Biomedical subjects
Publications and source records attributed to J Seelig.
Explore the source record for details and available documents.
The crystalline lipovitellin/phosvitin complex has a molecular weight of 456 000 and contains nearly 100 molecules of bound phospholipid. Earlier work using electron microscopy and three-dimensional image reconstruction methods established the symmetrical dimeric nature of this lipoprotein, but the organization of the lipid was unknown. Under conditions where the lipoprotein is in solution, the high-resolution 31P nuclear magnetic resonance (NMR) spectra contain two well-resolved peaks which can be assigned to phosphoserine moieties in both lipovitellin and phosvitin and to the phospholipid microdomains. The spin-lattice relaxation times, T1, for the phosphoserines and the phospholipid head groups are distinctly different, with the serine phosphates having faster reorientation rates. 31P NMR spectra of crystalline lipoprotein contain a broad symmetric component with a chemical shielding anisotropy of about -50 ppm. By obtaining 31P MNR spectra from several modified forms of the lipoprotein as well as from the extracted lipids, it is shown that the chemical shielding anisotropy is characteristic of phospholipid in a bilayer arrangement. As a further check on 31P NMR results, samples of the lipovitellin/phosvitin complex containing deuterium-labeled 1,2-dielaidoyl-sn-glycero-3-phosphocholine were studied by 2H NMR methods. The resulting spectrum has characteristics similar to those obtained from model lipid systems in a lamellar state. The results of the 31P and 2H NMR experiments correlate with the low-resolution model of the crystalline lipovitellin complex obtained by diffraction studies. It is proposed that each subunit of lipovitellin contains a microdomain of phospholipid in a bilayer-like arrangement.
Lipid/protein and lipid/lipid interactions in the yolk lipoprotein complex from Xenopus laevis were examined by introducing a series of lipid spin-labels into the complex and observing the electron spin resonance spectra as a function of the position of the label along the lipid chains, temperature, pH, and charge on the lipid polar head group. Analyses of the spectra show that, in addition to the expected component arising from lipid associated with protein, a second component with increased segmental flexibility and the greater temperature dependence characteristic of lipid/lipid interactions is observed. These spin-labeling data and supporting compositional data indicate that much of the lipid is organized into a lipid-rich region or pool, consistent with the earlier model derived from electron microscopy and diffraction data and with companion 31P and 2H nuclear magnetic resonance data reported in the preceding paper [Banaszak, L. J., & Seelig, J. (1982) Biochemistry (preceding paper in this issue)]. The bilayer-like component exhibits a greater restriction of motion compared to vesicles of the isolated lipids at the same temperature, as would be expected for a relatively small lipid pool. Phospholipids exchange between the two motionally distinguishable environments. The equilibrium binding undergoes a shift between these two environments as a function both of pH and of the charge on the phospholipid polar head group. This shift in average binding affinity is opposite in direction to that reported for membrane proteins and implicates negatively charged groups on the protein that repel negatively charged phospholipids. This effect is greatly reduced by alkaline phosphatase treatment, suggesting that some of the lipid binding sites are in close proximity to phosphorylated residues on the protein.
Explore the source record for details and available documents.
Explore the source record for details and available documents.
Glycerol selectively deuterated at various positions was synthesized and supplied to the growth medium of Escherichia coli strain T131 GP, which is defective in endogenous glycerol synthesis as well as glycerol degradation and lacks the ability to synthesize cardiolipin. The procedure enables the stereospecific labeling of the membrane phospholipids (approximately 80% phosphatidylethanolamine, approximately 20% phosphatdylglycerol). Deuterium magnetic resonance spectra were obtained for cell membranes and lipid dispersions either from total lipid extractions or from purified phosphitidylglycerol or -ethanolamine. When glycerol deuterated at various positions was used, all resonances of the phospholipid glycerol backbone and the terminal glycerol moiety in phosphatidylglycerol could be assigned. The results indicate that the molecular conformation of the glycerol backbone is independent of the phospholipid species investigated and is also not altered by the presence of high amounts of membrane proteins. For the quantitative interpretation of the deuterium magnetic resonance splittings, a model is proposed which assumes essentially free rotation around the glycerol C(2)-C(3) bond combined with an asymmetric and restricted jump process around the C(1)-C(2) bond. This model is compatible with known X-ray structures of phospholipids molecules. The two deuterons of both the glycerol backbone C(1) and C(3) segments were found to be magnetically inequivalent. Stereoselective monodeuteration eliminated one set of quadrupole splittings in both cases.
The two-subunit cytochrome c oxidase from Paracoccus denitrificans contains two heme a groups and two copper atoms. However, when the enzyme is isolated from cells grown on a commonly employed medium, its electron paramagnetic resonance (EPR) spectrum reveals not only a Cu(II) powder pattern, but also a hyperfine pattern from tightly bound Mn(II). The pure Mn(II) spectrum is observed at -40 degrees C; the pure Cu(II) spectrum can be seen with cytochrome c oxidase from P. denitrificans cells that had been grown in a Mn(II)-depleted medium. This Cu(II) spectrum is very similar to that of cytochrome c oxidase from yeast or bovine heart. Manganese is apparently not an essential component of P. denitrificans cytochrome c oxidase since it is present in substoichometric amounts relative to copper or heme a and since the manganese-free enzyme retains essentially full activity in oxidizing ferrocytochrome c. However, the manganese is not removed by EDTA and its EPR spectrum responds to the oxidation state of the oxidase. In contrast, manganese added to the yeast oxidase or to the manganese-free P. denitrificans enzyme can be removed by EDTA and does not respond to the oxidation state of the enzyme. This suggests that the manganese normally associated with P. denitrificans cytochrome c oxidase is incorporated into one or more internal sites during the biogenesis of the enzyme.
For confirmation of some general aspects of phospholipid conformation in membranes and extension of previous neutron diffraction studies on dipalmitoyllecithin, measurements have now been made on 1,2-dipalmitoyl-sn-glycero-3-phosphoethanolamine (DPPE) in the gel phase by the same method. Three selectively deuterated samples were investigated; in one of the specimens the first methylene segment close to the glycerol backbone in both chains was deuterated, and in the other two samples one of the methylene segments in the phosphoethanolamine group was replaced by CD2. Together with the undeuterated DPPE, these probes were investigated at very low water content (about 1.5--2 molecules of water per lipid) as oriented samples at 25 degrees C. The intensities of the first 12 reflections were collected and phased, and the mean positions of the segments were determined. The results confirm the idea that the conformation of a DPPE molecule in the gel state is very similar to the crystal structure of rac-1,2-dilauroyl-sn-glycero-3-phosphoethanolamine. The two main features are (1) the chains remain in all all-trans conformation having an axial displacement of about 3--4 A, (2) the zwitterionic dipoles in the head groups of both compounds are found to be aligned almost parallel to the bilayer surface. The main advantage of the method results in the fact that the combination of neutron scattering with selectively deuterated probes allows the determination of the mean label position to an accuracy of up to +/- 1 A.
The structural properties of the phosphoglycerol polar head group in bilayers of 1,2-dipalmitoyl-sn-glycero-3-phosphoglycerol have been studied with deuterium and phosphorus-31 nuclear magnetic resonance. For this purpose, 1,2-dipalmitoyl-sn-glycero-3-phosphoglycerol was deuterated chemically or biosynthetically at all three segments of the glycerol head group. Both the D and the L configurations of the glycerol head group have been synthesized, and the correct stereochemical configuration of the polar group was verified by an enzymtic assay, as direct measurement of the optical rotation was insufficiently sensitive to distinguish between these diastereomers. The phosphatidylglycerol sodium salts were dispersed in excess buffer (0.1 M NaCl, pH 7.0), and the bilayer properties were characterized by small-angle X-ray diffraction and differential scanning calorimetry. The deuterium quadrupole splittings, delta vQ, and the phosphorus-31 chemical shielding anisotropy, delta sigma, were measured as a function of temperature in the range 20-60 degrees C, yielding the following results. (1) Well-resolved deuterium signals were obtained for all head-group segments and could be assigned unambiguously. (2) Almost identical spectra were obtained for 1,2-dipalmitoyl-sn-glycero-3-phospho-1'-glycerol (natural L,D configuration) and 1,2-dipalmitoyl-sn-glycero-3-phospho-3'-glycerol (L,L configuration), suggesting very similar head-group motions and orientations for both diastereomers. (3) The spatial anisotropy of motion and the segmental fluctuations of the negatively charged phosphoglycerol are similar to those of the zwitterionic phosphocholine and phosphoethanolamine head groups but differ distinctly from those of phosphoserine which also carries a net negative charge. (4) The motional inequivalence of geminal deuterons in 1,2-dipalmitoyl-sn-glycero-3-phospho-3'-glycerol was demonstrated by synthesis of a stereospecifically monolabeled analogue.
Explore the source record for details and available documents.
Explore the source record for details and available documents.
Explore the source record for details and available documents.
Explore the source record for details and available documents.
Explore the source record for details and available documents.
Explore the source record for details and available documents.
Explore the source record for details and available documents.
Explore the source record for details and available documents.
Explore the source record for details and available documents.