Mahendra Kumar Jain

Effect of small molecules on the dipalmitoyl lecithin liposomal bilayer: III. Phase transition in lipid bilayer

SummaryThe effect of more than ninety lipid-soluble compounds on the phase transition behavior ofdl-α-dipalmitoyl lecithin bilayer has been examined by differential scanning calorimetry. The type of effect on the phase transition profile depends on the nature of the additive, whereas the extent of the effect depends on the concentration. The compounds examined include uncouplers, alkanols, fatty acids, detergents, organic solvents, ionophores, inorganic ions, and some commonly used spin-labelled and fluorescent membrane probes. A qualitatively distinct effect of several of these additives on the phase transition behavior of bilayer provides a method of determining the nature of the perturbation they induce in the bilayer organization. The observations are consistent with the hypothesis that the type of effect induced by an additive on the phase transition profile of the bilayer is related to the position of localization of the additive along the thickness of the bilayer. At least four different types of modified transition profiles that are related to changes in bilayer fluidity can be distinguished. These correspond to the localization of the additive in phosphorylcholine (type D), glycerol backbone (type B), C1–C8 methylene (type A), C9–C16 methylene (type C) region of the bilayer. A possible relationship between the type of phase transition profiles of modified liposomes and the physiological effects of drugs is also discussed.

Effects of garlic extract and of three pure components isolated from it on human platelet aggregation, arachidonate metabolism, release reaction and platelet ultrastructure.

We studied the effect of the methanol extract of garlic bulbs (EOG) and of three pure components isolated from it (F1, F2, F3), on human platelet aggregation induced by ADP, epinephrine, collagen, thrombin, arachidonate, PAF, and the ionophore A-23187. Incubation of PRP with EOG, either in methanol or in homologous PPP, inhibits platelet aggregation induced by all of the above mentioned agonists. F1, F2, and F3 also inhibit platelet aggregation, however, F3 was about four times more potent. Addition of EOG or F3 to platelets that have already been irreversibly aggregated by 10 microM ADP, induces rapid deaggregation. Inhibition of aggregation was still present after three hours. The inhibitory effect persisted even after the treated platelets were Gel-Filtered (GFP) or separated from plasma through a metrizamide gradient and resuspended in new homologous PPP. Thrombin-induced release of ATP from GFP was inhibited by 75-80% after EOG or F3 treatment. Incorporation of [3-H]-arachidonate by intact platelets was decreased by 50-60% in treated platelets. However, platelets incubated with the inhibitors after incorporation of radiolabeled arachidonate, although did not aggregate, produced, after thrombin activation similar amounts of radiolabeled TXB2 and lipoxygenase products as the controls. Electron microscopy of inhibited platelets, in the presence of thrombin, showed no degranulation but an increase of spherical forms. Our results suggest that the effects described might be mediate by a perturbation of the physicochemical properties of the plasma membrane rather than by affecting arachidonate or calcium metabolism in the cells. Chemical structures of F1, F2 and F3 have been provisionally assigned: F1 is diallytrisulfide, F2 is 2-vinyl-1,3-dithiene, and F3 is most probably allyl 1,5-hexadienyltrisulfide.

Long-Range Order in Biomembranes

Publisher Summary The central problem of membrane structure and its correlation with physiological and biochemical functions is to define the organization of constituent molecules. The existence of bilayers in biomembranes is established by a variety of physicochemical techniques. It has been shown that the subtleties of organizational and phase characteristics of the bilayer arise from the segmental motion and the transverse, rotational, and lateral mobilities of constituent lipids. These molecular features of lipids in the bilayer organization account for dielectric, viscoelastic, partitioning, and passive permeability characteristics. Experimental evidence indicates that the membrane lipids not only create a barrier to the free entry and exit of molecules into and out of the cell, but lipids also provide a matrix in/on which biochemical reactions can take place; through which certain metabolites can pass selectively; and with which recognition, adhesion, aggregation and fusion of cells can be mediated.

Interaction of phospholipase A2 and phospholipid bilayers.

Binding of phospholipase A2 from porcine pancreas and from Naja melanoleuca venom to vesicles of 1,2-di(tetradecyl)-rac-glycero-3-phosphocholine (diether-PC14) is studied in the presence and absence of 1-tetradecanoyl-sn-glycero-3-phosphocholine and myristic acid. The bound enzyme coelutes with the vesicles during gel filtration through a nonequilibrated Sephadex G-100 column, modifies the phase transition behavior of bilayers, and exhibits an increase in fluorescence intensity accompanied by a blue shift. Using these criteria it is demonstrated that the snake-venom enzyme binds to bilayers of the diether-PC14 alone. In contrast, the porcine enzyme binds only to ternary codispersions of dialkyl (or diacyl) phosphatidylcholine, lysophosphatidylcholine and fatty acid. Binding of pig-pancreatic enzyme to vesicles of the diether-PC14 could not be detected even after long incubation (up to 24 H) below, at, or above the phase-transition temperature, whereas the binding in the presence of products is almost instantaneous and observed over a wide temperature range. Thus incorporation of the products in substrate dispersions increases the binding affinity rather than increase the rate of binding. The results are consistent with the hypothesis that the pancreatic enzyme binds to defect sites at the phase boundaries in substrate bilayers induced by the products. The spectroscopically obtained hyperbolic binding curves can be adequately described by a single equilibrium by assuming that the enzyme interacts with discrete sites. The binding experiments are supported by kinetic studies.

Identification of a human cDNA clone for lysosomal type Ca2+-independent phospholipase A2 and properties of the expressed protein.

A Ca2+-independent phospholipase A2 (PLA2) maximally active at pH 4 and specifically inhibited by the transition-state analogue 1-hexadecyl-3-trifluoroethylglycero-sn-2-phosphomethanol (MJ33) was isolated from rat lungs. The sequence for three internal peptides (35 amino acids) was used to identify a 1653-base pair cDNA clone (HA0683) from a human myeloblast cell line. The deduced protein sequence of 224 amino acids contained a putative motif (GXSXG) for the catalytic site of a serine hydrolase, but showed no significant homology to known phospholipases. Translation of mRNA produced from this clone in both a wheat germ system and Xenopus oocytes showed expression of PLA2 activity with properties similar to the rat lung enzyme. Apparent kinetic constants for PLA2 with dipalmitoylphosphatidylcholine as substrate were Km = 0.25 mM and Vmax = 1.89 nmol/h. Activity with alkyl ether phosphatidylcholine as substrate was decreased significantly compared with diacylphosphatidylcholine. Significant lysophospholipase, phospholipase A1, or 1-O-alkyl-2-acetyl-sn-glycero-3-phosphocholine acetylhydrolase activity was not observed. Enzyme activity was insensitive to p-bromophenacyl bromide, bromoenol lactone, trifluoromethylarachidonoyl ketone, mercaptoethanol, and ATP, but was inhibited by MJ33 and diethyl p-nitrophenyl phosphate, a serine protease inhibitor. SDS-polyacrylamide gel electrophoresis with autoradiography of the translated [35S]methionine-labeled protein confirmed a molecular mass of 25.8 kDa, in good agreement with the enzyme isolated from rat lung. By Northern blot analysis, mRNA corresponding to this clone was present in both rat lung and isolated rat granular pneumocytes. These results represent the first molecular cloning of a cDNA for the lysosomal type Ca2+-independent phospholipase A2 group of enzymes.

Origin of the latency phase during the action of phospholipase A2 on unmodified phosphatidylcholine vesicles

The reaction progress curve for the action of pig-pancreatic phospholipase A2 on dimyristoylphosphatidylcholine vesicles is characterized under a variety of conditions. The factors that regulate the rate of hydrolysis during the presteady-state phase determine the latency period. The results demonstrate that the accelerated hydrolysis following the latency phase of the reaction progress curve is due to the product-assisted binding of the enzyme to the substrate bilayer by chaning the number of bindings sites and therefore the binding equilibrium. A critical mole fraction of products appears to be formed in the substrate bilayers before the steady-state phase of hydrolysis begins. The latency phase shows a minimum at the phase-transition temperature of the substrate vesicles; however, we did not observe a significant binding of the enzyme to pure substrate bilayers even at the phase-transition temperature. The rate of binding of the enzyme is found to be fast and the rate of desorption of the bound enzyme is very slow compared to the latency phase. The rate of redistribution of products between substrate bilayers is rather slow. These observations demonstrate that during the latency phase of the action of phospholipase A2, a critical mole fraction of products is formed in the substrate bilayer.

Kinetics of interfacial catalysis by phospholipase A2 in intravesicle scooting mode, and heterofusion of anionic and zwitterionic vesicles.

In this and the following three papers we examine the kinetics of action of pig pancreatic phospholipase A2 on vesicles of anionic phospholipids without any additives. The results provide the first unequivocal demonstration of interfacial catalysis in intravesicle scooting mode. In this paper we describe the conditions in which the action of pig pancreatic phospholipase A2 on DMPMe (ester) vesicles in the absence of any additive commences without a latency. Under these conditions the free monomer substrate concentration is insignificant; the bilayer enclosed vesicle organization remains intact even when all the substrate in the outer monolayer has been hydrolyzed; the rate of intervesicle exchange and the rate of transbilayer movement (flip-flop) of molecules is negligibly slow; and the rate of fusion of vesicles is insignificant. Thus an enzyme molecule bound to one vesicle hydrolyzes all the DMPMe molecules in the outer monolayer of the vesicle by a first-order process with a rate constant of 0.6 per min at 30 degrees C; or viewed another way, one enzyme molecule in a DMPMe vesicle can hydrolyze all the available substrate molecules at the rate of 3000 per min. At low anion concentrations excess substrate vesicles are not hydrolyzed unless the rate of intervesicle exchange of the bound enzyme is stimulated by anions in the aqueous phase. Higher calcium concentrations promote not only homofusion of DMPMe vesicles but also heterofusion of DMPMe and DMPC vesicles. It is proposed that calcium-induced isothermal lateral phase separation in DMPMe vesicles induces defects in the bilayer organization, and such defects are the sites for phospholipase A2 binding and for heterofusion with DMPC (ester) vesicles which do not have such sites.

Role of Cholesterol in Biomembranes and Related Systems

Publisher Summary This chapter examines the molecular aspects of interaction between cholesterol and phospholipids. It reviews mainly how these subtle interlipid interactions modify the gross functions of membranes. In the solid state, cholesterol is not readily dispersed in water or electrolyte solutions that could occur naturally. Yet, because cholesterol is integrated into all cells and most body fluids in nonparticulate form, some very efficient methods of microdispersion and phase change must operate biologically. Solubilization of cholesterol can be achieved in ternary systems with lecithin as the amphiphile, in which case, a complex, stable mesophase develops at a certain critical concentration. Evidence for the interaction of cholesterol with phospholipids to modify a bilayer has come from a variety of sources. Studies on sonicated dispersions of phospholipid in water indicate that the dispersions containing cholesterol are heavier, more asymmetrical, less hydrated, and more rigid. These changes are reflected in various properties of the liposomes. The effect of cholesterol on phospholipid dispersions in benzene is not so significant. However, available evidence does suggest that cholesterol is incorporated into the phospholipid aggregates. The chapter describes the evidence concerning possible interaction of cholesterol with phospholipids in the aqueous phase from the use of physical techniques.

Binding of phospholipase A2 to zwitterionic bilayers is promoted by lateral segregation of anionic amphiphiles

Catalytic action of phospholipase A2 is appreciably influenced by the organization and dynamics of bilayers of glycerophosphocholines (Apitz-Castro et al. (1988) Biochim. Biophys. Acta 688, 341-348). However, such effects of the quality of the interface are not observed with bilayers of glycerophosphoryl methanol and other anionic phospholipids (Jain et al. (1986) Biochim. Biophys. Acta 860, 435-447). Such differences between the catalytic susceptibility of zwitterionic versus anionic bilayers are due to a large difference in the affinity of the enzyme for these interfaces. Binding to phospholipase A2 to zwitterionic interfaces can be promoted in the presence of certain anionic additives. For example in the pre-steady-state phase of hydrolysis, segregation of the nacently produced products of hydrolysis could promote binding of phospholipase A2 to regions of higher anionic charge density in the zwitterionic interface. In this paper we show that the dynamics of segregation of the nacently produced products of hydrolysis in zwitterionic bilayers can be readily followed by monitoring the fluorescence intensity of the cationic dye NK-529 (Yu and Jain (1989) Biochim. Biophys. Acta 980, 15-22). The fluorescence emission characteristics of NK-529 change appreciably due to self-quenching of the bound dye molecules as the fatty acid molecules segregate in the bilayer. The kinetics of segregation of fatty acids during the course of hydrolysis of bilayers of zwitterionic phospholipids by phospholipase A2 exhibits an unequivocal correlation with a variety of phenomena that are observed during the transition from the pre-steady-state phase to the steady-state phase of hydrolysis in the reaction progress curves as a function of temperature and in the presence of lipophilic additives.

Cationic peptide antimicrobials induce selective transcription of micF and osmY in Escherichia coli.

Cationic antimicrobial peptides, such as polymyxin and cecropin, activated transcription of osmY and micF in growing Escherichia coli independently of each other. The micF response required the presence of a functional rob gene. It is intriguing that in this and other assays an identical response profile was also seen with hyperosmotic salt or sucrose gradient, two of the most commonly used traditional food preservatives. The osmY and micF transcription was not induced by hypoosmotic gradient, ionophoric peptides, uncouplers, or with other classes of membrane perturbing agents. The antibacterial peptides did not promote transcription of genes that respond to macromolecular or oxidative damage, fatty acid biosynthesis, heat shock, or depletion of proton or ion gradients. These and other results show that the antibacterial cationic peptides induce stasis in the early growth phase, and the transcriptional efficacy of antibacterial peptides correlates with their minimum inhibitory concentration, and also with their ability to mediate direct exchange of phospholipids between vesicles. The significance of these results is developed as the hypothesis that the cationic peptide antimicrobials stress growth of Gram-negative organisms by making contacts between the two phospholipid interfaces in the periplasmic space and prevent the hyperosmotic wrinkling of the cytoplasmic membrane. Broader significance of these results, and of the hypothesis that the peptide mediated contacts between the periplasmic phospholipid interfaces are the primary triggers, is discussed in relation to antibacterial resistance.