In a range of low ionic strength, aqueous dispersions of the anionic phospholipid DMPG (dimyristoylphosphatidylglycerol) have a transparent intermediate phase (IP, between T(m)(on) congruent with 20 degrees C and T(m)(off) congruent with 30 degrees C) between the turbid gel and fluid membrane phases, evidenced in turbidity data. Small angle x-ray scattering results on DMPG dispersions show that, besides the bilayer peak present in all phases, a peak corresponding to a mesoscopic structure at approximately 400 A is detected only in IP. The dependence of this peak position on DMPG concentration suggests a correlation in the bilayer plane, consistent with the stability of vesicles in IP. Moreover, observation of giant DMPG vesicles with phase contrast light microscopy show that vesicles "disappear" upon cooling below T(m)(off) and "reappear" after reheating. This further proves that although vesicles cannot be visualized in IP, their overall structure is maintained. We propose that the IP in the melting regime corresponds to unilamellar vesicles with perforations, a model which is consistent with all described experimental observations. Furthermore, the opening of pores across the membrane tuned by ionic strength, temperature, and lipid composition is likely to have biological relevance and could be used in applications for controlled release from nanocompartments.

Dimyristoylphosphatidylglycerol (DMPG) has been extensively studied as a model for biological membranes, since phosphatidylglycerol is the most abundant anionic phospholipid in prokaryotic cells. At low ionic strengths, this lipid presents a peculiar thermal behavior, with two sharp changes in the light scattering profile, at temperatures named here T(on)(m) and T(off)(m). Structural changes involved in the DMPG thermal transitions are here investigated by small angle X-ray scattering (SAXS), and compared to the results yielded by differential scanning calorimetry (DSC) and electron spin resonance (ESR). The SAXS results show a broad peak, indicating that DMPG is organized in single bilayers, for the range of temperature studied (10-45 degrees C). SAXS intensity shows an unusual effect, starting to decrease at T(on)(m), and presenting a sharp increase at T(off)(m). The bilayer electron density profiles, obtained from modeling the SAXS curves, show a gradual decrease in electron density contrast (attributed to separation between charged head groups) and in bilayer thickness between T(on)(m) and T(off)(m). Results yielded by SAXS, DSC and ESR indicate that a chain melting process starts at T(on)(m), but a complete fluid phase exists only for temperatures above T(off)(m), with structural changes occurring at the bilayer level in the intermediate region.

A study has been made of e.p.r. signals due to Mo(V) in reduced sulphite oxidase (EC 1.8.3.1) from chicken liver. Reduction by SO3(2-), or photochemically in the presence of a deazaflavin derivative, produces spectra indistinguishable from one another. Three types of spectra from the enzyme were distingusihed and shown to correspond to single chemical species, since they could be simulated at both 9 and 35 GHz by using the same parameters. These were the low-pH form of the enzyme, with gav. 1.9805, the high-pH form, with gav. 1.9681 and a phosphate complex, with gav. 1.9741. The low-H form shows interaction with a single exchangeable proton, with A(1H)av. (hyperfine coupling constant) = 0.98 mT, probably in the form of an MoOH group. Parameters of the signals are compared with those for signals from xanthine oxidase and nitrate reductase. The signal from the phosphate complex of sulphite oxidase in unique among anion complexes of Mo-containing enzymes in showing no hyperfine coupling to protons. There is no evidence for additional weakly coupled protons or nitrogen nuclei in the sulphite oxidase signals. The possibility is considered that the enzymic mechanism involves abstraction of a proton and two electrons from HSO3- by a Mo = O group in the enzyme.

The pineal hormone melatonin (5-methoxy-N-acetyl-tryptamine) has been reported to participate in important physiological processes. Although some of its biological actions seem to depend on a protein receptor at the membrane surface, melatonin is known to interact with a large variety of tissues and cells, suggesting that the molecule may not necessarily interact through a specific membrane receptor at a specific cell. Most discussions of melatonin activity have assumed that the molecule is highly hydrophobic. Contrary to belief, the present work shows that melatonin is soluble in a purely aqueous medium up to 5 x 10(-3) M and describes a new method of melatonin preparation which shows the high hydrophilicity of the molecule. The results presented will affect the current biological hypothesis on the need of a melatonin carrier in the blood stream or the mechanisms which allow the hormone to cross the cell membrane and interact at the level of the nucleus.

The interaction of chloride, fluoride and phosphate ions with the molybdenum centre of sulphite oxidase in the pH range 6.2 to 9.6 has been studied by e.p.r. of Mo(V) in the enzyme reduced by sulphite. Detailed studies were made from e.p.r. spectra recorded at about 120K and more limited studies from spectra of liquid samples at about 295K and also from enzyme activity measurements. Interconversion between low-pH and high-pH Mo(V) e.p.r. signal-giving species [described by Lamy, Gutteridge & Bray (1980) Biochem. J. 185, 397-403] is influenced by chloride concentration, a 10-fold increase in concentration (in the range of about 1 mM to 100 mM) causing an increase of about 1 pH unit in the apparent pK for the conversion. This suggests that chloride is a constituent of the low-pH species. In support of this, high concentrations of fluoride modified the e.p.r. spectrum. Partial conversion to a Mo(V) species, in which F- has presumably replaced Cl- and showing hyperfine coupling of A(19F)av. 0.5mT, is indicated. It is proposed that interconversion between high-pH and low-pH species is of the form: (formula; see text) No evidence that Cl- is essential for enzymic activity was found. Data relating to equilibria amongst low-pH, high-pH and also the phosphate species are presented. Depending on pH and on concentrations of Cl- and H2PO4-, one, two, or all three species may be present. Qualitatively, under appropriate conditions, the phosphate species tends to replace some or all of the low-pH species. Quantitative analysis by a computer procedure permitted an appropriate scheme to be deduced and equilibrium constants to be evaluated. Studies on the e.p.r. signals at 295K indicated that similar equilibria applied in liquid solution, but with some changes in the values of the constants. The structure of the molybdenum centre in its various states and the nature of the enzymic reaction are discussed.

Melatonin, the chief hormone of the pineal gland, has been reported to interact with a variety of different cells. This ubiquitously acting hormone has been found to interact with protein receptors both at the cell membrane and in the nucleus. Moreover, melatonin was recently shown to be a very potent hydroxyl radical scavenger. The present work focuses on the interaction of melatonin with pure lipid bilayers. It is shown that melatonin can cross multilamellar lipid vesicles, which are used here as model systems for the lipid phase of biological membranes. Thus, the data prove that melatonin can easily pass through the cell membrane and bath every part of the cell, as previously suggested in the literature. Melatonin lipid association constant was calculated based on the change of the hormone fluorescence intensity due to its penetration into the hydrophobic lipid phase. Though melatonin was recently shown to be highly soluble in aqueous media, its lipid association constant is rather high, indicating that the biological action of the hormone is likely to be at the membrane level, either via its interaction with membrane receptors, and/or as a lipoperoxidation radical scavenger.

No abstract

We investigate the bilayer pre-transition exhibited by some lipids at temperatures below their main phase transition, and which is generally associated to the formation of periodic ripples in the membrane. Experimentally we focus on the anionic lipid dipalmytoylphosphatidylglycerol (DPPG) at different ionic strengths, and on the neutral lipid dipalmytoylphosphatidylcholine (DPPC). From the analysis of differential scanning calorimetry traces of the two lipids we find that both pre- and main transitions are part of the same melting process. Electron spin resonance of spin labels and excitation generalized polarization of Laurdan reveal the coexistence of gel and fluid domains at temperatures between the pre- and main transitions of both lipids, reinforcing the first finding. Also, the melting process of DPPG at low ionic strength is found to be less cooperative than that of DPPC. From the theoretical side, we introduce a statistical model in which a next-nearest-neighbor competing interaction is added to the usual two-state model. For the first time, modulated phases (ordered and disordered lipids periodically aligned) emerge between the gel and fluid phases as a natural consequence of the competition between lipid-lipid interactions.