X-ray diffraction studies of oriented dilauroyl phosphatidylcholine bilayers in the L delta and L alpha phases.

X-ray diffraction studies on oriented multilayers of dilauroyl phosphatidylcholine in the lyotropic liquid-crystalline L alpha phase and a not previously reported mono-domain three-dimensional L delta phase at two temperatures (293 and 343 K) and various relative humidities (0-100%) are described. Absolute one-dimensional electron-density profiles of the different structural phase bilayers were constructed to a resolution of 4 A using direct methods (e.g. swelling and triplet structure-invariant relationships) to solve for the phase problem. The absolute electron-density distributions clearly demonstrate differences between the two structural phases of dilauroyl phosphatidylcholine bilayers. In addition, the various structural properties of the two different phases have been quantified. In the case of the L delta phase, the structural quantities (e.g. volumes of the terminal methyl group and headgroup, and the number of waters) are examined for the first time.

Constant helical pitch of the gramicidin channel in phospholipid bilayers.

X-ray diffraction has been applied in measuring the helical pitch of the gramicidin channel in oriented bilayers of dilauroylphosphatidylcholine (DLPC) and dimyristoylphosphatidylcholine (DMPC) at a polypeptide concentration of 9.1 mol %. The diffraction data show the helical pitch of gramicidin to be 4.7 +/- 0.2 A in both gel and liquid-crystalline phase bilayers, with and without monovalent cations. In addition, the width of the reflection due to the pitch of the helical gramicidin channel is consistent with a five turn helix.

Location of two antioxidants in oriented model membranes. Small-angle x-ray diffraction study.

Small-angle x-ray diffraction has been applied in locating either butylated hydroxytoluene (BHT) or delta-tocopherol and their brominated analogues at a concentration of 40 mol% in oriented bilayers of dipalmitoylphosphatidylcholine (DPPC) or DPPC + 15 mol% cholesterol at 20 degrees C. Phases were determined using swelling experiments with structure factors plotted in reciprocal space, creating a relatively smooth curve as the amount of water between the bilayers was changed. Continuous Fourier transforms were also calculated using sampling theory (Shannon, C. E. 1949. Proc. Inst. Radio Engrs. NY. 37:10-21) to further test the consistency of the phase assignments. Fourier synthesis of structure factors resulted in absolute electron density profiles for different bilayers to a resolution of 5-6 A. In addition, difference Patterson maps were constructed to confirm the positions of the bromine atoms in the unit cell. Analysis of the data indicates the following: (a) The BHT molecules are dispersed throughout the alkyl-chain region in DPPC samples with and without cholesterol. (b) The chromanol ring of delta-tocopherol is in the vicinity of the glycerol backbone-headgroup region in samples of DPPC or DPPC + 15 mol% cholesterol. (c) Difference Patterson maps confirm the localization of bromine atoms in the various delta-tocopherol samples and lack of bromine localization in the various BHT samples.

High-resolution electron density profiles reveal influence of fatty acids on bilayer structure.

Small-angle x-ray diffraction studies were performed on gel phase-oriented bilayers of dipalmitoylphosphatidylcholine (DPPC) and DPPC containing 40 mol% of either palmitic acid (PA) or palmitic acid brominated at the 2-position (BPA). Oriented samples were prepared using a method developed by us, which is as simple as powder sample preparations while offering all the advantages of oriented samples made by traditional methods. Phases were determined using swelling experiments with structure factors plotted in reciprocal space, creating a relatively smooth curve as the amount of water between the bilayers was changed. Continuous Fourier transforms were also calculated to further test the consistency of the phase assignments. The diffraction data were used to calculate absolute electron density profiles for different bilayers to a resolution of 5-6 A. Analysis indicates the following: (a) The electron density profiles for the three preparations are virtually identical in the hydrocarbon chain region. (b) There is a decrease in the electron density of the glycerol backbone-headgroup region and d-space in DPPC-PA compared to DPPC. (c) The bromine of fatty-acid brominated at the 2-position is in the vicinity of the glycerol backbone. (d) The bilayer thickness of DPPC containing either brominated or unbrominated fatty acid remains relatively constant with increased levels of hydration, unlike DPPC bilayers.

Palmitoyl carnitine can exist in lamellar and hexagonal phases.

Carnitine is a necessary cofactor for the transport of fatty acids across the inner mitochondrial membrane. The behaviour in a buffer solution of the acyl carnitine, palmitoyl carnitine, has been investigated. Palmitoyl carnitine is amphiphilic and resembles, in some structural respects, a lysolecithin. X-ray diffraction studies showed that palmitoyl carnitine in buffer at a low temperature formed a gel-phase bilayer with fully interdigitated hydrocarbon chains perpendicular to the bilayer surface. The bilayer thickness increased slightly as water content increased due to a reorientation of the carnitine portion. As temperature was increased a transition occurred to a hexagonal (H1) phase with buffer solution separating cylinders of palmitoyl carnitine. The transition occurred over a broad temperature range of approximately 20 degrees C.

Antioxidant levels in germinating soybean seed axes in relation to free radical and dehydration tolerance.

The axis of soybean seeds suffer dehydration injury if they are dried to 10% moisture at 36 hours of imbibition, but tolerate this stress if dried at 6 hours of imbibition. Deesterification of membrane phospholipids has been correlated with the increased permeability and increased lipid phase transition temperatures of membranes from dehydration injured tissues. Deesterification, measured as increased free fatty acid:phospholipid and decreased phospholipid:sterol ratios, occurred primarily when the tissue was in the dry state and did not change significantly (P

Simulation of dehydration injury to membranes from soybean axes by free radicals.

Smooth microsomal membranes were isolated from axes of soybean (Glycine max L. Merr.) seeds at the dehydration-tolerant (6 hours of imbibition) and dehydration-susceptible (36 hours of imbibition) stages of development and were exposed to free radicals in vitro using xanthine-xanthine oxidase as a free radical source. Wide angle x-ray diffraction studies indicated that the lipid phase transition temperature of the microsomal membranes from the dehydration-tolerant axes increased from 7 to 14 degrees C after exposure to free radicals, whereas those from the dehydration-susceptible axes increased from 9 to 40 degrees C by the same free radical dose. The increased phase transition temperature was associated with a decrease in the phospholipid:sterol ratio, and an increase in the free fatty acid:phospholipid ratio. There was no significant change in total fatty acid saturation, which indicated that free radical treatment induced deesterification of membrane phospholipid, and not a change in fatty acid saturation. Similar compositional and structural changes have been previously observed in dehydration-injured soybean axes suggesting that dehydration may induce free radical injury to cellular membranes. Further, these membranes differ in their susceptibility to free radical injury, presumably reflecting compositional differences in the membrane since these membranes were exposed to free radicals in the absence of cytosol.

Association between Membrane Phase Properties and Dehydration Injury in Soybean Axes.

Axes of soybean seeds are tolerant to dehydration at 6 hours of imbibition, but susceptible to dehydration injury if dried at 36 hours of imbibition. Smooth microsomal membranes were isolated from axes imbibed for 6 hours (dehydration tolerant state) and 36 hours (dehydration susceptible state) before and after dehydration treatment. The phase properties and the lipid composition of the membrane fraction were investigated. Wide angle x-ray diffraction patterns of microsomal membranes from axes imbibed for 6 or 36 hours indicated a liquid-crystalline to gel phase transition at approximately 7 degrees C. Membranes from axes dehydrated at 6 or 36 hours of imbibition and rehydrated for 2 hours exhibited a phase transition at 7 degrees C and 47 degrees C, respectively. Changes in fatty acid saturation did not account for the changes in phase properties. However, the increased phase transition temperature of the membranes from dehydration injured axes was associated with an increase in free fatty acid:phospholipid molar ratio and a decrease in phospholipid:sterol ratio. These results suggests that dehydration prompted a deesterification of the linkage between glycerol and fatty acid side chains of the phospholipid molecules in the membrane. The resultant increase in free fatty acid content in the membrane is thought to alter the fluidity and phase properties of the membrane and contribute to dehydration injury.

X-ray diffraction from striated muscles and nerves in normal and dystrophic mice.

The structure of striated muscle (thick and thin filaments, filament lattice, and collagen), peripheral nerve myelin, and tendon collagen were studied in tissues from dystrophic and normal mice using small-angle x-ray diffraction. There were increases in the amount of disorganized tissue in the dystrophic mice, and the time course of the changes was monitored over the first 42 weeks of life. As the dystrophic mice became older, the contractile apparatus of the muscles appeared to atrophy, while the amount of collagen increased. In general, the molecular structure and packing appeared to remain unchanged as the disease progressed, although changes in the relative amounts and the organization of proteins were noted. In both normal and dystrophic mice, the collagen periodicity (65.7 nm) was 2% smaller when detected in muscle tissue compared with that detected in tendon tissue.

Electron density distribution of dry avian tendon.

The electron density profile along the unit cell of dry native collagen is determined. The method used is a modification of one that has been used successfully for wet native collagen. Uranyl acetate and phosphotungstic acid were used for isomorphous addition in phase determination and were located by electron microscopy. Structure factor magnitudes for dry collagen with and without the heavy metals were obtained from X-ray diffraction data. The first 9 orders were investigated. Standard Argand diagrams provided a unique solution for the phase of each. The resultant profile suggests that significant conformation changes occur in both the non-helical ends and the triple-helix portions of the molecules.

Effect of Dehydration on Leakage and Membrane Structure in Lotus corniculatus L. Seeds.

Membrane damage as a result of dehydration was studied in Lotus corniculatus L. cv. Carroll seeds which had been pregerminated for 0, 12, and 24 hours prior to dehydration. During reimbibition, desiccation-tolerant (0- and 12-hour) seeds leaked relatively low quantities of all solutes (total electrolytes, potassium, phosphate, sugar, amino acid, and protein). Desiccation-sensitive (24-hour) seeds leaked higher levels, but evidence of selective permeability remained. Membrane damage was not manifested as a complete removal of the diffusion barrier, although its permeability properties were dramatically altered. Consequently, the plasmalemma was not ruptured or torn by the dehydration treatment, but a more subtle structural alteration occurred.The possibility that seed membranes form a hexagonal rather than a lamellar phase at moisture contents below 20% was investigated by x-ray diffraction. Phospholipids were extracted from desiccation-tolerant (0-hour) and desiccation-sensitive (24-hour) seeds and hydrated to 5, 10, 20, and 40% water. This phospholipid-water system was examined using low-and wide-angle x-ray diffraction and was found to be exclusively lamellar, even at 5% water. Consequently, membrane damage and the leakage of cytoplasmic solutes from seeds cannot be explained by the formation of a hexagonal phase by membrane phospholipids.

Skin collagen has an unusual d-spacing.

Small angle X-ray diffraction patterns show a d-spacing of 65 +/- 0.5 nm for the collagen in wet intact skin of amphibian larvae (tadpole) as well as from that of nature frogs, chickens and mice. The collagen in these tissues is largely collagen I which exhibits a d-spacing of 67 +/- 0.5 nm in wet intact unstretched tendons. The d-spacing of the skin collagen did not decrease on drying, while it is well known that that of tendon collagen does decrease on drying. The reasons for the decreased d-spacing in the normal skin are not known but we suggest that the different glycosaminoglycan content of skin may be an important factor.

Experimental confirmation of calculated phases and electron density profile for wet native collagen.

An experimental procedure is developed to phase the reflections obtained in x-ray diffraction investigations of collagen in native wet tendons. Phosphotungstic acid was used for isomorphous addition in phase determination and was located by electron microscopy. Structure factors (with phases) were obtained from the electron microscopy data for the heavy metal. Structure-factor magnitudes for collagen with and without the heavy metal were obtained from the x-ray diffraction data. The first 10 orders were investigated. Standard Argand diagrams provided two solutions for each of these, except the weak sixth order. In each case, one of the two possible solutions agrees well with the phases proposed on theoretical grounds by Hulmes et al. The present results suggest that their other proposed phases are probably correct. An electron density profile along the unit cell of the fibril is presented that shows a distinct step, as expected on the basis of the hole-overlap model. The overlap region is 48% of the length of the unit cell.

Structural deterioration of tendon collagen in genetic muscular dystrophy.

The structure of gastrocnemius tendons from chickens with genetically induced muscular dystrophy has been studied by low-angle X-ray diffraction. Compared with normal samples there is poor alignment of collagen within the tendons. This difference is quite pronounced at eight weeks when the affected birds are still in comparatively good physical condition. Similar changes have been reported for birds with nutritionally induced muscular dystrophy (Bartlett, M. W., Egelstaff, P. A., Holden, T. M., Stinson, R. H. and Sweeny, P. R. (1973) Biochim. Biophys. Acta 328, 213-220).