Soybean Phospholipids Research Articles

Isolation of H +-translocating ATPase in tonoplast of Tradescantia virginiana L. leaf cells

The tonoplast of Tradescantia virginiana L. was prepared from leaf cells and then solubilized with deoxycholate (DOC) and n-octyl-β- d-glucoside ( n-OG). Three major polypeptides (68, 60, 16 kDa) and several other minor components were isolated. These polypeptides were reconstituted in soybean phospholipids (asolectin). The H + pump activity was investigated with the reconstituted system as well as with the tonoplast. In both cases, the quinacrine–fluorescence quenching was observed in the presence of ATP–Mg 2+, indicating the H + pumping. The H + pump activity was inhibited by gramicidin D, a channel-forming ionophore, and by KNO 3, an inhibitor specific to tonoplast-type (V-type) H +-ATPase.

Structure and function in rhodopsin: asymmetric reconstitution of rhodopsin in liposomes.

We report on preparation of rhodopsin proteoliposomes with the cytoplasmic domain of rhodopsin facing the exterior of the proteoliposomes. Rhodopsin purified from rod outer segments of bovine retinae by immunoaffinity chromatography in octyl glucoside was reconstituted into liposomes prepared from soybean phospholipids by detergent dialysis. The orientation of rhodopsin in the liposomes was determined by susceptibility of its C terminus to papain and the endoproteinase, Asp-N, followed by SDS/PAGE, which showed that the cytoplasmic domain in at least 90% of rhodopsin faced the exterior of the proteoliposomes. By using escape of (32)P-KP(i) encapsulated in the proteoliposomes as the assay, the half-life of the proteasomes was approximately 8 days. After light activation, rhodopsin in proteoliposomes showed the rate of decay of metarhodopsin II and the initial rate of transducin activation comparable with the rates of rhodopsin in rod outer segment membranes. This finding demonstrates the functional capability of rhodopsin in proteoliposomes for kinetic studies of protein-protein interactions.

Influence of lecithin on mitochondrial DNA and age-related hearing loss.

Lecithin is a polyunsaturated phosphatidylcholine (PPC), which are high energy functional and structural elements of all biologic membranes. PPC play a rate-limiting role in the activation of numerous membrane-located enzymes, including superoxide dismutase and glutathione, which are important antioxidants protecting cell membranes from damage by reactive oxygen species (ROS). ROS-induced damage to mitochondrial DNA may lead to reduced mitochondrial function in the cochlea and resultant hearing loss. The effects of lecithin on aging and age-associated hearing loss were studied in rats by measuring hearing sensitivities using auditory brainstem responses (ABR). In addition, mitochondrial function as a measure of aging was assessed by determining mitochondrial membrane potentials using flow cytometry and by amplifying mitochondrial DNA deletions associated with aging. Harlan-Fischer rats aged 18 to 20 months (n = 14) were divided into 2 groups. The experimental group was supplemented orally for 6 months with lecithin, a purified extract of soybean phospholipid (Nutritional Therapeutics, Allendale, NJ). The data obtained were compared with the control group. ABRs were recorded at 2-month intervals and showed significant preservation of hearing sensitivities in the treated subjects. Flow cytometry revealed significantly higher mitochondrial membrane potentials in the treated subjects, suggesting preserved mitochondrial function. Finally, the common aging mitochondrial DNA deletion (mtDNA(4834)) were amplified from brain and cochlear tissue including stria vascularis and auditory nerve. This specific deletion was found significantly less frequent in all tissues in the treated group compared with the controls. These experiments support our hypothesis and provide evidence that lecithin may preserve cochlear mitochondrial function and protect hearing loss associated with aging.

Production of structured phospholipids by lipase-catalyzed acidolysis: optimization using response surface methodology

Structured phospholipids were produced by lipase-catalyzed acidolysis between soybean phospholipids and free fatty acids. For this purpose three commercially available immobilized lipases were examined and Lipozyme TL IM was finally selected for further optimization. Incorporation of acyl donors, including caprylic acid, conjugated linoleic acid (CLA), eicosapentaenoic acid (EPA), and docosahexaenoic acid (DHA), into phospholipids was examined. Different apparent incorporation rates were observed for the individual fatty acids. The main reason was attributed to the purity of the starting acyl donor mixtures, for example the starting material for CLA contained only 77% CLA. The nature of the phospholipids also affected the incorporation rates of caprylic acid catalyzed by Lipozyme TL IM. The following order of reactivity was observed phosphatidylcholine>phosphatidylethanolamine>phosphatidic acid>phosphatidylinositol. Lipozyme TL IM-catalyzed acidolysis in a solvent-free system was optimized using response surface methodology (RSM). A three-level five-factor fractional factorial design with star points was adopted. The five major factors chosen were lipase dosage ( L d, wt.% based on substrates), water addition ( W c, wt.% based on lipase), reaction time ( T i, h), reaction temperature ( T e, °C), and substrate ratio ( S r, caprylic acid/phospholipids, mol/mol). Acidolysis was influenced by these factors in the following order: T i> L d> S r> T e, whereas water addition had no effect on the incorporation in the range of 2–4 wt.%. The best fitting quadratic model was determined by regression and backward elimination. Based on the fitted model, the optimal reaction conditions for the production of structured phospholipids by Lipozyme TL IM-catalyzed acidolysis between soybean phospholipids and caprylic acid were found to be T i, 70 h; L d, 30 wt.%; T e, 57 °C, S r, 5.5/1 mol/mol, and no water addition. At these experimental conditions, 39% incorporation of caprylic acid can be obtained.

Synergy in K+/H+ exchange across phospholipid vesicle membranes with combinations of valinomycin and chlorophenolsThe T-jump experiments described in this work were carried out during James Hudson's visit to Tata Institute of Fundamental Research, Mumbai 400 005, India during 2000–2001.

The protonophoric mobilities of mixtures of chlorophenols A1 and A2, inferred from a study of the decay of the pH difference across soybean phospholipid vesicular membrane (=ΔpH), have shown that there is a significant synergy for certain combinations of A1 and A2 in the presence of valinomycin (VAL). The results have shown that in addition to a term proportional to [VAL]0[Ai]0, a term proportional to the product of the concentrations [VAL]0[A1]0[A2]0 (with A1 = A2 giving a quadratic term in the “two species case”) contributes to the ΔpH relaxation rate (=1/τ). The magnitudes of these terms depend on the choice of chlorophenols. The former term has been assigned to the well-known mechanism in which the anion A− and the metal ion K+ are back transported across the membrane in the form of the ternary complex VAL–K+–A−. The latter term has been assigned to the less known catalyzed transport mechanism in which A1− is transported from the polar region of the membrane to the non-polar region in the form of the electro-neutral complex VAL–K+–A1− in a fast step followed by translocation through the non-polar region as A1− after dissociation from the ternary complex assisted by a complexing A2− from the polar region. The translocation of VAL–K+ across the membrane facilitates the compensating charge flux of K+ ions in this mechanism. The processes characterized in this work are useful in improving our understanding of the toxicity of uncouplers and uncoupler mixtures and may also help in the development of appropriate analytical tools.

Conversion of degummed soybean oil to biodiesel fuel with immobilized Candida antarctica lipase

Crude soybean oil did not undergo methanolysis with immobilized Candida antarctica lipase but degummed oil did. Therefore, the substance that was removed in the degumming step was estimated to inhibit the methanolysis of soybean triacylglycerols (TAGs). The main components of soybean gum are phospholipids (PLs), and soybean PLs actually inhibited the methanolysis reaction. In addition, PLs were detected in chloroform/methanol (MeOH) extracts from the immobilized lipase preparation that had been used in the methanolysis of crude soybean oil. These results showed that PLs were at least one of the inhibitory substances in methanolysis of TAGs. The inhibition may due to the interference of the interaction of the lipase molecule with substrates by PLs bound on immobilized preparation. These findings indicated that degummed oil has to be used as a substrate for enzymatic methanolysis. Indeed, three-step methanolysis successfully converted 93.8% degummed soybean oil to its corresponding methyl esters, and the lipase could be reused for 25 cycles without any loss of the activity.

Modification of Soybean Phospholipids by Enzymatic Transacylation.

Modified glycerophospholipids with expected novel physiological functions were prepared by introducing different acyl groups into soybean phospholipids. The method was based on the enzymatic transesterification of phospholipids with monoacylglycerol (MG) or diacylglycerol (DG). The transacylation ratios reached 46-59% and nearly quantitative recovery of phospholipids was obtained in the case of transacylation of phospholipids with MGs having C12 to C18 saturated and monounsaturated acyl groups. With C18:2-MG or C18:3-MG, however, transacylation ratios were less than ca. 20% MGs with C4 to C8 saturated acyl groups also gave very low transacylation ratios, but the ratios were moderately improved by using DGs with C6 and C8 acyl groups. It is considered from a series of the results that higher transacylated phospholipids might be obtained y repeating transacylations owing to be able to recover the phospholipids quantitatively after the reaction because MG with longer acyl groups than C10 or DG with C8 acyl groups acted as acyl donors and replacement for essential water, to suppress the simultaneous hydrolysis, in an enzymatic reaction.

Expressing and purifying membrane transport proteins in high yield

Structural analysis of native or recombinant membrane transport proteins has been hampered by the lack of effective methodologies to purify sufficient quantities of active protein. We addressed this problem by expressing a polyhistidine tagged construct of the cardiac sodium–calcium exchanger (NCX1) in Trichoplusia ni larvae (caterpillars) from which membrane vesicles were prepared. Larvae vesicles containing recombinant NCX1-his protein supported NCX1 transport activity that was mechanistically not different from activity in native cardiac sarcolemmal vesicles although the specific activity was reduced. SDS-PAGE and Western blot analysis demonstrated the presence of both the 120 and 70 kDa forms of the NCX1 protein. Larvae vesicle proteins were solubilized in sodium cholate detergent and fractionated on a chelated Ni 2+ affinity chromatography column. After extensive washing, eluted fractions were mixed with soybean phospholipids and reconstituted. The resulting proteoliposomes contained NCX1 activity suggesting the protein retained native conformation. SDS-PAGE revealed two major bands at 120 and 70 kDa. Purification of large amounts of active NCX1 via this methodology should facilitate biophysical analysis of the protein. The larva expression system has broad-based application for membrane proteins where expression and purification of quantities required for physical analyses is problematic.

Fractionation of soybean phospholipids by preparative high-performance liquid chromatography with sorbents of various particle size

Normal-phase high-performance liquid chromatography was used on a preparative scale to seperate phosphatidylethanolamine (PE), phosphatidylinositol (PI) and phosphatidylcholine (PC) phospholipids from soybean. Separation was achieved using mixtures of three solvents, hexane, methanol and isopropanol. The optimized mobile phase compositions were experimentally determined while operating in a linear gradient mode using 15, 5–20, 25–40, and 40–63 μm preparative particles as well as 4 μm analytical particles. A gradient mobile phase was established on a commmercially available analytical Nova-Pak column such that hexane linearly decreased from 85 to 0 as isopropanol and methanol linearly increased in two gradient steps from 10 to 30 and 5 to 70 respectively. The total run time was 25 min at a flow-rate of 1.5 ml/min. A slight change in mobile phase composition was required to increase the resolution of phospholipids. The 15 μm particle size gave the best separation of the preparative particle sizes examined based on their resolutions between PE and PI and PI and PC. Finally, the retention factors of PE and PC were correlated in terms of mobile phase composition.

Effect of phosphatidylcholine on skin permeation of indomethacin from gel prepared with liquid paraffin and hydrogenated phospholipid

The effects of hydrogenated and unhydrogenated phosphatidylcholine (HPC, PC) on the permeation of indomethacin (IM) through hairless rat skin were investigated using liquid paraffin (LP) and a gel prepared with LP and hydrogenated soybean phospholipid (HSL). IM solubility at 95°C increased in proportion to the concentration of HPC or PC, whereas solubility at 37°C did not increase with HPC. IM showed no permeation until 10 h from LP without HPC/PC, but permeated at rates of ≈5 and 10 μg/cm 2 within 10 h from LP with HPC and PC, respectively. The permeation from the gel with various formulations (HSL, 15%; PC/HPC, 0–5%; IM, 0.5–2%) was determined. Permeation rates were 1.7–4.8 μg/cm 2 per h and were proportional to the skin concentration. Skin concentration was correlated to the release rate from the gel. We concluded that IM was solubilized by phospholipids, high activity in the vehicle led to high partition of IM in skin, and permeation increased due to a high skin concentration.

Effect of microsome–liposome fusion on the rotational mobility of cytochrome P450IIB4 in rabbit liver microsomes

Membrane fusion of microsomes with soybean phospholipid vesicles was performed at pH 6.5 to investigate the effect of lipid-enrichment in the membrane on the rotational mobility of cytochrome P450. Rotational diffusion of cytochrome P450 in the microsomal membrane of phenobarbital-induced rabbit liver was measured by detecting the decay of absorption anisotropy after photolysis of the heme CO complex by a vertically polarized laser flash. The fusion procedures yielded three separate fractions upon sucrose density gradient centrifugation with lipid-to-protein ratio in weight (L/P) as follows: 1.5 in the bottom fraction, 2.2 in the middle fraction, and 3.9 in the top fraction. In each fraction, co-existence of mobile and immobile cytochrome P450 was observed. The percentage of rotationally mobile P450 (with the mean rotational relaxation time of φ=505–828 μs) in each of the different bands was found to be 59% in the bottom fraction, 61% in the middle fraction, and 68% in the top fraction. This increase in mobile population of P450 due to lipid-enrichment indicates that aggregated proteins in microsomal membranes dissociate with increasing L/P which is inversely proportional to the protein concentration in the membrane. With freeze-fracture electron microscopy, it was shown that the average distance increased between intramembrane particles by lipid-enrichment. Thus, the significant immobile population (32%) of P450 in microsomal membranes can be explained by nonspecific protein aggregation which is a consequence of the low L/P of 0.8. The decrease in the mobile population in the bottom fraction compared with intact microsomes was shown to be due to the pH 6.5 incubation used for fusion.

Effect of phloretin on ionophore mediated electroneutral transmembrane translocations of H+, K+ and Na+ in phospholipid vesicles

Rates of M+/H+ exchange (M+=K+, Na+) across phospholipid membranes by ionophore mediated electroneutral translocations and transports through channels could either increase or decrease or change negligibly on adding the polar molecule phloretin to the membrane. The changes depend on pH, the concentration and choice of M+ and choice of ionophore/channel. Such diverse behaviours have been inferred from studies on the decay of the pH difference across soybean phospholipid vesicular membrane (=ΔpH). The transporters used in this study are (a) the exchange ionophores: nigericin, monensin; (b) combinations of alkali metal ion carriers, valinomycin or nonactin with weak acids carbonyl cyanide m-chlorophenylhydrazone or 2,4-dinitrophenol and (c) channels formed by gramicidin A. All the diverse results can be rationally explained if we take note of the following. (i) The rate limiting steps are associated with the transmembrane translocations involving the rate limiting species identified in the literature. (ii) Phloretin in the membrane decreases the apparent M+ dissociation constant, KM, of the M+ bound ionophores/channels which has the effect of increasing the concentration of these species. (iii) The concentrations of H+ bound ionophores/channels decrease on adding phloretin. (iv) Phloretin inhibits ternary complex formation (involving valinomycin or nonactin, M+ and an anion) by forming 1:2 complexes with valinomycin–M+ or nonactin–M+. (v) On adding 6-ketocholestanol to the membrane (instead of phloretin) KM increases. The decreases/increases in KM mentioned above are consistent with the consequences of a hypothesis in which phloretin decreases and 6-ketocholestanol increases the positive internal membrane dipole potential.

Complexing of basic pancreatic proteinase inhibitor with soybean phospholipid multilamellar vesicles.

The formation of complexes of basic pancreatic proteinase inhibitor (BPTI) with multilamellar vesicles (MLV) from six preparations of soybean phospholipids of various composition was studied. When BPTI, a non-membrane protein, interacts with MLV, the vesicles aggregate, forming a precipitate of protein-lipid complexes. The BPTI content in the protein-lipid complexes increases with decreasing pH of the medium and on addition of negatively charged components into the lipid mixture. The protein-induced aggregation of the phospholipid vesicles is suggested to be mainly determined by electrostatic forces. The antiproteinase activity of BPTI in the complexes was rather low but increased up to 70% of the initial activity on addition of an ionic detergent (sodium deoxycholate).

Proteoliposomes Colocalized with Endogenous Mitochondria in Mouse Fertilized Egg

Colocalization of mitochondria is the first step of intermitochondrial interaction or fusion in a cell. Here, we showed colocalization between exogenous mitochondria and endogenous ones or between exogenous proteoliposomes and endogenous mitochondria in mouse fertilized eggs by confocal laser microscopy. Isolated mitochondria from mouse liver and proteoliposomes containing mitochondrial membrane were directly labeled with red fluorescent aliphatic marker, PKH26, which is incorporated into lipid membrane, and then were microinjected into fertilized mouse eggs. Exogenous mitochondria appeared to be almost colocalized with endogenous mitochondria at the 4- and 8-cell stages, when mitochondria were stained with Rhodamine 123 (green fluorescent marker). On the contrary, when liposomes consisted of soy bean phospholipid were microinjected into the eggs as a control, their localization was different from that of endogenous mitochondria. Next, the submitochondrial particles and proteoliposomes were microinjected. Both the proteoliposomes and the submitochondrial particles appeared to colocalize with endogenous mitochondria at the 4-cell stage. These results suggest the existence of a factor that makes liposomes colocalize with mitochondria. Such a proteoliposome would be useful for the development of mitochondrial gene transfer techniques.

An ethylene-induced cDNA encoding a lipase expressed at the onset of senescence.

A cDNA clone encoding a lipase (lipolytic acyl hydrolase) expressed at the onset of petal senescence has been isolated by screening a cDNA expression library prepared from carnation flowers (Dianthus caryophyllus). The cDNA contains the lipase consensus sequence, ITFAGHSLGA, and encodes a 447-amino acid polypeptide with a calculated molecular mass of 50.2 kDa that appears to be a cytosolic protein. Over-expression of the clone in Escherichia coli yielded a protein of the expected molecular weight that proved capable of deesterifying fatty acids from p-nitrophenylpalmitate, tri-linolein, soybean phospholipid, and Tween in both in vitro and in situ assays of enzyme activity. The abundance of the lipase mRNA increases just as carnation flowers begin to senesce, and expression of the gene is also induced by treatment with ethylene. Southern blot analyses of carnation genomic DNA have indicated that the lipase is a single copy gene. The lipase gene is also expressed in carnation leaves and is up-regulated when the leaves are treated with ethylene. Deesterification of membrane lipids and ensuing loss of membrane structural integrity are well established early events of plant senescence, and the expression pattern of this lipase gene together with the lipolytic activity of its cognate protein indicate that it plays a fundamentally central role in mediating the onset of senescence.

The Effect of Lipid Environment and Retinoids on the ATPase Activity of ABCR, the Photoreceptor ABC Transporter Responsible for Stargardt Macular Dystrophy

ABCR is a photoreceptor-specific ATP-binding cassette transporter that has been linked to various retinal diseases, including Stargardt macular dystrophy, and implicated in retinal transport across rod outer segment (ROS) membranes. We have examined the ATPase and GTPase activity of detergent-solubilized and reconstituted ABCR. 3-[(3-Cholamidopropyl)dimethylammonio]-1-propanesulfonic acid-solubilized ABCR had ATPase and GTPase activity (K(m) approximately 75 micrometer V(max) approximately 200 nmol/min/mg) that was stimulated 1.5-2-fold by all-trans-retinal and dependent on phospholipid and dithiothreitol. The K(m) for ATP decreased to approximately 25 micrometer after reconstitution, whereas the V(max) was strongly dependent on the lipid used for reconstitution. ABCR reconstituted in ROS phospholipid had a V(max) for basal and retinal activated ATPase activity that was 4-6 times higher than for ABCR in soybean or brain phospholipid. This enhanced activity was mainly due to the high phosphatidylethanolamine (PE) content of ROS membranes. PE was also required for retinoid-stimulated ATPase activity. ATPase activity of ABCR was stimulated by the addition of N-retinylidene-PE but not the reduced derivative, retinyl-PE. ABCR expressed in COS-1 cells also exhibited retinal-stimulated ATPase activity similar to that of the native protein. These results support the view that ABCR is an active retinoid transporter, the nucleotidase activity of which is strongly influenced by its lipid environment.

Characterization of a P-type Na+-ATPase of a Facultatively Anaerobic Alkaliphile, Exiguobacterium aurantiacum

A facultatively anaerobic alkaliphile, Exiguobacterium aurantiacum, possesses a P-type Na(+)-stimulated ATPase in the membrane (Koyama, N. (1999) Curr. Microbiol. 39, 27-30). In this study, we attempted to purify and characterize the enzyme. The ATPase appears to consist of a single polypeptide with an apparent molecular mass of 100 kDa. The enzyme exhibited an optimum pH for activity at around 9. The enzyme was strongly inhibited by vanadate (50% inhibition observed at 3 microm) and forms an acylphosphate intermediate, suggesting a P-type ATPase. The enzyme, when reconstituted into soybean phospholipid vesicles, exhibited ATP-dependent (22)Na(+) uptake, which was completely inhibited by gramicidin. The reconstituted vesicles exhibited a generation of membrane potential (positive, inside). The enzyme is likely to be involved in an electrogenic transport of Na(+).

Effect of tranilast oily gel on carrageenin-induced granulation in rats.

Tranilast (TL) oily gels consisting of hydrogenated soybean phospholipid and fatty-acid ester were prepared, and the inhibitory effect of the gels on the growth of granulation tissue were evaluated in a carrageenin-induced rat granulation model. By the application of 0.1 and 0.2% TL oily gel, the weight of granulation tissue was significantly reduced to 64 and 55%, respectively, of control value. Furthermore, these gels reduced their respective hydroxyproline content to 64 and 51% of the control. On the other hand, the inhibitory effect of 10% TL ointments, which are clinically used for the treatment of keloids and hypertrophic scars as hospital preparations, was much lower than that of the oily gels. In addition, the application of 0.1 and 0.2% oily gel led to high concentration (0.1% gel, 168+/-18 microg/g; 0.2% gel, 221+/-16 microg/g) of TL in the dermis as compared with the 10% TL ointments. These results suggest that TL oily gels may be a useful topical formulation for the treatment of keloids and hypertrophic scars.

Determination of retention factors of phospholipids in gradient HPLC

The mechanism of separation of solutes under gradient elution conditions has been studied. Separation of a mixture of soybean phospholipids (phosphatidylethanolamine, phosphatidylinositol, and phosphatidylcholine) in the ternary mobile phase: methanol, hexane, isopropanol was considered as an example. An analytical expression in terms of the composition of the ternary mobile phase was obtained to calculate retention factors in two-step gradient modes. The following new polynomial equation was derived: logk=A+BF+CF2+DG+EG2 where A, B, C, D, and E are empirical coefficients; F and G are volume fractions of isopropanol and methanol respectively in the mobile phase based on hexane. The coefficients for phospholipids were obtained under gradient conditions.

Fractionation of soybean phospholipids by high‐performance liquid chromatography with an evaporative light‐scattering detector

AbstractPhosphatidylcholine (PC) and phosphatidylethanolamine (PE) from 23 soybean lines with a wide range of fatty acid compositions were resolved into seven fractions by high‐performance liquid chromatography (HPLC). Fraction identities were assigned from fatty acid compositions determined by gas chromatography (GC). A mass detector, i.e., an evaporative light‐scattering detector, was used for HPLC quantification. The detector response was a power function of PC and PE concentrations. Various correction methods were applied to the detector response to obtain the best agreement between phospholipid (PL) fatty acid compositions determined by GC and that calculated from the corrected HPLC fraction percentages. The corrected HPLC fraction composition also was compared with that calculated from stereospecific distribution data using a 1‐random‐2‐random hypothesis. Correlation between PL‐fatty acid and HPLC‐fraction percentages showed that genetic modification of soybean oil composition caused changes in PL species, which alter physical properties and may alter the physiological functions of PL in biomembranes.