Donald L. Melchior

Lipid Phase Transitions and Regulation of Membrane Fluidity in Prokaryotes

Publisher Summary Normal cell function requires fluid membrane bilayers. In addition to selecting mixtures that form stable bilayers for their membrane lipids, prokaryotes maintain their membranes in a fluid state under varying environmental conditions. This chapter discusses the reason for the cells requirement of fluid membranes; and presents some of the strategies they use to maintain their membranes in a fluid state. The roles different lipid classes play in membrane function is considered from the perspective of their physical behavior. The properties of membrane lipids in water are also described. Because of the hydrophobic effect, lipid molecules aggregate in water to minimize the contact of nonpolar regions with water while allowing the contact of head groups with water. The membrane bilayers of most organisms are entirely or mostly fluid at physiological temperatures, but at lower temperatures undergo a major reversible change of state. Membrane bilayers are composed of many different lipids and melt over a wide range of temperatures. Membranes from a variety of prokaryotes crystallized at lower than physiological temperature. The lipid molecules act to keep bilayers in a stable state despite changes in environmental conditions, but are not themselves bilayer formers. The functions of the membrane bilayer as acceptors and sinks are interrelated; and the two functions provide a sensitive, unified means to control the fatty acid composition and hence the physical state of biological membranes, whether fatty acids are supplied exogenously in the growth medium or are endogenously biosynthesized by the organism.

The preparation and characterization of liposomes containing X-ray contrast agents

Unilamellar phospholipid vesicles loaded with the water-soluble, ionic X-ray contrast agent diatrizoate (Hypaque, Renografin) were manufactured by reverse-phase evaporation for use as organ-enhancing agents in X-ray computed tomography. Encapsulation efficiency was determined as a function of various diatrizoate concentrations in vesicles of varying lipid composition. Loss of encapsulated diatrizoate over 24 h was examined in vesicles composed of several egg phosphatidylcholine/cholesterol ratios. Size estimates for loaded vesicles were obtained by negative-stain electron microscopy, Millipore filtration and light microscopy. Intravenous in vivo injection of loaded vesicles in the rat resulted in significant enhancement of both spleen and liver on subsequent scans. Vesicles were similarly prepared with the water-soluble, nonionic agent metrizamide (Amipaque). Encapsulation efficiency was determined, and in vivo behavior was observed.

The physical state of quick-frozen membranes and lipids

Lipid bilayers and biomembranes produce nearly identical calorimeter scans regardless of whether they are slowly cooled under near-equilibrium conditions or rapidly frozen at rates used in freeze-fracture electron microscopy. Except for the melting of ice at 273 K, for both cooling regimens no significant thermal events occur from 100 K to the usual gel to liquid crystal transition. The gel to liquid crystal transition itself is somewhat altered by rapid cooling when bilayers contain mixed lipid species. Combined with X-ray diffraction studies, the results indicate that quickly frozen bilayers are crystalline, but that the crystalline domains are quite small or otherwise disordered. In contrast to the behavior of lipids in bilayers, hexagonal-phase calcium cardiolipin easily forms a glass upon cooling.

The influence of saturated fatty acid modulation of bilayer physical state on cellular and membrane structure and function

Cultured chick fibroblasts supplemented with stearic acid in the absence of serum at 37 degrees C degenerate and die in contrast to cells grown at 41 degrees C which appear normal in comparison with controls. These degenerative effects at 37 degrees C are alleviated by addition to stearate-containing media of fatty acids known to fluidize bilayers. These observations suggest that cell degeneration at 37 degrees C may involve alterations in the physical state of the membrane. Fatty acid analysis of plasma membrane obtained from stearate-supplemented cells clearly demonstrates the enrichment of this fatty acid species into bilayer phospholipids. Moreover, the extent of enrichment is similar in cells grown at both 37 and 41 degrees C. Stearate enrichment at either temperature does not appear to alter significantly membrane cholesterol or polar lipid content. Fluorescence anisotropy measurements for perylene and diphenylhexatriene incorporated into stearate-enriched membranes reveals changes suggestive of decreased bilayer fluidity. Moreover, analysis of temperature dependence of probe anisotropy indicates that a similarity in bilayer fluidity exists between stearate-enriched membranes at 41 degrees C and control membranes at 37 degrees C. Calorimetric data from liposomes prepared from polar lipids isolated from these membranes show similar melting profiles, consistent with the above lipid and fluorescence analyses. Arrhenius plot of stearate-enriched membrane glucose transporter function reveals breaks which coincide with the main endotherm of the pure phospholipid phase transition, indicating the sensitivity of the transporter to this transition which is undetectable in these native bilayers. These data suggest the existence of regions of bilayer lipid microheterogeneity which affect integral enzyme function, cell homeostasis and viability.

Modulation of red blood cell sugar transport by lyso-lipid

The in vitro presentation to red blood cells of specific lysolipids in amounts comparable to lysolipid levels in serum is shown to markedly influence protein-mediated glucose transport. Lysolipids were introduced exogenously into cell membranes by incubating erythrocytes in buffer containing varying concentrations of lysolipid (under 3.2 microM). The transport-modulating potency of the lysolipids was found to be dependent both on headgroup and hydrocarbon chain. MPL (monopalmitoyl lecithin, L-alpha-lysopalmitoylphosphatidylcholine) had the greatest influence on sugar transport. 15 min incubation of red cells in MPL suspensions sufficed for 99% association of the lysolipid with the cell membranes. This association correlated with altered red-cell sugar transport. At MPL/bilayer lipid molar ratios as low as 0.03%, MPL was found to act as a reversible, hyperbolic, mixed-type inhibitor of exchange D-glucose exit (both Km(app) and Vmax for transport are reduced). Dissociation of MPL from the membrane results in the recovery of original transport activity. MPL at 1.5.10(-17) mol MPL/red cell was found to reduce Ki(app) for D-glucose inhibition of cytochalasin B binding to the glucose carrier protein in red cell ghost membranes. Our findings demonstrate that red-cell membrane-exogenous lysolipid associations can significantly modify protein mediated sugar transport. The simplest explanation of our findings is a direct interaction of lysolipid with the transport protein.

Alterations in red blood cell sugar transport by nanomolar concentrations of alkyl lysophospholipid.

Acyl lysolipids presented in vitro to red blood cells in amounts comparable to blood serum levels inhibit protein-mediated glucose transport (Naderi, A., Carruthers, A. and Melchior, D.L. (1989) Biochim. Biophys. Acta 985, 173-181). In this study, an alkyl lysolipid (2-O-methyl-1-O-octadecyl-sn-glycero-3- phosphocholine; ALP), was found to be an order of magnitude more effective in inhibiting sugar transport than the most potent acyl lysolipid. Bilayer concentrations of ALP as low as 5 ALP molecules per transporter (0.1 mol% of total membrane lipid) result in a 50% inhibition of transport activity. ALP acts as a competitive inhibitor of exchange L-glucose transport, of CCB binding to the glucose transporter and of D-glucose inhibition of CCB binding to the transporter. Inhibition of zero-trans sugar uptake by ALP is noncompetitive. The two enantiomers of ALP show a different ability to inhibit sugar transport. The action of ALP is consistent with a mechanism in which ALP interacts with a transmembrane portion of the sugar transport molecule resulting in a competitive displacement of D-glucose or cytochalasin B from the cytosolic facing side of the transport molecule. The simplest explanation of our findings is a direct interaction of the ALP molecule with the transport protein.

A firefly luciferase assay for subnanomolar concentrations of amphipathic substances

A sensitive assay is described for accurately quantitating subnanomolar aqueous concentrations of a wide variety of amphipathic and hydrophobic biological materials. This paper extends a luciferase-luciferin method previously used to measure aqueous concentrations of anesthetics to a variety of hormones, metabolites, and membrane active agents. The assay can cover analyte ranges from picomolar to micromolar. The sensitivity of the assay is shown to correlate with the hydrophobic nature of the analyte. The mechanism of the assay appears to result from competition of analyte with luciferin for a hydrophobic binding site on the luciferase molecule. This assay allows measurement of the partitioning of analytes into lipid bilayers from aqueous solution.

The Fluorosome™ technique for investigating membrane on- and off-loading of drugs by β-CD and sonicated SUV

The application of the Fluorosome technique to test drug delivery systems is described. Fluorosomes, egg phosphatidylcholine liposomes with bilayer embedded fluorophores, were employed to investigate the ability of sonicated small unilamellar vesicles (sSUV) and β‐cyclodextrins (β‐CD) to deliver drugs into or extract drugs from the fluorosomes phospholipid bilayer. The addition of phloretin to a fluorosome suspension resulted in fluorescence reduction reflecting phloretin entering the bilayer and quenching fluorophore fluorescence. Subsequent addition of sSUV to phloretin pretreated fluorosomes showed an increase in fluorescence reflecting phloretin extraction from the fluorosome membrane. Sequential additions of β‐estradiol loaded β‐CD to fluorosomes as well as the addition of β‐estradiol alone resulted in fluorescence reduction due to β‐estradiol insertion into the membrane. Further addition of pure β‐CD resulted in a fluorescence increase indicating β‐estradiol extraction from the fluorosome membrane.

Some Uses of Differential Scanning Calorimetry in Biomembrane Research

The application of Differential Scanning Calorimetry (DSC) to biological problems is relatively recent. DSC is proving to be a powerful approach not only for investigating structural problems, but for studies on physiological processes as well. DSC has had its most notable biological successes in studies of lipid containing systems (1,2). The intent of this chapter is to briefly describe some calorimetric approaches our laboratory has used to obtain information on these systems.