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Biomedical subjects

E Oldfield

Publications and source records attributed to E Oldfield.

At least 109 records · Page 6Linked to original sources
PubMed1979

Deuterium nuclear magnetic resonance investigation of dimyristoyllecithin--dipalmitoyllecithin and dimyristoyllecithin--cholesterol mixtures.

Deuterium nuclear magnetic resonance (NMR) spectra of 1,2-dimyristoyl-3-sn-phosphatidylcholines (DMPCs) specifically deuterated in the 2-chain at one of positions 2', 3', 6', or 14' have been obtained by the quadrupole-echo Fourier transform method at 34.1 MHz (corresponding to a magnetic field strength of 5.2 T) or the pure material as a function of temperature, and in the presence of either 1,2-dipalmitoyl-3-sn-phosphatidylcholine (DPPC) or cholesterol as a function of temperature and composition. The results with pure DMPC and DMPC--DPPC mixtures indicate that a sharp, intense deuterium resonance is characteristic of fluid-phase lipids, whereas a broad resonance is characteristic of solid-phase lipids. There is shown to be good agreement between the deuterium NMR derived DMPC--DPPC phase diagram and that derived by using other techniques. The deuterium NMR results obtained with the DMPC--cholesterol system are not interpreted in terms of a phase diagram. They do indicate, however, that the transition breadth is increased considerably and the temperature at which the lipid chains "solidify" is depressed by the addition of cholesterol to the DMPC bilayer. The particular nature of the increase and the depression is found to be dependent on where the label is located on the lipid.

Cholesterol↗
PubMed1978

Spectroscopic studies of specifically deuterium labeled membrane systems. Nuclear magnetic resonance investigation of the effects of cholesterol in model systems.

Deuterium nuclear magnetic resonance spectra of dimyristoylphosphatidylcholines specifically labeled in positions 2', 3', 4', 6', 8', 10', 12', and 14', of the 2 chain, of an N-deuteriomethylphosphatidylcholine, and of cholesterol-3alpha-d1, have been obtained by the Fourier transform method at 5.46 and 3.52 T on two "home-built" widebore superconducting magnet spectrometers, as a function of temperature and composition. Data on the specifically deuterium-labeled cholesterol molecule (in nonsonicated membrane systems) permits an estimate of the most probable angle of tilt of the sterol in the membrane, and evaluation of the order parameter (Salpha) describing rigid body motions in the bilayer. Segmental order parameters derived from the data presented allow calculation of individual chain segment projections onto the director axis and, consequently, estimation of effective chain length. It is shown that mathematical models which include chain tilt as well as those which neglect this type of rigid body motion give essentially identical results when applied to the dimyristoylphosphatidylcholine and dimyristoylphosphatidylcholine-cholesterol bilayer systems (in excess water, between 23 and 60 degrees C). Results of calculations of chain length and membrane thickness of a dimyristoylphosphatidylcholine-30 mol % cholesterol membrane system at 23 degrees C give excellent agreement when compared with recent high-resolution neutron diffraction data obtained on specifically deuterium labeled lecithin-cholesterol systems. No evidence for formation of lecithin-cholesterol complexes having lifetimes of approximately 30 ms has been found. Below the pure-lipid gel-liquid crystal phase transition temperature Tc but in the presence of cholesterol, we have obtained further evidence for 1-chain/2-chain nonequivalence. At 10 degrees C, the 2' segment of the 2 chain, but not the 2' segment of the 1 chain or the 3', 6', or 12' segments of the 2 chain, is broadened almost beyond detection. These results are in agreement with similar effects reported recently for the dipalmitoylphosphatidylcholine-cholesterol system and may indicate a bent configuration for the 2 chain, in the lecithin-cholesterol system. Further cooling below Tc results in loss of the 1-chain 2'-position signal intensity plus 2-chain 3', 6', and 12' signals simultaneously. The increase in length of the 2 chain of dimyristoylphosphatidylcholine upon addition of 30 mol % cholesterol of 23 degrees C is about 2.3 A. Addition of cholesterol to a choline-labeled lecithin results in complex behavior of the head group deuterium quadrupole splitting as a function of temperature, and cholesterol mole fraction. Above approximately 20 mol % cholesterol, the main effect is a decrease in quadrupole splitting as cholesterol content increases, the opposite effect to that observed with hydrocarbon chains.

Cholesterol↗
PubMed1978

Deuterium nuclear magnetic resonance investigation of the effects of proteins and polypeptides on hydrocarbon chain order in model membrane systems.

Deuterium Fourier-transform nuclear magnetic resonance spectra have been obtained of 1-myristoyl 2-(14,14,14-trideutero)myristoyl phosphatidylcholine bilayers at 34.1 MHz by using the quadrupole echo pulse technique. Thereby, we have investigated the effects upon the deuterated dimyristoyl phosphatidylcholine bilayers of the following proteins and polypeptides: gramicidin A, bacteriophage f1 coat protein, beef brain myelin proteolipid apoprotein, cytochrome b(5), and cytochrome c oxidase (ferrocytochrome c:oxygen oxidoreductase, EC 1.9.3.1). Above T(c), the transition temperature between the gel and liquid crystal phases, the quadrupole splitting of the deuterium-labeled methyl group is reduced or collapsed in the presence of protein or polypeptide. No evidence has been found for ordered "boundary lipid." Below T(c), the spectra show that the hydrocarbon chains are prevented from crystallizing by the protein (or polypeptide) incorporated in the membrane. Similar disordering effects above T(c) are also seen when an unsaturated lipid, 1-(16,16,16-trideutero)palmitoyl 2-palmitoleyl phosphatidylcholine is complexed with cytochrome oxidase.

Journal Article↗
PubMed1976

Deuterium magnetic resonance spectroscopy of isotopically labeled mammalian cells.

Choline completely deuterated in the methyl groups has been incorporated into two mammalian cell systems. The first was the chemically transformed mouse fibroblast LM cell line, grown in suspension culture. The second system consisted of mitochondria from Sprague-Dawley rats which were fed upon a choline-deficient diet enriched with deuterated choline chloride. Two classes of deuterium nuclear magnetic resonance (NMR) signal were obtained from each system. The first class of signal exhibited no residual quadrupole coupling constant and was assigned to the natural abundance of 2H in water, together with free labeled choline. The second class of signal had a residual quadrupole coupling of about 1 HKz and was assigned to 2H-labeled choline headgroups of phospholipids. These results are in conflict with a previous study of deuterated rat liver mitochondrial membranes. Deuterium magnetic resonance spectra can be obtained on mammalian membranes that have incorporated specific deuterium-labeled compounds and, therefore, provides a very powerful method for studying the dynamic structure of membranes.

Animals↗
PubMed1975

Studies of individual carbon sites of hemoglobins in solution by natural abundance carbon 13 nuclear magnetic resonance spectroscopy.

Proton-decoupled natural abundance 13C NMR spectra of carbon monoxide hemoglobins were recorded at 15.18 MHz by the Fourier transform method, under conditions of spectrometer sensitivity sufficient for detection of individual carbon resonances. The aromatic region of each spectrum contains broad bands of methine carbon resonances, and some relatively narrow peaks arising from nonprotonated carbons. Resonances of heme carbons were detected in spectra of carbon monoxide hemoglobins, but not in spectra of ferrihemoglobin (as a result of paramagnetic effects). Spectra of carbon monoxide hemoglobins from various species yielded only a few well resolved individual carbon resonances, most notably those of Cgamma of tryptophan residues. A comparison of the spectra of human adult, human fetal, chicken AII, and bovine fetal hemoglobins yielded specific assignments for all resonances of Cgamma of tryptophan residues. In the cases of human fetal, chicken AII, and bovine fetal hemoglobins, each tryptophan yielded a completely resolved individual carbon resonance. The chemical shift difference between the resonances of Cgamma of Trp-130beta and Cgamma of Trp-37beta is about 6 ppm. The chemical shift difference between Trp A12[14]alpha and Trp A12[15]beta is 1 ppm or less. A comparison of the chemical shifts of analogous tryptophan residues of the four carbon monoxide hemoglobins suggests very similar conformations in solution.

Animals↗
PubMed1975

Studies of individual carbon sites of proteins in solution by natural abundance carbon 13 nuclear magnetic resonance spectroscopy. Relaxation behavior.

The aromatic regions in proton-decoupled natural abundance 13C Fourier transform nuclear magnetic resonance spectra (at 14.2 kG) of small native proteins contain broad methine carbon bands and narrow nonprotonated carbon resonances. Some factors that affect the use of natural abundance 13C Fourier transform NMR spectroscopy for monitoring individual nonprotonated aromatic carbon sites of native proteins in solution are discussed. The effect of protein size is evaluated by comparing the 13C NMR spectra of horse heart ferrocytochrome c, hen egg white lysozyme, horse carbon monoxide myoglobin, and human adult carbon monoxide hemoglobin. Numerous single carbon resonances are observed in the aromatic regions of 13C NMR spectra of cytochrome c, lysozyme, and myoglobin. The much larger hemoglobin yields few resolved individual carbon resonances. Theoretical and some experimental values are presented for the natural linewidths (W), spin-lattice relaxation times (T1), and nuclear Overhauser enhancements (NOE) of nonprotonated aromatic carbons and Czeta of arginine residues. In general, the 13C-1H dipolar mechanism dominates the relaxation of these carbons. 13C-14N dipolar relaxation contributes significantly to 1/T1 of C epsilon2 of tryptophan residues and Czeta of arginine residues of proteins in D2O. The NOE of each nonprotonated aromatic carbon is within experimental error of the calculated value of about 1.2. As a result, integrated intensities can be used for making a carbon count. Theoretical results are presented for the effect of internal rotation on W, T1, and the NOE. A comparison with the experimental T1 and NOE values indicates that if there is internal rotation of aromatic amino acid side chains, it is not fast relative to the over-all rotational motion of the protein.

Arginine↗
PubMed1975

Studies of individual carbon sites of proteins in solution by natural abundance carbon 13 nuclear magnetic resonance spectroscopy. Strategies for assignments.

Natural abundance 13C Fourier transform NMR spectra (at 15.18 MHz, in 20-mm sample tubes) of aqueous native proteins yield numerous narrow single carbon resonances of nonprotonated aromatic carbons. Techniques for the assignment of these resonances are presented. Each technique is applied to one or more of the following proteins: ferricytochrome c from horse heart and Candida krusei, ferrocytochrome c and cyanoferricytochrome c from horse heart, lysozyme from hen egg white, cyanoferrimyoglobins from horse and sperm whale skeletal muscle, and carbon monoxide myoglobin from horse. In all of the protein spectra we have examined, methine aromatic carbons give rise to broad bands. Studies of the narrow resonances of nonprotonated aromatic carbons of proteins are facilitated by removal of these broad bands by means of the convolution-difference method, preferably from spectra recorded under conditions of noise-modulated off-resonance proton decoupling. We present a summary of the chemical shift ranges for the various types of nonprotonated aromatic carbons of amino acid residues and hemes of diamagnetic proteins, based on our results for hen egg white lysozyme, horse heart ferrocytochrome c, horse carbon monoxide myoglobin, and carbon monoxide hemoglobins from various species...

Animals↗
PubMed1973

Cytochrome c: observation of numerous single-carbon sites of the reduced and oxidized species by means of natural-abundance 13C nuclear magnetic resonance spectroscopy.

Proton-decoupled, natural-abundance (13)C nuclear magnetic resonance spectra (obtained at 15.18 MHz by the Fourier transform method) of aqueous ferrocytochrome c, ferricytochrome c, and mixtures of both species were recorded. The 18 nonprotonated aromatic carbons of amino-acid residues and the 16 nonprotonated aromatic carbons of the heme yielded 22 narrow single-carbon resonances and 6 narrow two-carbon resonances in the spectrum of ferrocytochrome c. Only some of these resonances were detected in the spectrum of ferricytochrome c. Fast electron transfer between ferrocytochrome c and ferricytochrome c produced chemical exchange effects in spectra of mixtures of the two species: 16 nonprotonated aromatic carbons yielded narrow exchange-averaged resonances as a consequence of their small natural linewidths in both redox states and the small changes in their chemical shifts (relative to the reciprocal of the lifetime between electron exchange) when going from the reduced to the oxidized species. These peaks were assigned to carbons situated far from the iron atom. Their fast exchange behavior was used to establish a one-to-one correspondence between resonances in spectra of the two redox states. The other 18 nonprotonated aromatic carbons yielded exchange-broadened resonances as a consequence of large chemical-shift differences between the diamagnetic and paramagnetic species, and/or large paramagnetic broadening of the resonances of ferricytochrome c. We assigned these resonances (only one of which was identified in the spectrum of ferricytochrome c alone) to carbons that are near the iron atom: C(zeta) of Tyr 67, C(gamma) of His 18, and the 16 nonprotonated carbons of the porphyrin ring. Tentative specific assignments for C(zeta) of Tyr 67 (in the spectra of both redox forms) and for C(zeta) of His 18 (in the spectrum of ferrocytochrome c) are also presented.

Amino Acids↗