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R J Cushley

Publications and source records attributed to R J Cushley.

At least 37 records · Page 2Linked to original sources
PubMed1990

Dynamic structure of the lower density lipoproteins. I. Incorporation of high levels of labelled lipids into very low and low density lipoproteins.

Selectively labelled lipids have been incorporated into the surface monolayer of human serum low density lipoprotein (LDL) and very low density lipoprotein (VLDL). From 3 to 17 mol% of phosphatidylcholine, selectively deuterated at various positions along the sn-2-acyl chain, was transferred from unilamellar vesicles to VLDL using a partially purified phosphatidylcholine transfer protein. Selectively deuterated palmitic acids were incorporated into LDL (6-20 mol%) and into VLDL (7-10 mol%). Electron microscopy, light scattering, and 31P nuclear magnetic resonance indicated that particle size remained unchanged. Gel exclusion chromatography and chemical analysis showed no difference in hydrodynamic properties and only slight alteration to particle component ratios. Biological activity of labelled VLDL was measured from the rate of cholesterol esterification by cultured J774A.1 cells. Effect of labelling LDL was evaluated by monitoring LDL uptake and degradation by cultured human skin fibroblasts. In all cases the lipoproteins containing labels were indistinguishable from their native counterparts.

Animals↗
PubMed1990

Dynamic structure of the lower density lipoproteins. II. Deuterium NMR studies of the monolayer of very low and low density lipoproteins.

The order of phosphatidylcholine (PC) acyl chains in the surface monolayer of very low density lipoproteins (VLDL) and low density lipoproteins (LDL) has been determined from 2H nuclear magnetic resonance order parameters, SCD, using selectively deuterated PC or palmitic acids. From the computer simulated line shapes, we find two distinct phospholipid domains within the amphiphilic monolayer of both VLDL and LDL. In the more ordered domain of LDL, SCD was approximately 0.3 for the "plateau" chain region. The SCD values of VLDL particles are similar to those of LDL for the 5,6- and 11,12-positions, hence we suggest the organization of the more ordered region of VLDL and LDL are similar. The domain of low order in LDL comprises less than 10% of the phospholipid molecules (we do not distinguish between PC and sphingomyelin), having approximately the same order (SCD less than 0.1) as egg PC - sphingomyelin unilamellar vesicles. In VLDL, the domain of low order comprises between approximately 10 and approximately 20% of the phospholipid molecules and the entire acyl chain is in an essentially isotropic environment (SCD less than 0.02). We prepared VLDL-sized microemulsions composed of egg PC, deuterated PC, and triolein to test whether the apoproteins were responsible for creating the two differently organized domains in VLDL and LDL. Surprisingly, these protein-free particles also showed two domains of different order at two temperatures. The high order region, however, is less ordered than in VLDL and LDL. We explain two surface domains of PC in terms of lipid organization and the unique interactions of lipids in the various lipoprotein particles.

Chemical Phenomena↗
PubMed1988

Lipid-lipid interactions in reconstituted high-density lipoproteins by deuterium nuclear magnetic resonance.

Lipid-lipid interactions between the core and monolayer have been studied by using reconstituted high-density lipoproteins (rHDLs) composed of apoHDL3 with either dipalmitoylphosphatidylcholine (DPPC) or egg phosphatidylcholine (egg PC) as the monolayer and either cholesteryl oleate (CO) or triolein (TO) as the core. The effect of the monolayer on the core was observed by deuterium nuclear magnetic resonance (2H NMR) studies of rHDLs containing the core component cholesteryl [18, 18, 18-2H3] oleate [( 2H3]CO) or tri[16,16-2H2]oleoylglycerol [( 2H6]TO) surrounded by a monolayer of either DPPC or egg PC as a function of temperature. The reverse effect, that of the core on the monolayer, was examined by both 2H and 31P NMR studies of rHDLs containing [5,5-2H2]PC in the presence of CO or TO as a function of temperature. The 2H NMR line widths of [2H3]CO and [2H6]TO were considerably broader and showed a greater temperature dependence in rHDLs containing DPPC than in those containing egg PC. Similarly, the C-2H order parameters of [2H2]PC were higher and showed a greater temperature dependence in rHDLs containing CO than in those containing TO. In contrast, the 31P NMR line widths were identical for both [2H2]-PC/CO/apoHDL3 and [2H2]PC/TO/apoHDL3 at 25 and 6 degrees C, showing only a slight temperature dependence. Thus, acyl chains of both the monolayer and core components show increased order when in contact with neighboring lipids of higher order. The data demonstrate a direct effect of core cholesteryl esters and triglycerides with the phospholipid monolayer of HDL.

Deuterium↗
PubMed1988

Deuterium nuclear magnetic resonance study of the interaction of branched chain compounds (phytanic acid, phytol) with a phospholipid model membrane.

Deuterium nuclear magnetic resonance (2H-NMR) spectra have been determined for 50 wt% aqueous dispersions of 1-palmitoyl(stearoyl)-2-[2H31]palmitoyl-sn-glycero-3-phosphocho lin e (PC-d31) containing 20 mol% of the isoprenoid compounds phytol or phytanic acid over the temperature range -5-55 degrees C. Concentration effects of the isoprenoid compounds are also reported. First moments (M1) and order parameters were calculated from the spectra. 20 Mol% of either branched chain compound causes an approximate 9% increase in the mean order parameter SCD. Significant effects are seen on the PC-d31 phase behavior. 20 Mol% of either branched chain compound causes the gel to liquid crystalline onset temperature (Ts) to drop to 28 degrees C from 38 degrees C for PC-d31 alone, as seen from the temperature dependent M1 values. The melting range ([Tl--Ts]) is congruent to 1.5 degrees C for PC-d31 and congruent to 11 degrees C for PC-d31 containing 20 mol% of the branched chain compounds. This is in direct contrast to their straight chain analogues, hexadecanol and palmitic acid, which have been shown to elevate the phase transition temperature. The isoprenoid compounds cause significant disruption of the gel phase, forcing nearest neighbor phospholipid chains apart. Transverse relaxation times (T2e, the time constant for decay of the quandrupolar echo) have been determined over the temperature range -5-50 degrees C. Possible explanation for the effect of the isoprenoid compounds on the dynamic structure of phospholipids in the bilayer are proffered.

Calorimetry, Differential Scanning↗
PubMed1988

Soluble complex formation between low-density lipoprotein and glycosaminoglycans. A 2H and 31P-NMR, and quasi-elastic light scattering study.

Soluble complex formation between LDL and heparin (HEP) and chondroitin sulfate (CS) has been studied by 2H- and 31P-NMR and light scattering. The 2H-NMR linewidths of [2H]HEP and [2H]C4S increase substantially upon binding to LDL, with the [2H]HEP linewidths broader at low glycosaminoglycan (GAG)/low density lipoprotein (LDL) ratios. Preliminary analysis of the bound C2H3 group correlation times suggests that the observed linewidths are determined by the complex size, and that both [2H]GAGs have similar motions when bound to LDL. The 31P-NMR data demonstrate that large LDL-HEP complexes (diameter approx. 50 nm) are formed only over a narrow range of HEP concentrations, whereas the size of LDL-CS complexes increases continuously over the range of CS concentrations studied, reaching values of 32-35 nm for both C4S and C6S. At the lower protein concentrations studied by light scattering (less than or equal to 1 mg/ml), the same trends are observed, although the mean diameters are less than those estimated by 31P-NMR. Soluble complex formation was unaffected by the presence of 2 mM Ca2+. Dilution studies demonstrate that complex size varies with protein concentration. The binding of GAGs to LDL was also examined by HEP-CS competition studies. HEP has the higher affinity while no differences in binding could be detected between C4S and C6S.

Calcium↗
PubMed1987

Phospholipid/cholesterol membranes containing n-alkanols: a 2H-NMR study.

The influences of 1-octanol and 1-decanol on aqueous multilamellar dispersions of 1-hexadecanoyl(octadecanoyl)-2-[2H31]hexadecanoyl-sn-glycero -3-phosphorylcholine (PC-d31)/cholesterol (3:1) have been examined using 2H-NMR. The gel to liquid crystalline phase transition of the PC-d31/cholesterol dispersion is modulated by the addition of 1-alkanol, which reduces the onset temperature and increases the width of the transition. 1-Octanol has a greater effect on the transition onset and completion temperatures than does 1-decanol, as determined from analysis of the temperature-dependent 2H-NMR spectra. 2H-NMR C-2H bond order parameters as a function of phospholipid acyl chain position at 60 degrees C, where all dispersions are fully liquid crystalline, have been calculated from the depaked spectra. 1-Decanol reduces the phospholipid order by only 2%. This can be attributed to the lower effective cholesterol concentration in the 1-alkanol/PC-d31/cholesterol dispersions. 1-Octanol, however, reduces the phospholipid order by 10% at 60 degrees C. Correlations between the effects of 1-octanol and 1-decanol on phospholipid order parameters and phospholipid/cholesterol phase transitions are discussed.

1,2-Dipalmitoylphosphatidylcholine↗
PubMed1987

Surface diffusion in human serum lipoproteins.

From the viscosity dependence of the 31P NMR signals, the diffusion coefficients DT of phospholipid molecules in the surface monolayer of HDL, LDL and VLDL have been determined. DT for HDL3 and HDL2 are found to be 2.3 X 10(-8) cm2/s and 1.8 X 10(-8) cm2/s, respectively. These values are similar to values reported for diffusion of phospholipid molecules in phospholipid bilayers above the gel to liquid crystalline phase transition temperature. Viscosity dependence of [16,16,16-2H3]phosphatidylcholine incorporated into HDL2 yielded a value similar to that determined by 31P (DT = 1.9 X 10(-8) cm2/s). Slower diffusion coefficients were measured for LDL2 and VLDL. VLDL had a value DT = 9.1 X 10(-9) cm2/s. The diffusion coefficient for LDL2 was 1.4 X 10(-9) cm2/s. Thus, diffusion of phospholipids in LDL2 is a full order of magnitude slower at 25 degrees C than diffusion of phospholipids in the HDLs.

Diffusion↗
PubMed1986

Orientational order of cholesteryl oleate in low-density lipoprotein observed by 2H-NMR.

Cholesteryl oleate, selectively deuterated at various positions along the acyl chain, has been incorporated into fresh human serum low-density lipoprotein (LDL2). Temperature-dependent 2H-NMR spectra were recorded between 15 and 45 degrees C. For deuterons at C-2' and C-5' of the acyl chain, two 2H-NMR spectral components, a broad and a narrow signal, are observed. This is interpreted as reflecting the coexistence of two cholesteryl ester regions in the LDL2 core which possess different degrees of order. The C-2H bond order parameters, SCD, are approx. 0.12-0.20 for the more ordered region and approx. 0.04-0.06 for the less ordered region. Longitudinal relaxation times, T1, of deuterated cholesteryl oleate are found to increase between C-8' and the terminal -C2H3 group, which is consistent with an increased rate of chain motion toward the free ends of the ester acyl chains.

Cholesterol Esters↗
PubMed1986

Deuterium NMR study of the interaction of alpha-tocopherol with a phospholipid model membrane.

The effects of 5, 10, and 20 mol % incorporation of alpha-tocopherol (vitamin E) on 50 wt % aqueous multilamellar dispersions of sn-2-substituted [2H31]palmitoylphosphatidylcholine (PC-d31), a saturated, deuterated phospholipid prepared from egg lysophosphatidylcholine, have been studied by deuterium nuclear magnetic resonance (2H NMR) and differential scanning calorimetry (DSC). Moment analysis of the 2H NMR spectra as a function of temperature and DSC heating curves demonstrate that the main gel to liquid-crystalline phase transition is progressively broadened and its onset temperature lowered by increasing concentrations of alpha-tocopherol. Below the transition temperature (40 degrees C) for PC-d31 bilayers, the 2H NMR spectra indicate that acyl chain motion is increased by addition of alpha-tocopherol and that this effect extends to lower temperatures with higher alpha-tocopherol content. Above the transition, average carbon-deuterium bond order parameters calculated from the first spectral moment establish that alpha-tocopherol increases acyl chain ordering within the PC-d31 bilayer by as much as 17% at 20 mol % incorporation. Profiles of order parameter vs. chain position, constructed from 2H NMR spectra following application of the depaking technique, show that despite higher order the general form of the profile is not significantly altered by alpha-tocopherol.

Calorimetry, Differential Scanning↗
PubMed1986

A deuterium NMR study of labelled n-alkanol anesthetics in a model membrane.

The 2H-NMR spectra of 50 wt.% aqueous multilamellar dispersions of dipalmitoylphosphatidylcholine (DPPC) containing either selectively deuterated 1-decanol (25 mol%) or [2H17]-1-octanol (25 mol%) have been measured as a function of temperature. Both alkanols are potent anesthetics. A detailed carbon-deuterium bond order parameter profile of 1-decanol in liquid crystalline phospholipid dispersions at 50 degrees C was determined from the quadrupolar splittings of 1-decanols deuterated at eight different positions. A maximum order parameter SCD = 0.20 was obtained for [5,5-2H2]-1-decanol, with labels at both ends of the 1-decanol exhibiting reduced order parameters. Explanations for the reduced order towards the hydroxyl group of 1-decanol are discussed in terms of either increased amplitudes of motion or geometric effects due to hydrogen bonding. By comparing the order parameter profile of sn-2 chain deuterated phosphatidylcholine dispersions containing 25 mol% 1-decanol (J.L. Thewalt, S.R. Wassall, H. Gorrissen and R.J. Cushley, Biochim. Biophys. Acta, 817 (1985) 355) with the profile of deuterated 1-decanol in DPPC, we estimate that decanol is approximately parallel to the C-3 to C-13 region of the phosphatidylcholine's sn-2 chain. Variation of the spectral moments M1 with temperature indicates that both 1-decanol and 1-octanol are sensitive to the packing of the lipid in which they are dissolved. Below the phase transition temperature, the 2H-NMR spectra of either 1-decanol (selectively deuterated) or 1-octanol (perdeuterated) are broad powder patterns, characteristic of axially symmetric rotation about the alcohol's long axis. This is in contrast to the 2H-NMR spectra obtained from deuterated phosphatidylcholine under similar conditions, which implies that the phospholipid acyl chain conformations are more restricted than those of the alcohol at these temperatures. From the M1 behavior of the various alkanol chain segments with temperature, the gel to liquid crystalline phase transition is seen to initiate in the middle of the DPPC/1-alkanol bilayer.

Anesthetics↗
PubMed1985

Deuterium NMR study of the effect of n-alkanol anesthetics on a model membrane system.

The effects of 25 mol% incorporation of two anesthetics, 1-octanol and 1-decanol, on a deuterated, saturated phospholipid in 50 wt% aqueous multilamellar dispersions have been studied by 2H-NMR spectroscopy and differential scanning calorimetry (DSC). The phospholipid used is sn-2 substituted '[2H31]-palmitoylphosphatidylcholine' (PC-d31). DSC thermograms demonstrate that PC-d31 has phase behavior qualitatively similar to that of dipalmitoylphosphatidylcholine, with a pretransition at 31 degrees C and a main gel to liquid crystalline transition at 40 degrees C. Analysis of the temperature-dependent 2H-NMR spectra in terms of the first moment, which is extremely sensitive to the phospholipid phase, shows that 1-octanol and 1-decanol depress and broaden the main transition. This is confirmed by DSC, which shows that the pretransition is eliminated by the 1-alkanols. The carbon-deuterium bond order of the phospholipid deuterated acyl chains, in the presence and absence of 1-alkanols, was determined from deuterium quadrupolar splittings. Spectra were analyzed using the depaking technique. A 1-alkanol concentration of 25 mol% had no significant effect on the profile of the carbon-deuterium bond order parameter SCD along the phospholipid acyl chain at 50 degrees C. Thus, it appears that the liquid crystalline phase is able to accommodate large amounts of linear anesthetic molecules without substantial effect on molecular ordering within the membrane bilayer. Preliminary results show that the transverse relaxation rates of the acyl chain segments are significantly decreased by the presence of 1-octanol or 1-decanol.

1-Octanol↗
PubMed1985

Chain dynamics of selectively deuterated fatty acids in high-density lipoproteins studied by deuterium NMR.

Deuterium order parameters have been determined for approximately 5 mol% selectively deuterated palmitic acid incorporated into the outer monolayer of high-density lipoproteins (HDL3). The values are SCD = 0.38 for [2,2-2H2]palmitic acid, 0.38 for [4,4-2H2]palmitic acid, 0.37 for [5,5,6,6-2H4]palmitic acid, 0.23 for [11,11,12,12-2H4]palmitic acid, and 0.05 for [16,16,16-2H3]palmitic acid. Comparison of the acyl chain order parameters in HDL3 with acyl chain order parameters determined recently [Parmar, Y.I., Wassall, S.R., & Cushley, R.J. (1984) J. Am. Chem. Soc. 106, 2434-2435] for approximately 5 mol% deuterated palmitic acid in sonicated unilamellar vesicles, composed of the same ratio of phosphatidylcholine/sphingomyelin (85/15 w/w) found in HDL3, shows that acyl chain order in the HDL3 monolayer is approximately 3-5 times higher than in the vesicle bilayer. The acyl chain order in the lipoprotein monolayer is approximately 1.5-2 times higher than in the bilayer of phosphatidylcholine multilamellar dispersions. Deuterium longitudinal relaxation times have been measured for deuterated palmitic acid in HDL3, and the values T1 approximately 16 ms for C2H2 and 170 ms for C2H3 groups are a factor of more than 2 times smaller than found in phospholipid bilayers.

Deuterium↗
PubMed1984

Deuterium magnetic resonance study of the interaction between chondroitin sulfate and human plasma low density lipoproteins.

[2H]Chondroitin sulfate was prepared by partial N-deacetylation of chondroitin sulfate (via hydrazinolysis) followed by treatment with [2H6]acetic anhydride. 2H NMR spectra of [2H]chondroitin sulfate in the presence of human plasma low density lipoprotein provide evidence for a soluble complex stoichiometry of 3 (and possibly 2) lipoproteins per polysaccharide molecule, and allow a rough estimation of the dissociation constant Kd.

Chondroitin↗
PubMed1983

Molecular motion and conformation of cholesteryl esters in reconstituted high density lipoprotein by deuterium magnetic resonance.

Reconstituted high density lipoprotein has been prepared by sonication and preparative ultracentrifugation of mixtures containing the apoprotein of high density lipoprotein, egg phosphatidylcholine, cholesteryl oleate, and acyl chain deuterated cholesteryl palmitate in aqueous buffer. The resulting structures have a size and chemical composition very similar to native high density lipoprotein. Deuterium NMR spectra and longitudinal relaxation times were obtained at approximately 25 degrees C. The variation of the 2H NMR line width with chain position is consistent with an average conformation such that the ester acyl chain is extended. In addition, 2H NMR line widths and longitudinal relaxation times indicate that the ester acyl chains possess significant mobility.

Cholesterol Esters↗
PubMed1983

Carbon-13 nuclear magnetic resonance study of spin labelled cholesteryl ester in model membranes.

Carbon-13 NMR longitudinal relaxation times for unilamellar vesicles of egg phosphatidylcholine (PC) in aqueous dispersion have been measured following the incorporation of spin labelled cholesteryl palmitate. The spin label induced relaxation rates, 1/T1,SL, for fatty acyl chain carbons show that the C5 segment of the cholesteryl ester acyl chain is located near the C1 and C2 segments of the phospholipid acyl chains. A greater spin label induced enhancement of relaxation rate was observed for the inner vesicle layer than for the outer, and is attributed to a higher ester incorporation and/or tighter lipid packing in the inner layer.

Cholesterol Esters↗
PubMed1982

Deuterium magnetic resonance of triacylglycerols in phospholipid bilayers.

Aqueous dispersions of egg-phosphatidylcholine and egg-phosphatidylcholine/30 mol% cholesterol containing deuterated tripalmitin or triolein were studied at approx. 25 degrees C by 2H-NMR. Incorporation of tripalmitin into egg-phosphatidylcholine bilayers was found to be less than 0.1 mol%, while the incorporation of triolein is approx. 2.5 mol% in the absence and approx. 0.7 mol% in the presence of cholesterol. The profile of order parameter versus chain position for deuterated triolein suggests that the oleoyl chains of the triacylglycerol have an average orientation such that the C2 chain segments and the segments in the vicinity of the C9-C10 double bond are tilted with respect to the bilayer normal, while all other segments are parallel to the bilayer normal. Longitudinal relaxation times were also determined and indicate that the acyl chains of triolein have a motional behaviour similar to that of phospholipid acyl chains in the bilayer.

Animals↗
PubMed1981

Deuterium nuclear magnetic resonance study of the interaction of substrates and inhibitors with soybean lipoxygenase.

Deuterium NMR spectra for a series of selectively deuterated substrates and inhibitors in the presence of lipoxygenase-1 (EC 1.13.11.12) are presented. Extrapolation of the 2H NMR line widths yielded transverse relaxation rates for the bound inhibitors [2H21]dodecanoic acid (protonated at the 2,2-position), [2,2-2H]dodecanoic acid, and [12,12,12-2H]dodecanoic acid which are 1/T2,bd = 5.0 X 10(3), 1.12 X 10(4), and 1.16 X 10(3) s-1, respectively. The substrates [9,10,12,13-2H]linoleic acid and [11,11-2H]linoleic acid had 1/T2,bd = 8.2 X 10(3) and 7.95 X 10(3) s-1, respectively. Kinetic measurements established Ki = 1.5 X 10(-3) M for dodecanoic acid (lauric acid) inhibition of lipoxygenase when the substrate is linoleic acid (Km = 2.6 X 10(-5) M). Lipoxygenase, with Mr 102,000, is predicted to have a rotational correlation time tau c - 1.2 X 10(-7) s, yielding a 1/T2,bd = 1.56 X 10(4) s-1 for tightly bound ligand. Hence, the correlation times of the selectively deuterated inhibitors indicate internal motions are present in the bound species.

Kinetics↗