Three amino-terminal heme-less peptides, two heme-containing amino-terminal fragments, one central fragment, and three carboxy-terminal fragments were studied. The electron spin resonance spectra of phospholipids spin-labeled at the C5 position of the fatty acid chain indicate that both amino-terminal and carboxy-terminal fragments of the apocytochrome c molecule cause a restriction of motion of the lipids, whereas the heme-containing peptides and protein have less effect. In addition, a second motionally more restricted lipid component, which is observed for apocytochrome c interacting with phosphatidylserine dispersions containing lipids spin-labeled at the C12 or C14 position [Görrissen, H., Marsh, D., Rietveld, A., & de Kruijff, B. (1986) Biochemistry 25, 2904-2910], was observed both on binding the carboxy-terminal fragments and on binding of the amino-terminal fragments of the precursor protein. Interestingly, even a small water-soluble peptide consisting of the 24 carboxy-terminal residues gave rise to a two-component spectrum, with an outer hyperfine splitting of the restricted lipid component of 59 G, indicating a considerable restriction of the chain motion. This suggests that both the carboxy- and amino-terminal parts of the protein penetrate into the center of the bilayer and cause a strong perturbation of the fatty acyl chain motion. The implications of these findings for the mechanism of apocytochrome c translocation across membranes are discussed.

Animals↗