Formation and characterization of mixed micelles of the nonionic surfactant Triton X-100 with egg, dipalmitoyl, and dimyristoyl phosphatidylcholines

Mixed micelles of the nonionic surfactant Triton X-100 and egg phosphatidylcholine were isolated by column chromatography on 6% agarose and by centrifugation at 35,000g. It was found that egg phosphatidylcholine bilayers are able to incorporate Triton X-100 at molar ratios of Triton to phospholipid below about 1:1, whereas above a molar ratio of about 2:1 Triton/phospholipid all of the phospholipid is converted into mixed micelles. Mixed micelles at a molar ratio of about 10:1 Triton/phospholipid were found to be in the same size range as pure micelles of Triton X-100. The formation of mixed micelles with dipalmitoyl phosphatidylcholine at room temperature, when the phospholipid is below its thermotropic phase transition, is shown to require relatively high concentrations of Triton X-100. The point at which dimyristoyl phosphatidylcholine bilayers are converted to mixed micelles was found to be less clear cut than with egg phosphatidylcholine, but above a molar ratio of about 2:1 Triton/phospholipid, all of this phospholipid is also in mixed micelles. The relevance of these results to the solubilization of membrane-bound proteins with Triton X-100 and the action of phospholipase A₂, which hydrolyzes phosphatidylcholine when it is in mixed micelles with Triton X-100, is discussed.     

Interaction of D-beta-hydroxybutyrate apodehydrogenase with phospholipids.

The interaction of a soluble homogeneous preparation of D-beta-hydroxybutyrate apodehydrogenase with phospholipid was studied in terms of restoration of enzymic activity and complex formation. The purified apoenzyme, which is devoid of lipid, is inactive. It is reactivated specifically by the addition of lecithin or mixtures of phospholipids containing lecithin. Mitochondrial phospholipid, i.e. the mixture of phospholipids in mitochondria, reactivates with the highest specific activity (approximately 100 micromol of DPN reduced/min/mg at 37 degrees and with the greatest efficiency (2.5 to 4 mol of lecithin/mol of enzyme subunit). Each of the lecithins of varying chain length and unsaturation reactivated the enzyme, albeit to differing extents and efficiencies. In general, lecithins containing unsaturated fatty acid moieties reactivated better than those containing the comparable saturated lipid. Optimal reactivation can be obtained for the various lecithins when they are microdispersed together with phosphatidylethanolamine. When the lecithins are added microdispersed together with both phosphatidylethanolamine and cardiolipin, maximal efficiency is obtained. Also, PC6:0 and 8:0 reactivate as soluble molecules, so that a phospholipid bilayer is not necessary to reactivate the enzyme. Complex formation was studied using gel exclusion chromatography. It can be shown that each of the phospholipids which reactivate combines with the apoenzyme. Mitochondrial phospholipid, which reactivates the best, binds most effectively; PC8:0, which reactivates with poor efficiency, can be shown to bind with low affinity, and negligible binding occurs at concentrations which do not reactivate the enzyme. Since the apoenzyme is apparently homogeneous and devoid of phospholipid or detergents, it would appear that reactivation does not involve reversal of inhibition such as by removal of a regulatory subunit or detergent from the catalytic subunit. Rather, we conclude that phospholipid is a necessary and integral portion of this enzyme whose active form is a phospholipid-protein complex. The apoenzyme also forms a complex with phosphatidylethanolamine and/or cardiolipin, which do not reactivate enzymic activity. Salt dissociates such complexes in contrast with the lecithin-apoenzyme complex. Binding of phospholipid is a necessary but not sufficient requisite for enzymic activity. The same energies of activation are obtained from Arrhenius plots for the membrane-bound enzyme and for the purified soluble enzyme reactivated with mitochondrial phospholipid or different lecithins. This observation is compatible with the view that the purified enzyme has not been adversely modified in the isolation. Furthermore, essentially the same energies of activation were obtained for saturated lecithins below their transition temperatures and for unsaturated lecithins above their transition temperatures. Hence, there is no indication that a lipid phase transition occurs to influence the activity of this enzyme.     

Effect of membrane fluidity and fatty acid composition on the prothrombin-converting activity of phospholipid vesicles.

Vesicles composed of phospholipids with different fatty acyl side chains have been utilized to examine the importance of the nonpolar membrane region for the prothrombin-converting activity of procoagulant phospholipid vesicles. Membranes composed of phosphatidylserine (PS) and phosphatidylcholine (PC) with unsaturated fatty acyl side chains were more active in prothrombin activation than membranes composed of phospholipids with saturated fatty acyl chains. This phenomenon was observed above the phase transition temperature, i.e., on membranes in the liquid-crystalline state. The prothrombin-converting activity of saturated phospholipids approached the activity of unsaturated phospholipids at high factor Va concentrations, which is indicative for a less favorable equilibrium constant for prothrombinase assembly on membrane surfaces composed of saturated phospholipids. The difference between saturated and unsaturated phospholipids was annulled on membranes with high mole percentages of PS. This may result from a compensating contribution of electrostatic forces to the binding equilibria involved in prothrombinase assembly. Additional effects on the prothrombin-converting activity were observed when membranes containing saturated phospholipids were studied below their phase transition temperature. In agreement with Higgins et al. [(1985) J. Biol. Chem. 260, 3604-3612], we found that the time required for the assembly of prothrombinase from membrane-bound factors Xa and Va is considerably prolonged on solid membranes. However, we also observed an effect of membrane fluidity on the steady-state rate of prothrombin activation. Kinetic experiments at saturating factor Va concentrations showed that the transition from the liquid-crystalline to the gel state caused a more than 9-fold decrease of the kcat of prothrombin activation without affecting the Km for prothrombin.(ABSTRACT TRUNCATED AT 250 WORDS)     

Microsomal cholesterol ester hydrolase of rat brain: lipids as effector of enzymatic activity

Evidence is presented that lipid plays an important role in the function of the microsomal cholesterol ester hydrolase of rat brain. The catalytic activity was almost completely lost when most of cholesterol and up to 70% of phospholipids were removed from lyophilized microsomes by extraction with chloroform at ?20 °C. The activity was completely restored when the chloroform-extracted lipid was added back to the assay mixture in the amount equal to the original concentration. Cholesterol or individual phospholipid alone was not effective in reconstituting the lost enzymatic activity. Effective restoration of the activity required addition of cholesterol and a phospholipid. Among the phospholipids tested, phosphatidylserine was the most effective, followed by ethanolamine phospholipids and phosphatidylcholine. The apparent V was dependent on the amount of the lipid added, while the K_m for the substrate, cholesteryl oleate, remained relatively constant, indicating that the effect of the added lipid was primarily on the reaction rate and not on the affinity of the enzyme to the substrate. The similar lipid dependence was observed with the Triton X-100-solubilized enzyme preparation. When the lipid phase of the microsomal membrane was perturbed, the enzyme became unstable when heated at 50 °C and its activity showed a discontinuity in the Arrhenius plots. Therefore, not only the concentration of the added lipid but also the physical state of the lipid phase around the enzyme appeared to be important for the activity of the rat brain microsomal cholesterol ester hydrolase.     

An improved method for the isolation and assay of the acid lipase from human liver.

An improved method for the isolation and assay of the lysosomal acid lipase from human liver has been developed. Over 90% of the enzymatic activity was extracted in soluble form by brief homogenization of frozen tissue with the nonionic surfactant, Triton X-100. With cholesterol, [1-14C]oleate and 4-methylumbelliferyl plamitate as substrate in emulsions with the amphoteric surfactant, N-tetradecyl-N,N,-dimethyl-3-ammonio-1-propanesulfonate, and ethanol, an apparent V of 1.9 nmol . min-1 . mg-1 protein was obtained with the radioactive substrate and 29 nmol . min-1 . mg-1 protein with the fluorogenic substrate analog, respectively. The released radioactivity-labelled oleic acid was quantitated by selective extraction with a new biphasic solvent system containing carbon tetrachloride and hexane. This assay procedure offers the advantages over other procedures that subcellular fractionation of the tissue is not required for the isolation of the cellular fractionation of the tissue is not required for the isolation of the enzyme; the enzymatic activity toward these emulsions is much greater than previously reported for other methods of substrate solubilization and cholesterol esters with saturated and unsaturated fatty acids can be employed as substrate since both types of fatty acids can be efficiently partitioned and quantitated with this solvent system.     

Membrane-surfactant interactions. The role of surfactant in mitochondrial complex III-phospholipid-Triton X-100 mixed micelles

Complex III (ubiquinol-cytochrome c reductase) was purified from beef heart mitochondria in the form of protein-phospholipid-Triton X-100 mixed micelles (about 1:80:100 molar ratio). Detergent may be totally removed by sucrose density gradient centrifugation, and the resulting lipoprotein complexes retain full enzyme activity. In order to understand the role of surfactant in the mixed micelles, and the interaction of Triton X-100 with integral membrane proteins and phospholipid bilayers, both the protein-lipid-surfactant mixed micelles and the detergent-free lipoprotein system were examined from the point of view of particle size and ultrastructure, enzyme activity, tryptophan fluorescence quenching, 31P NMR, and Fourier transform infrared spectroscopy. The NMR and IR spectroscopic studies show that surfactant withdrawal induces a profound change in phospholipid architecture, from a micellar to a lamellar-like phase. However, electron microscopic observations fail to reveal the existence of lipid bilayers in the absence of detergent. We suggest that, under these conditions, the lipid:protein molar ratio (80:1) is too low to permit the formation of lipid bilayer planes, but the relative orientation and mobility of phospholipids with respect to proteins is similar to that of the lamellar phase. Protein conformational changes are also detected as a consequence of surfactant removal. Fourier transform infrared spectroscopy indicates an increase of peptide beta-structure in the absence of Triton X-100; changes in the amide II/amide I intensity ratio are also detected, although the precise meaning of these observations is unclear. Tryptophanyl fluorescence quenching by acrylamide shows that a significant fraction of the Trp residues sensing the quencher become less readily available to it in the absence of surfactant. The temperature dependence of enzyme activity (expressed in the form of Arrhenius plots) is also different in the presence and absence of detergent. The effects of surfactant removal do not appear to be readily reversible upon readdition of Triton X-100.     

PROPERTIES OF MEMBRANE-BOUND DOPAMINES-HYDROXYLASE IN CHROMAFFIN GRANULES FROM BOVINE ADRENAL MEDULLA

Abstract— Properties of membrane-bound and soluble dopamine-β-hydroxylase were studied. Both enzyme forms have identical affinities for tyramine as the substrate. Arrhenius plots of the membrane-bound activity displayed a discontinuity at 29°C, the activation energy changing from 20,500 cal/mol below 29°C to 9500 cal/mol above 29°C. The soluble enzyme, like the purified enzyme, did not show discontinuities in Arrhenius plots, the activation energies being 18,500 cal/mol and 16,500 cal/mol respectively. The membrane-bound enzyme showed a discontinuity in the p K^m for tyramine versus reciprocal temperature plot, with a transition at 29°C, whereas the soluble enzyme failed to show such transition.
The membrane-bound dopamine-β-hydroxylase is solubilized by Triton X-100 as well as by lysolecithin. Of lecithin, lysophosphatidyl ethanolamine and phosphatidyl serine, lysolecithin was the only phospholipid to induce solubilization of membrane-bound dopamines β-hydroxylase. The 29°C-transition was not removed by treatment either with lysolecithin or with Triton X-100 used at concentration of up to 2%. This could indicate a solubilization of dopamine-β-hydroxylase with an associated lipid moiety which cannot be dispersed from the enzyme molecule and which still affects the activation energy and Michaelis constant for tyramine.
Results are discussed in terms of the relationship between lipid organization and enzyme activities; the significance of the solubilization by lysolecithin during exocytosis is considered in terms of the exocytosis process and fate of the chromaffin granule.     

Kinetic analysis of the Ca2+-dependent, membrane-bound, macrophage phospholipase A2 and the effects of arachidonic acid

The kinetics of the Ca2+-dependent, alkaline pH optimum, membrane-bound phospholipase A2 from the P388D1 macrophage-like cell line were studied using various phosphatidylcholine (PC) and phosphatidylethanolamine (PE) substrates. This enzyme exhibits "surface dilution kinetics" toward PC in Triton X-100 mixed micelles, and the "dual phospholipid model" was found to adequately describe its kinetic behavior. With substrate in the form of sonicated vesicles, the dual phospholipid model should give rise to Michaelis-Menten type kinetics. However, the hydrolysis of dipalmitoyl-PC, 1-palmitoyl-2-oleoyl-PC, and 1-stearoyl-2-arachidonoyl-PC vesicles exhibited two distinct activities. Below 10 microM, the data appeared to follow Michaelis-Menten behavior, while at higher concentrations, the data could best be fit to a Hill equation with a Hill coefficient of 2. These PCs had Vmax values for the low substrate concentration range of 0.2-0.6 nmol min-1 mg-1 and Km values of 1-2 microM. At the high substrate concentration range, the Vmax values were between 5 and 7 nmol min-1 mg-1. PC containing unsaturated fatty acids had an apparent Km, determined from the Hill equation, of about 15 microM, while the apparent Km of dipalmitoyl-PC was 0.6 microM. When 70% glycerol was included in the assays, a single Michaelis-Menten curve was obtained for both dipalmitoyl-PC and 1-stearoyl,2-arachidonoyl-PC. Possible explanations for these kinetic results include reconstitution of the membrane-bound phospholipase A2 in the phospholipid vesicle or the enzyme has tow distinct phospholipid binding function. The kinetics for both dipalmitoyl-PC and dipalmitoyl-PE hydrolysis in vesicles was very similar, indicating that the enzyme does not greatly prefer one of these head groups over the other. The enzyme also showed no preference for arachidonoyl containing phospholipid. Enzymatic activity toward PC containing saturated fatty acids was linear to about 15% hydrolysis while the hydrolysis of PC containing unsaturated fatty acids was linear to only about 5%. This loss of linearity was due to inhibition by released unsaturated fatty acids. Arachidonic acid was found to be a competitive inhibitor of dipalmitoyl PC hydrolysis with a K1 of 5 microM. This tight binding suggests a possible in vivo regulatory role for arachidonic acid. Three compounds of the arachidonic acid cascade, prostaglandin F2 alpha, 6-keto-prostaglandin F1 alpha, and thromboxane B2, showed no inhibition of enzymatic activity.     

Phospholipase A2 activity towards phosphatidylcholine in mixed micelles: surface dilution kinetics and the effect of thermotropic phase transitions

Phospholipase A₂ will act on dipalmitoyl phosphatidylcholine as substrate when the phospholipid is part of a mixed micelle with Triton X-100 at a molar ratio of Triton to phospholipid of 2:1 or greater. Kinetic studies at high molar ratios of Triton X-100 to phospholipid are reported and show that the binding of phospholipase A₂ to substrate depends on the total concentration of Triton X-100 and phospholipid, but that the rate of enzymatic catalysis decreases proportionally to the Triton X-100 concentration. These results are interpreted in terms of a model involving surface dilution kinetics. The relationship of this model to that of competitive inhibition is discussed. In addition, the activity of phospholipase A₂ towards dipalmitoyl phosphatidylcholine and dimyristoyl phosphatidylcholine at different temperatures is reported, and the results show a direct effect of the thermotropic phase transition of dipalmitoyl phosphatidylcholine on enzymatic activity.     

Amphiphile dependency of the monomeric and dimeric forms of acetylcholinesterase from human erythrocyte membrane

Human erythrocyte membrane-bound acetylcholinesterase was converted to a monomeric species by treatment of ghosts with 2-mercaptoethanol and iodoacetic acid. After solubilization with Triton X-100, the reduced and alkylated enzyme was partially purified by affinity chromatography and separated from residual dimeric enzyme by sucrose density gradient centrifugation in a zonal rotor. Monomeric and dimeric acetylcholinesterase showed full enzymatic activity in presence of Triton X-100 whereas in the absence of detergent, activity was decreased to approx. 20% and 15%, respectively. Preformed egg phosphatidylcholine vesicles fully sustained activity of the monomeric species whereas the dimer was only 80% active. The results suggest that a dimeric structure is not required for manifestation of amphiphile dependency of membrane-bound acetylcholinesterase from human erythrocytes. Furthermore, monomeric enzyme appears to be more easily inserted into phospholipid bilayers than the dimeric species.     

Identification of novel membrane-bound phospholipase D from Streptoverticillium cinnamoneum, possessing only hydrolytic activity

A membrane-bound phospholipase D (PLD) has been identified and isolated in a soluble form from an actinomycete, Streptoverticillium cinnamoneum. The enzyme has a monomeric structure with a molecular size of about 37 kDa, being the smallest among the enzymes so far reported. The enzyme catalyzes the hydrolysis of phosphatidylethanolamine and phosphatidylserine as preferred substrates, but not the transphosphatidylation reaction of their phospholipid groups to ethanol. Together with the absence of immunochemical cross-reactivity, these enzymatic properties demonstrate that the membrane-bound enzyme is distinct from the extracellular enzyme recently characterized and cloned from the same bacterial strain [C. Ogino et al., J. Biochem. 125 (1999) 263–269] and is therefore regarded as a novel prokaryotic PLD.     

Antibodies to liposomal phosphatidylserine and phosphatidic acid

Polyclonal antisera to phosphatidylserine or phosphatidic acid were induced in rabbits by injecting liposomes containing phosphatidylserine or phosphatidic acid and lipid A. Adsorption of antisera with liposomes containing different phospholipids revealed that some degree of reactivity with one or more phospholipids other than the immunizing phospholipid was often observed. However, cross-reactivity with other phospholipids was not a universal phenomenon, and one antiserum to phosphatidylserine failed to cross-react (i.e., was not adsorbed) with liposomes containing other phospholipids. All of the antisera were inhibited by soluble phosphorylated haptens (e.g., phosphocholine but not choline), but one of the antisera to phosphatidylserine was inhibited both by phosphoserine and by serine alone. Liposomal membrane composition influenced the activity of antiserum to phosphatidylserine. Regardless of whether unsaturated (beef brain) or saturated (dimyristoyl) phosphatidylserine was used in the immunizing liposomes, the antisera reacted more vigorously with liposomes containing unsaturated than saturated phosphatidylserine. We conclude that liposomes containing lipid A can serve as vehicles for stimulating polyclonal antisera to phosphatidylserine and phosphatidic acid. Although cross-reactivity with certain other phospholipids can be observed, sera from selected animals apparently can exhibit a high degree of specific activity to the immunizing phospholipid antigen.     

Effects of reconstitution of membrane-bound lipoprotein lipase and dispersity of substrate on its reaction characteristics

Reaction characteristics of a membrane-bound lipoprotein lipase acting on a hydrophobic substrate were investigated in aggregated structures—lipid bilayers of liposomes and mixed micelles of Triton X-100. The enzyme activity was enhanced with increases in Triton X-100 and phospholipid concentrations in micellar and liposomal structures. This higher activity was found to be due to both the solubilization state of the hydrophobic substrate and the hydrophobic interactions of the enzyme with either phospholipid or Triton X-100 molecules as a result of its incorporation into the aggregated systems. The enzyme reconstituted into lipid bilayers of liposomes prepared from 15 mM DMPC in the presence of 0.05% Triton X-100 showed a further 1.5-fold higher activity in comparison with the activity without reconstitution in micelles of 1.0% Triton X-100. These results indicate the necessity of the bilayer structure to retain the membrane-bound enzyme in an active conformation.     

Partial purification of glycerophosphate acyltransferase from Escherichia coli.

Glycerophosphate acyltransferase, a membrane-bound enzyme catalyzing the initial step of phospholipid biosynthesis in Escherichia coli, has been extracted with Triton X-100, a nonionic detergent, and purified 20- to 40-fold. This preparation is free from lysophosphatidate acyltransferase. Glycerophosphate acyltransferase is inactive in detergent extracts, but can be reconstituted by the addition of phospholipid. Under such conditions, the enzyme is associated with phospholipid. The sole product of the reaction with acyl coenzyme A as substrate is 1-acyl-sn-glycero-3-phosphate. Furthermore, the enzyme shows a marked preference for saturated fatty acyl conenzyme A, implying that this enzyme is responsible for the predominance of saturated moieties in position 1 of E. coli phospholipids. Acyltransferase from two mutants, plsA and plsB, was partially purified and characterized. Results support the view that plsB is a structural gene for the acyltransferase, but suggest that the plsA gene product is not directly involved in phospholipid biosynthesis.     

Effect of triton X-100 on the activity and solubilization of rat liver mitochondrial phosphatidylserine decarboxylase

It was shown that, among ionic and nonionic detergents tested, only Triton X-100 was able to stimulate the activity of rat liver phosphatidylserine decarboxylase, whereas other detergents were without effect or were inhibitory. The solubilization procedure of phosphatidylserine decarboxylase from mitochondrial membranes with Triton X-100 was elaborated. The dependence of the solubilized decarboxylase on the Triton X-100 to phosphatidylserine ratio and the inhibitory effect of Triton X-100 at its molar ratio to phospholipid higher than 5.6 was observed. No divalent cation requirement and no dependence of the ionic strength for the solubilized enzyme were observed. Kinetic parameters were determined.     

Protein kinase C activation by cis-fatty acid in the absence of Ca2+ and phospholipids

Protein kinase C has been shown to be a phospholipid/Ca2+-dependent enzyme activated by diacylglycerol (Nishizuka, Y. (1984) Nature 308, 693-697; Nishizuka, Y. (1984) Science 225, 1365-1370). We have reported that unsaturated fatty acids (oleic acid and arachidonic acid) can activate protein kinase C independently of Ca2+ and phospholipid (Murakami, K., and Routtenberg, A. (1985) FEBS Lett. 192, 189-193). This study shows that other cis-fatty acids such as linoleic acid also fully activate protein kinase C in the same manner. None of the saturated fatty acids (C:4 to C:18) nor the detergents (sodium dodecyl sulfate and Triton X-100) tested here were as effective as oleic acid. Unlike oleic acid, these detergents strongly inhibited protein kinase C activity induced by Ca2+/phosphatidylserine (PS) and diacylglycerol. Lowering the critical micelle concentration of oleic acid by increasing ionic strength also strongly inhibited oleic acid activation of protein kinase C activity. Dioleoylphosphatidylserine activated protein kinase C effectively (Ka = 7.2 microM). On the other hand, dimyristoylphosphatidylserine, which contains saturated fatty acids at both acyl positions, failed to activate protein kinase C even in the presence of Ca2+. These observations suggest that: protein kinase C activation by free fatty acid is specific to the cis-form and is not due to their detergent-like action, cis-fatty acid activation is due to the direct interaction of protein kinase C with the monomeric form of cis-fatty acids and not with the micelles of fatty acids, and cis-fatty acids at acyl positions in PS are also important for Ca2+/PS activation of protein kinase C.     

Reconstitution of Saccharomyces cerevisiae phosphatidylserine synthase into phospholipid vesicles. Modulation of activity by phospholipids

Membrane-associated phosphatidylserine synthase was purified from Saccharomyces cerevisiae (Bae-Lee, M., and Carman, G. M. (1984) J. Biol. Chem. 259, 10857-10862) and reconstituted into phospholipid vesicles containing phosphatidylcholine/phosphatidylethanolamine/ phosphatidylinositol/phosphatidylserine. Reconstitution was performed by removing detergent from an octyl glucoside/phospholipid/Triton X-100/enzyme mixed micelle by Sephadex G-50 super-fine chromatography. The average diameter of the vesicles was 90 nm, and the enzyme was reconstituted asymmetrically with the active site facing outward. The enzymological properties of reconstituted phosphatidylserine synthase were determined in the absence of detergent. The enzyme was reconstituted into vesicles with phospholipid compositions approximating those of wild type and mutant strains of S. cerevisiae. Reconstituted activity was modulated by the phosphatidylinositol/phosphatidylserine ratio in the vesicles. The modulation of activity observed in the vesicles is enough to account for some of the fluctuations in the phosphatidylserine content in vivo.     

Different effects of Triton X-100, deoxycholate, and fatty acids on the kinetics of glutathione peroxidase and phospholipid hydroperoxide glutathione peroxidase

The effects of Triton X-100, deoxycholate, and fatty acids were studied on the two steps of the ping-pong reaction catalyzed by Se-dependent glutathione peroxidases. The study was carried out by analyzing the single progression curves where the specific glutathione oxidation was monitored using glutathione reductase and NADPH. While the "classic" glutathione peroxidase was inhibited only by Triton, the newly discovered "phospholipid hydroperoxide glutathione peroxidase" was inhibited by deoxycholate and by unsaturated fatty acids. The kinetic analysis showed that in the case of glutathione peroxidase only the interaction of the lipophilic peroxidic substrate was hampered by Triton, indicating that the enzyme is not active at the interface. Phospholipid hydroperoxide glutathione peroxidase activity measured with linoleic acid hydroperoxide as substrate, on the other hand, was not stimulated by the Triton concentrations which have been shown to stimulate the activity on phospholipid hydroperoxides. Furthermore a slight inhibition was apparent at high Triton concentrations and the effect could be attributed to a surface dilution of the substrate. Deoxycholate and unsaturated fatty acids were not inhibitory on glutathione peroxidase but inhibited both steps of the peroxidic reaction of phospholipid hydroperoxide glutathione peroxidase, in the presence of either amphiphilic or hydrophilic substrates. This inhibition pattern suggests an interaction of anionic detergents with the active site of this enzyme. These results are in agreement with the different roles played by these peroxidases in the control of lipid peroxide concentrations in the cells. While glutathione peroxidase reduces the peroxides in the water phase (mainly hydrogen peroxide), the new peroxidase reduces the amphyphilic peroxides, possibly at the water-lipid interface.     

Miscibility of Sphingomyelins and Phosphatidylcholines in Unsaturated Phosphatidylcholine Bilayers

Polyunsaturated phospholipids are common in biological membranes and affect the lateral structure of bilayers. We have examined how saturated sphingomyelin (SM; palmitoyl and stearoyl SM (PSM and SSM, respectively)) and phosphatidylcholine (PC; dipalmitoyl PC and 1-palmitoyl-2-stearoyl PC (DPPC and PSPC, respectively)) segregate laterally to form ordered gel phases in increasingly unsaturated PC bilayers (sn-1: 16:0 and sn-2: 18:1...22:6; or sn-1 and sn-2: 18:1…22:6). The formation of gel phases was determined from the lifetime analysis of trans-parinaric acid. Using calorimetry, we also determined gel phase formation by PSM and DPPC in unsaturated PC mixed bilayers. Comparing PSM with DPPC, we observed that PSM formed a gel phase with less order than DPPC at comparable bilayer concentrations. The same was true when SSM was compared with PSPC. Furthermore, we observed that at equal saturated phospholipid concentration, the gel phases formed were less ordered in unsaturated PCs having 16:0 in sn-1, as compared to PCs having unsaturated acyl chains in both sn-1 and sn-2. The gel phases formed by the saturated phospholipids in unsaturated PC bilayers did not appear to achieve properties similar to pure saturated phospholipid bilayers, suggesting that complete lateral phase separation did not occur. Based on scanning calorimetry analysis, the melting of the gel phases formed by PSM and DPPC in unsaturated PC mixed bilayers (at 45 mol % saturated phospholipid) had low cooperativity and hence most likely were of mixed composition, in good agreement with trans-parinaric acid lifetime data. We conclude that both interfacial properties of the saturated phospholipids and their chain length, as well as the presence of 16:0 in sn-1 of the unsaturated PCs and the total number of cis unsaturations and acyl chain length (18 to 22) of the unsaturated PCs, all affected the formation of gel phases enriched in saturated phospholipids, under the conditions used.     

Effect of active dry wine yeast storage upon viability and lipid composition

Alterations in the plasma membrane of Active Dry Wine Yeast (ADWY) produce deleterious effects upon yeast fermentation activity. The aim of this study was to evaluate the changes in viability mediated by plasma membrane alterations induced by poor ADWY storage conditions. Yeast recovery was analyzed after growth in optimal medium. The effects upon vitality were measured by impedance variation. Cell membrane functionality was measured by anisotropy and cell lipid composition was also determined. The results showed a reduction in viability of up to 57–87% in the stored yeast. The storage effect increased saturated fatty acids, and reduced unsaturated fatty acids and phospholipid contents. The stored yeast recovery was related to membrane functionality and significant increases in unsaturated fatty acid, sterol and phospholipid concentrations. Good vitality was positively correlated to high unsaturated fatty acid, phosphatidylserine and phosphatidylethanolamine concentrations and negatively to high saturated fatty acid and phosphatidylcholine contents.