Simultaneous extraction of order parameters and orientational distribution fuctions from 31[superscript]P NMR spectra of magnetically partially oriented phospholipid bilayers

Order parameter profiles extracted from the NMR spectra of model membranes are a valuable source of information about their structure and molecular motions. To al1alyze powder spectra the de-Pake-ing (numerical deconvolution) ~echnique can be used, but it assumes a random (spherical) dist.ribution of orientations in the sample. Multilamellar vesicles are known to deform and orient in the strong magnetic fields of NMR magnets, producing non-spherical orientation distributions. A recently developed technique for simultaneously extracting the anisotropies of the system as well as the orientation distributions is applied to the analysis of partially magnetically oriented 31p NMR spectra of phospholipids. A mixture of synthetic lipids, POPE and POPG, is analyzed to measure distortion of multilamellar vesicles in a magnetic field. In the analysis three models describing the shape of the distorted vesicles are examined. Ellipsoids of rotation with a semiaxis ratio of about 1.14 are found to provide a good approximation of the shape of the distorted vesicles. This is in reasonable agreement with published experimental work. All three models yield clearly non-spherical orientational distributions, as well as a precise measure of the anisotropy of the chemical shift. Noise in the experimental data prevented the analysis from concluding which of the three models is the best approximation. A discretization scheme for finding stability in the algorithm is outlined

The structural effects of divalent cations and insulin on phospholipid model membranes /

It is well accepted that structural studies with model membranes are of considerable value in understanding the structure of biological membranes. Many studies with models of pure phospholipids have been done; but the effects of divalent cations and protein on these models would make these studies more applicable to intact membrane. The present study, performed with above view, is a structural analysis of divalent io~cardio1ipin complexes using the technique of x-ray diffraction. Cardiolipin, precipitated from dilute solution by divalent ionscalcium, magnesium and barium, contains little water and the structure formed is similar to the structure of pure cardiolipin with low water content. The calcium-cardiolipin complex forms a pure hexagonal type II phase that exists from 40 to 400 C. The molar ratio of calcium and cardiolipin in the complex is 1 : 1. Cardiolipin, precipitated with magnesium and barium forms two co-existing phases, lamellar and hexagonal, the relative quantity of the two phases being dependent on temperature. The hexagonal phase type II consisting of water filled channels formed by adding calcium to cardiolipin may have a remarkable permeability property in intact membrane. Pure cardiolipin and insulin at pH 3.0 and 4.0 precipitate but form no organised structure. Lecithin/cardiolipin and insulin precipitated at pH 3.0 give a pure lamellar phase. As the lecithin/cardiolipin molar ratio changes from 93/7 to SO/50, (a) the repeat distance of the lamellar changes from 72.8 X to 68.2 A; (b) the amount of protein bound increases in such a way that cardiolipin/insulin molar ratio in the complex reaches a maximum constant value at lecithin/cardiolipin molar ratio 70/30. A structural model based on these data shows that the molecular arrangement of lipid and protein is a lipid bilayer coated with protein molecules. The lipid-protein interaction is chiefly electrostatic and little, if any, hydrophobic bonding occurs in this particular system. So, the proposed model is essentially the same as Davson-Daniellifs model of biological membrane.

Comparative study of bovine heart and bacillus subtilis cytochrome c oxidase vesicles and the influence of bulk pH on cytochrome c oxidase components

Studies on the steady state behavior of soluble cytochrome c oxidase are extensive. These studies have examined the influence of ionic strength and pH and may provide answers to questions such as the link between proton translocation and charge separation. The present study examined the influence of external bulk pH on ApH formation, biphasic kinetics, and steady state reduction of cytochromes c and a of cytochrome c oxidase in proteoliposomes. Bulk pH has an appreciable effect on ApH formation and steady state reduction levels of cytochromes c and 8. Bulk pH affected total Vmax and Km at the low affinity binding site of cytochrome c. This study also examined the influence of bovine serum albumin and free fatty acids on proton pumping activity in bovine heart proteoliposomes. Proton pumping activity decreased after treatment with BSA, and was subsequently reinstated after further treatment with FFA. Much study in the superfamily of haem/copper oxidases has recently been devoted to the bacterial oxidases. The present study has examined some protein composition characteristics and bioenergetic features of Bacillus subtilis cytochrome caa3 oxidase. Results provide evidence for the structural composition of the enzyme in relation to the covalently bound cytochrome c to the oxidas~. Bioenergetically, caa3 COV showed appreciable proton pumping activity. Steady state analysis of the caa3 COV showed significantly different cytochrome c and a reduction characteristics compared to the bovine enzyme.

Measurement of repulsive forces between charged phospholipid bilayers

Electrostatic forces between membranes containing charged lipids were assumed to play an important role in influencing interactions between membranes long before quantitative measurements of such forces were available. ~ur measurements were designed to measure electrostatic forces between layers of lecithin charged with lipi~s carrying ionizable head groups. These experiments have shown that the interactions between charged lipid bila.yere are dominated by electrostatic forces only at separations greater than 30 A. At smaller separations the repulsion between charged bilayers is dominated by strong hydration forces. The net repulsive force between egg lecithin bilayers containing various amounts of cherged lipids (phosphatidylglycerol (PG) 5,10 ano 50 mole%, phosphatidyli. nosi tol (PI) 10 mole% and sodium oleate (Na-Ol) 3,5 and 10 mole%, where mole% gives the ratio of the number of moles' of .charged lipid to the total number of moles of all lipids present in the sample) was stuoied with the help ('If the osmotic streas technique described by LeNeveu et aI, (1977). Also, the forces between pure PG were j_nvestigated in the same manner. The results have been plotted showing variation of force as a function of bilay- _ er separation dw• All curVes 90 obtained called force curves, were found to be similar in sha.pe, showing two distinct regions, one when dw<.30 A is a region cf very rapid iiivariation of force with separation ( it is the region dominated by hydre,tion force) and second when dw> 40 A is a region of very slow variation of force with separB.tion ( it is the region dominated by the electrostatic force). Between these two regions there exists a transition area in which, in most systems studied, a phase separation of lipids into fractions containing different amounts of charged groups, was observed. A qualitative analysis showed that our results were v/ell described by the simple electrostatic double -le.yer theory. For quantitative agreement between measured and calculated force curves however, the charge density for the calculations had to be taken as half of that given by the number density of charged lipids present in the lecithin bilayers. It is not clear at the moment what causes such low apparent degree of ionization among the charged head groups, and further study is needed in this area.

The effects of gramicidin on the structure of phospholipid assemblies /

Gramicidin is an antibiotic peptide that can be incorporated into the monolayers of cell membranes. Dimerization through hydrogen bonding between gramicidin monomers in opposing leaflets of the membrane results in the formation of an iontophoretic channel. Surrounding phospholipids, with various associated mechanical properties, have been shown to influence the gating properties of this channel. Conversely, gramicidin incorporation has been shown to affect the structure of spontaneously formed lipid assemblies. Using small-angle x-ray diffraction and model systems composed of phospholipids and gramicidin, the physical effects incurred by gramicidin incorporation were measured. The reverse hexagonal (H^) phase composed of dioleoylphosphatidylethanolamine (DOPE) monolayers decreased in lattice dimension with increasing incorporation of gramicidin. This indicated that gramicidin was adding negative curvature to the monolayers. In this system, gramicidin was measured to have an apparent intrinsic radius of curvature (Rop*™") of -7. 1 A. The addition of up to 4 mol% gramicidin in mixtures with DOPE did not result in the monolayers becoming stiffer, as indicated by unaltered bending moduli for each composition. Dioleoylphosphatidylcholine (DOPC) alone forms the lamellar (LJ phase when hydrated, but undergoes a transition into the H^ phase when mixed with gramicidin. The lattice repeat dimension decreases systematically with increased gramicidin content. Again, this indicated that gramicidin was adding negative curvature to the monolayers. At 12 mol% gramicidin in mixtures with DOPC, the apparent radius of intrinsic curvature of gramicidin (Rop*"^) was measured to be -7.4 A. This mixture formed monolayers that were very resistant to bending under osmotic pressure, with a measured bending modulus of 1 15 kT. The measurements made in this study demonstrate that peptides are able to modulate the spontaneous curvature and other mechanical properties of phospholipid assemblies.

Mechanism of tocopherol transfer by human α-tocopherol transfer protein (α-hTTP)

Vitamin E is a well known fat soluble chain breaking antioxidant. It is a general tenn used to describe a family of eight stereoisomers of tocopherols. Selective retention of a-tocopherol in the human circulation system is regulated by the a -Tocopherol Transfer Protein (a-TIP). Using a fluorescently labelled a-tocopherol (NBD-a-Toc) synthesized in our laboratory, a fluorescence resonance energy transfer (FRET) assay was developed to monitor the kinetics of ligand transfer by a-hTTP in lipid vesicles. Preliminary results implied that NBD-a-Toe simply diffused from 6-His-a-hTTP to acceptor membranes since the kinetics of transfer were not responsive to a variety of conditions tested. After a series of trouble shooting experiments, we identified a minor contaminant, E coli. outer membrane porin F (OmpF) that co-purified with 6-His-a-hTTP from the metal affinity column as the source of the problem. In order to completely avoid OmpF contamination, a GST -a-hTTP fusion protein was purified from a glutathione agarose column followed by an on-column thrombin digestion to remove the GST tag. We then demonstrated that a-hTTP utilizes a collisional mechanism to deliver its ligand. Furthennore, a higher rate of a-tocopherol transfer to small unilamellar vesicles (SUV s) versus large unilamellar vesicles (LUV s) indicated that transfer is sensitive to membrane curvature. These findings suggest that ahTTP mediated a-Toc transfer is dominated by the hydrophobic nature of a-hTTP and the packing density of phospholipid head groups within acceptor membranes. Based on the calculated free energy change (dG) when a protein is transferred from water to the lipid bilayer, a model was generated to predict the orientation of a-hTTP when it interacts with lipid membranes. Guided by this model, several hydrophobic residues expected to penetrate deeply into the bilayer hydrophobic core, were mutated to either aspartate or alanine. Utilizing dual polarization interferometry and size exclusion vesicle binding assays, we identified the key residues for membrane binding to be F 165, F 169 and 1202. In addition, the rates of ligand transfer of the u-TTP mutants were directly correlated to their membrane binding capabilities, indicating that membrane binding was likely the rate limiting step in u-TTP mediated transfer of u-Toc. The propensity of u-TTP for highly curved membrane provides a connection to its colocalization with u-Toc in late endosomes.

Order and membrane organization in chlorhexidine-lipid mixtures /

Formulations of a general bactericidal agent, chlorhexidine, mixed with a phospholipid at different concentrations are investigated using ^H NMR spectroscopy on a chain-deuterated lipid analog. Lipid-chlorhexidine formulation is known to release the drug into an aqueous medium slowly, maintaining a comparable concentration of the drug for up to four times longer than a direct aqueous solution. The NMR data does not support the proposed liposomal entrapment of chlorhexidine in lipid compartments. Complex thermal history of the lipid-chlorhexidine preparations is investigated in detail. In preparation for a counterpart measurement, using ^H NMR of deuterated chlorhexidine mixed with protonated lipid, the synthesis of a deuterated analog of chlorhexidine is performed.

Skeletal muscle fibre-type comparison of whole tissue and subcellular membrane phospholipids and fatty acids

Membranes are dynamic structures that affect cell structure and function. Compositional changes ofmembranes have been shown with the application of a perturbation; however these are limited to whole tissue analysis. The purpose of this thesis was to compare the phospholipid (PL) fatty acid (FA) composition of rat whole muscle (Wm) to 1) purified and non-purified subsarcolemmal (SS) mitochondria in soleus, plantaris, and red gastrocnemius, and 2) sarcolemma, transverse-tubules, SS and intermyofibrillar (IMF) mitochondria fix)m whole hindlimb. The major findings were that 1) contamination significantly altered the PL FA composition of the SS mitochondrial membrane fraction, 2) Wm and SS mitochondria compositions differed between muscle types, and 3) Wm did not accurately reflect the PL FA composition of any isolated subcellular membranes, with each being unique from each other. As such, the relevancy of the trends reported in the literature of the effects of perturbations on Wm may be limited.

Heterogeneity of photosystem II as it occurs in domain specific regions of the thylakoid membrane of spinach (spinacia oleracia L.)

Thylakoid membrane fractions were prepared from specific regions of thylakoid membranes of spinach (Spinacia oleracea). These fractions, which include grana (83), stroma (T3), grana core (8S), margins (Ma) and purified stroma (Y100) were prepared using a non-detergent method including a mild sonication and aqueous two-phase partitioning. The significance of PSlla and PSII~ centres have been described extensively in the literature. Previous work has characterized two types of PSII centres which are proposed to exist in different regions of the thylakoid membrane. a-centres are suggested to aggregate in stacked regions of grana whereas ~-centres are located in unstacked regions of stroma lamellae. The goal of this study is to characterize photosystem II from the isolated membrane vesicles representing different regions of the higher plant thylakoid membrane. The low temperature absorption spectra have been deconvoluted via Gaussian decomposition to estimate the relative sub-components that contribute to each fractions signature absorption spectrum. The relative sizes of the functional PSII antenna and the fluorescence induction kinetics were measured and used to determine the relative contributions of PSlla and PSII~ to each fraction. Picosecond chlorophyll fluorescence decay kinetics were collected for each fraction to characterize and gain insight into excitation energy transfer and primary electron transport in PSlla and PSII~ centres. The results presented here clearly illustrate the widely held notions of PSII/PS·I and PSlIa/PSII~ spatial separation. This study suggests that chlorophyll fluorescence decay lifetimes of PSII~ centres are shorter than those of PSlIa centres and, at FM, the longer lived of the two PSII components renders a larger yield in PSlIa-rich fractions, but smaller in PSIlr3-rich fractions.

Proton translocation by cytochrome c oxidase reconstituted into proteoliposomes

Cytochrome c oxidase .inserted into proteoliposomes translocates protons with a stoichiometry of approx-, imately 0.4-0.6 H+/e- in the presence of valinomycin plus pottasium. The existance .ofsuchproton translocation is .supportedby experiments with lauryl maltoside which abolished the pulses but~~d not inhibit cyt. c binding .or oxidase turnover. Pulses with K3FeCN6 did not induce acidification further supporting vectorial proton transport by cyt ..aa3 . Upon lowering the ionic strength and pulsing with ferrocytochrome c, H+/eratios increased. This increase is attributed to scaler proton release consequent upon cyt.c-phospholipid binding. Oxygen pulses at low ionic strength however did not exhibit this large scaler increase in H+/e- ratios.A-small increase was observed upon .02 pul'sing at·low ionic strengt.h. This increase was KeN and, ,pcep sensitive and thus possibly due to a redox linked scaler deprotonation. Increases in the H+/e- ratio also occurred ifp~lses ,were performed in the presence of nonactin rather.than valinomycin. The fluorescent pH indicator pyranine was internally trapped inaa3 conta~ning "proteoliposomes. Internal alkalinization, as mon,itored by pyranine fluorescence leads to a of approx.imately 0.35 units, which is proportional to electron flux. This internal alkalinization was also DCCD sensitive, being inhibited by approximately 50%. This 50% inhibition of internal alkalinization supports the existance of vectorial proton transport.

Structural effects of neutral lipids on the divalent cation-induced interactions of phosphatidylserine - containing bilayers

As Ca2+ and phosphatidylserine (PS) are known to induce the adhesion of bilayer vesicles and form collapsed multibilayer structures in vitro, it was the aim of this study to examine how that interaction and the resultant structures might be modified by neutral lipid species. X-ray diffraction data from multilamellar systems suggest that phosphatidylcholine (PC) and diacylglycerol (DG) might be in the collapsed phase up to a concentration of -30 mole % and that above this concentration these neutral lipids may modify Ca2+-induced bilayer interactions. Using large unilamellar vesicles and long incubations in excess Ca2+ to ensure equilibration, similar preliminary results were again obtained with PC, and also with phosphatidylethanolamine (PE). A combination of X-ray diffraction, thin-layer chromatography, density gradient centrifugation and freeze-fracture electron microscopy, used in conjunction with an osmotic stress technique, showed that (i) -30 mole % PC can be accomodated in the Ca(DOPS)2 phase; and (ii) higher PC levels modify Ca2+-induced bilayer interactions resulting in single lamellar phases of larger dimension and reduced tendency for REV collapse. Importantly, the data suggest that PC is dehydrated during the rapid collapse process leading. to Ca(DOPS)2 formation and exists with this dehydrated phase. Similar results were obtained using PS isolated from bovine brain. Preliminary studies using two different phosphatidylethanolamine (PE) species indicated accomodation by Ca(DOPS)2 of -25-30 mole 0/0 PE and bulk phase separation, of species favouring a non-bilayer phase, at higher levels. Significantly, all PS/PE vesicles appear to undergo a complete Ca2+-induced collapse, even with contents of up to 90 mole % PE. These data suggest that PE may have an important role in fusion mechanisms in vivo. In sum the data lend both structural and stoichiometric evidence for th~ existence of laterally segregated neutral lipid molecules within the same bilayers as PS domains exposed to Ca2+.

The pancreatic islet system of the rock bass /|nby Edward Francis Squires. -- 260 St. Catharines, Ont. : [s. n.],

The endocrine pancreas of the rock bass (Ambloplites rupestris) was examined by light and electron microscopy. Two cell types with staining properties similar to mammalian A and B cells, and a third, non-staining cell type were found in the spherical pancreatic islets that were surrounded by a connective tissue capsule and embedded in two small masses of exocrine tissue. From an analysis of the ultrastructure of the A and B cells, a secretory cycle for each of these cell types was proposed. The secretory cycle of the A cell consisted of three well defined stages: (1) A cell production stage: during which A granule formation occurred in the sacs of the Golgi apparatus and the cell was characterized by the presence of numerous secretory granules, some elements of lamellar endoplasmic reticulum, and a homogeneously granular nucleus. The cytoplasm contained few distended cisternae, variable numbers of free ribosomes, microtubules and small vesicles. (2) A cell release stage: during which the release of A granules occurred and the cell usually contained several large distended cisternae and variable numbers of secretory granules. Granule release mechanisms included exocytosis, by which individual granules were released into the extracellular space after their membranes fused with the plasmalemma, and emiocytosis, by which one or more granules were released into a large cisterna whose membrane fused with the plasmalemma and formed a pore through which the cisternal contents passed out of the cell. (3) A cell reorganization stage: during which the changeover from the release stage to the production stage occurred and the reorganization of organelles and membrane structures took place. The cell contained few secretory granules and numerous small endoplasmic reticular cisternae. The cytoplasm exhibited less electron density than either of the other two stages. The A granule after formation underwent a series of morphological changes which were described in four numerically identified phases. The secretory cycle of the B cell consisred of two stages: (1) B cell production stage: during which the B granule formation occurred in the sacs of the Go1gi apparatus. The cell was characterized by an irregular outline, the presence of numerous secretory granules, and an irregularly shaped nucleus which contained variable amounts of clumped chromatin. The cytoplasm contained moderate amounts of lamellar endoplasmic reticulum studded with ribosomes, several small vesicles, and an active Go1gi apparatus. (2) B cell release stage: during which the release of B granules occurred. The cell contained a rounded nucleus with dispersed chromatin, several distended endoplasmic reticular cisternae and a variable number of secretory granules. Granule release occu~ by emiocytosis and exocytosis similar to that found for the A cell.

The effect of lysobisphosphatidic acid (LBPA) on α-tocopherol transfer protein (α-TTP) binding to lipid membranes

The a-tocopherol transfer protein (a-TTP) is responsible for the retention of the atocopherol form of vitamin E in living organisms. The detailed ligand transfer mechanism by a-TTP is still yet to be fully elucidated. To date, studies show that a-TTP transfers a-tocopherol from late endosomes in liver cells to the plasma membrane where it is repackaged into very low density lipoprotein (VLDL) and released into the circulation. Late endosomes have been shown to contain a lipid known as lysobisphosphatidic acid (LBP A) that is unique to this cellular compartment. LBPA plays a role in intracellular trafficking and controlling membrane curvature. Taking these observations into account plus the fact that certain proteins are recruited to membranes based on membrane curvature, the specific aim of this project was to examine the effect of LBP A on a-TTP binding to lipid membranes. To achieve this objective, dual polarization interferometry (DPI) and a vesicle binding assay were employed. Whilst DPI allows protein binding affinity to be measured on a flat lipid surface, the vesicle binding assay determines protein binding affinity to lipid vesicles mimicking curved membranes. DPI analysis revealed that the amount of a-TTP bound to lipid membranes is higher when LBPA is present. Using the vesicle binding assay, a similar result was seen where a greater amount of protein is bound to large unilamellar vesicles (LUV s) containing LBP A. However, the effect of LBP A was attenuated when small unilamellar vesicles (SUVs) were replaced with LUVs. The outcome of this project suggests that aTTP binding to membranes is influenced by membrane curvature, which in turn is induced by the presence of LBP A.

Sarcolemmal lipid analysis from mechanically skinned rat muscle fibres.

Membrane lipid composition, which includes phospholipid (PL) headgroup, and fatty acid (FA) saturation, has been shown to affect cellular function. The sarcolemma (SL) membrane is integral to skeletal muscle function and health. Previous studies assessing SL lipid composition are limited as they have 1) restricted analysis to a PL level and neglected FA composition and 2) relied on aggressive membrane isolation procedures resulting in t-tubule and sarcoplasmic reticulum contamination and unknown levels of nuclear and mitochondrial contamination. Thus, to overcome these limitations, this study assessed a method of individually skinned skeletal muscle fibres as an alternative to analyze complete sarcolemmal membrane lipid composition. The major findings of this study were 1) complete SL lipid composition can be obtained 2) the SL had higher sphingomyelin content than previous studies and 3) the SL membrane had minimal nuclear and mitochondrial contamination and was void of contamination from sarcoplasmic reticulum and t-tubules.