PIPES Buffer Research Articles

Optimisation of in vitro measurement of available iron from different fortificants in citric fruit juices

The percentage of dialyzable ferrous and total iron were studied in a citric juice (pineapple and passion fruit) fortified with ferrous sulphate, micronised dispersible ferric pyrophosphate and ferrous bis-glycinate in similar concentrations (49.2 mg Fe/l). The in vitro method of Kapsokefalou and Miller (1991) [Kapsokefalou, M., & Miller, D. D. (1991). Effects of meat and selected food components on the valence of nonheme iron during in vitro digestion. Journal of Food Science, 56, 352–355.] was optimised for this matrix using 0.15 N PIPES buffer (pH 8.5) to adjust pH during pancreatic digestion. We also studied different pH values of Hepes buffer used in the measurement of iron concentrations with Ferrozine (chromogen solution). The maximum absorbances were obtained with a Hepes buffer pH value of 8.5. Ferrous sulphate was used as a reference salt due to its high bioavailability, although novel compounds, such as ferrous bis-glycinate and micronised dispersible ferric pyrophosphate, showed a high relative iron availability in this juice. Taking into account that percentage of dialysable ferrous iron is considered to be the more available fraction of total iron, the iron fortificant ferrous bis-glycinate proved to be more adequate for fortifing citric juices, giving a 10.7% of dialyzable ferrous iron. Moreover, the percentage of dialyzable total iron from ferrous bis-glycinate (31.0%) was statistically higher than those from ferrous sulphate and micronised ferric pyrophosphate (28.4% and 28.2%, respectively).

Turbidity as a probe of tubulin polymerization kinetics: A theoretical and experimental re-examination

We report here an examination of the validity of the experimental practice of using solution turbidity to study the polymerization kinetics of microtubule formation. The investigative approach proceeds via numerical solution of model rate equations to yield the time dependence of each microtubule species, followed by the calculation of the time- and wavelength-dependent turbidity generated by the calculated distribution of rod lengths. The wavelength dependence of the turbidity along the time course is analyzed to search for generalized kinetic regimes that satisfy a constant proportionality relationship between the observed turbidity and the weight concentration of polymerized tubulin. An empirical analysis, which permits valid interpretation of turbidity data for distributions of microtubules that are not long relative to the wavelength of incident light, is proposed. The basic correctness of the simulation work is shown by the analysis of the experimental time dependence of the turbidity wavelength exponent for microtubule formation in taxol-supplemented 0.1 M Pipes buffer (1 mM GTP, 1 mM EGTA, 1 mM MgSO 4, pH 6.4). We believe that the general findings and principles outlined here are applicable to studies of other fibril-forming systems that use turbidity as a marker of polymerization progress.

Na,K-ATPase from Mice Lacking the γ Subunit (FXYD2) Exhibits Altered Na+ Affinity and Decreased Thermal Stability

The gamma subunit of the Na,K-ATPase, a 7-kDa single-span membrane protein, is a member of the FXYD gene family. Several FXYD proteins have been shown to bind to Na,K-ATPase and modulate its properties, and each FXYD protein appears to alter enzyme kinetics differently. Different results have sometimes been obtained with different experimental systems, however. To test for effects of gamma in a native tissue environment, mice lacking a functional gamma subunit gene (Fxyd2) were generated. These mice were viable and without observable pathology. Prior work in the mouse embryo showed that gamma is expressed at the blastocyst stage. However, there was no delay in blastocele formation, and the expected Mendelian ratios of offspring were obtained even with Fxyd2-/- dams. In adult Fxyd2-/- mouse kidney, splice variants of gamma that have different nephron segment-specific expression patterns were absent. Purified gamma-deficient renal Na,K-ATPase displayed higher apparent affinity for Na+ without significant change in apparent affinity for K+. Affinity for ATP, which was expected to be decreased, was instead slightly increased. The results suggest that regulation of Na+ sensitivity is a major functional role for this protein, whereas regulation of ATP affinity may be context-specific. Most importantly, this implies that gamma and other FXYD proteins have their effects by local and not global conformation change. Na,K-ATPase lacking the gamma subunit had increased thermal lability. Combined with other evidence that gamma participates in an early step of thermal denaturation, this indicates that FXYD proteins may play an important structural role in the enzyme complex.

The Development of Marine Toxicity Identification Evaluation (TIE) Procedures Using the Unicellular Alga Nitzschia closterium

Unicellular algae are highly sensitive to a wide range of toxicants and have been used extensively in ecotoxicological testing. This, along with their ability to grow in very small test volumes over short test durations, make them ideal test organisms for use in Toxicity Identification Evaluations (TIEs). Despite this, microalgae have not previously been used in marine TIE studies. In this study, the marine diatom Nitzschia closterium was shown to be a highly suitable test organism after modification of the standard test protocol to reduce test volumes to 6 mL and test duration to 48 h. The alga was tolerant to the chemicals used in phase I of the standard USEPA TIE protocol, and physical TIE manipulations had no effect on algal growth. The cation exchange procedure, however, inhibited algal growth, while the anion exchange procedure stimulated growth, making these two procedures unsuitable for use with this species. Of the buffers trialed for the graduated pH procedure, 0.01 M PIPES buffer was found to be suitable for buffering at pH 7 because it maintained the required pH over the duration of the test and did not affect the growth or sensitivity of the algae to one reference toxicant (copper). A trial TIE on a secondary-treated sewage effluent for discharge into coastal waters showed that the developed protocols could successfully be used to identify ammonia as the major toxicant in the effluent.

Decrypting the Biochemical Function of an Essential Gene from Streptococcus pneumoniae Using ThermoFluor® Technology

The protein product of an essential gene of unknown function from Streptococcus pneumoniae was expressed and purified for screening in the ThermoFluor affinity screening assay. This assay can detect ligand binding to proteins of unknown function. The recombinant protein was found to be in a dimeric, native-like folded state and to unfold cooperatively. ThermoFluor was used to screen the protein against a library of 3000 compounds that were specifically selected to provide information about possible biological functions. The results of this screen identified pyridoxal phosphate and pyridoxamine phosphate as equilibrium binding ligands (K(d) approximately 50 pM, K(d) approximately 2.5 microM, respectively), consistent with an enzymatic cofactor function. Several nucleotides and nucleotide sugars were also identified as ligands of this protein. Sequence comparison with two enzymes of known structure but relatively low overall sequence homology established that several key residues directly involved in pyridoxal phosphate binding were strictly conserved. Screening a collection of generic drugs and natural products identified the antifungal compound canescin A as an irreversible covalent modifier of the enzyme. Our investigation of this protein indicates that its probable biological role is that of a nucleoside diphospho-keto-sugar aminotransferase, although the preferred keto-sugar substrate remains unknown. These experiments demonstrate the utility of a generic affinity-based ligand binding technology in decrypting possible biological functions of a protein, an approach that is both independent of and complementary to existing genomic and proteomic technologies.

A Neural Network Model in the Calibration of Glucose Sensor Based on the Immobilization of Glucose Oxidase into Polypyrrole Matrix

AbstractThe immobilization of enzymes on an electrode surface is of great importance in bioelectrochemistry. The entrapment of enzymes into a polymer matrix is simple and a speedy technique for the production of biosensors. This procedure of enzyme immobilization by electropolymerization has a great significance in fabrication of micro sensors in the preparation of multiplayer devices. In current study, glucose oxidase enzyme that is specific for the glucose determination was entrapped into polypyrrole matrix containing p‐benzoquinone in PIPES buffer and glucose sensitivity of the biosensor was investigated. Then, artificial neural network analysis was done for the nonlinear calibration plot. This implementation can be used for the sensor failure detection, as well. The estimation power of the neural network used in the direct and inverse calibration modelling was examined by statistical methods. It presented the good performance for the estimation power.

Microplate quantification of plant leaf superoxide dismutases

Superoxide dismutases (SODs) catalyze the dismutation of superoxide radicals in a broad range of organisms, including plants. Quantification of SOD activity in crude plant extracts has been problematic due to the presence of compounds that interfere with the dose–response of the assay. Although strategies exist to partially purify SODs from plant extracts, the requirement for purification limits the rapidity and practical number of assays that can be conducted. In this article, we describe modification of a procedure using o-dianisidine as substrate that permits relatively rapid quantification of SOD activity in crude leaf extracts in a microplate format. The method employs the use of a commercial apparatus that permits lysis of 12 tissue samples at once and the use of Pipes buffer to reduce interference from compounds present in crude leaf extracts. The assay provided a linear response from 1 to 50 units of SOD. The utility of the assay was demonstrated using tissue extracts prepared from a group of taxonomically diverse plants. Reaction rates with tissue extracts from two grasses were linear for at least 60 min. Tissues of certain species contained interfering compounds, most of which could be removed by ultrafiltration. The presence of plant catalases, peroxidases, and ascorbate in physiological quantities did not interfere with the assay. This approach provides a means to quantify SOD activity in relatively large numbers of plant samples provided that the possibility for the presence of interfering compounds is considered. The presence of interfering compounds in certain plant tissues necessitates caution in interpreting the effects of plant stresses on SOD.

Selective cyclooxygenase-2-inhibitors can modulate IgE-mediated histamine release in vitro

Rationale The aim of the study was to evaluate the effect of the non-steroidal selective cyclooxygenase (COX)-2 inhibitor celecoxib on IgE-dependent in vitro histamine release (HR) from blood basophils induced by anti-IgE or specific allergens. Methods The study was performed using the automated glass fiber-based whole blood leukocyte histamine release test. Celecoxib dissolved in DMSO was gradually diluted first in 50% of ethanol and then in pH 7.4 PIPES buffer. Eight five-fold dilutions of celecoxib were made in PIPES buffer ranging from 2.5×10 −4 M to 2.5×10 −11 M. Heparinized whole blood was obtained from healthy donors and from patients with sensitivity to allergens confirmed by clinical history, skin tests and RAST. Blood from each qualified patient was incubated with different concentrations of celecoxib during 0, 1, 2, 4, and 8 hours with subsequent challenge with anti-IgE or specific allergens. Results Incubation of blood with celecoxib during 0, 1, and 2 hours and subsequent stimulation with anti-IgE gave a concentration-dependent of moderate increase of HR while incubation for more time (4-8 hours) inhibited HR from blood basophils. Pre-incubation of blood from allergic patients with high concentrations of celecoxib during 4-8 hours and subsequent stimulation with specific allergens inhibited HR but there was no effect on HR after 1-2 hours of preliminary incubation at low concentrations of celecoxib. Conclusions Celecoxib can modulate basophil HR induced by IgE, suggesting that COX-2 inhibition may modulate allergic responses.

Inhibition of copper-catalyzed cysteine oxidation by nanomolar concentrations of iron salts

Problems caused by the presence of adventitious metals in buffers and reagents are well recognized in studies of metal-catalyzed oxidation reactions. In most cases, metal contamination leads to an increase in rate, and chelating agents are inhibitory. In the present study, however, the rate of copper-catalyzed oxidation of cysteine was found to be increased by buffer purification with Chelex resin or by addition of micromolar concentrations of the specific iron chelator desferrioxamine (DFO). These effects are attributable to inhibition of copper-catalyzed oxidation by adventitious iron. In purified buffer at pH 7.25, containing 0.4 μM copper, cysteine was oxidized at a rate of 32 μM/min. Addition of iron salts to this buffer caused a dose-related decrease in this rate, up to a maximum of 85%. A 50% decrease in rate was recorded at an iron concentration of only 11 nM. Other transition metals were without effect. Similar effects of purification or addition of DFO on the rate of cysteine oxidation were seen in Tris, glycylglycine, Mops, and Pipes buffers. Catalase decreased the rate of cysteine oxidation, but the sensitivity to iron was similar in the presence and absence of catalase. Copper-catalyzed oxidation of cysteamine and reduced glutathione was much less sensitive to inhibition by iron. Our results offer an explanation for the conflicting literature reports of the effects of chelating agents and catalase on cysteine oxidation, and emphasize the need for buffer purification or addition of DFO in studies concerned with the oxidation or cytotoxicity of this thiol. The exceptional sensitivity of copper-catalyzed cysteine oxidation to iron makes this an attractive system for monitoring the iron content of buffers, and may also have application for determining the free iron content of physiological fluids.

Demineralization of Dentin by Streptococcus mutans Biofilms Grown in the Constant Depth Film Fermentor

To develop a bacterial demineralization model, we grew Streptococcus mutans biofilms in a constant depth film fermentor (CDFF) and studied the effects of sucrose pulsing frequency (SPF) in time on dentin demineralization. S. mutans biofilms were grown in dentin specimens with grooves and on dentin surface specimens for 20 days. During the experiments, 2% sucrose was pulsed either 4 or 8 times per day for periods of 30 min. Diluted brain-heart infusion medium containing 25 mM PIPES buffer and 1.5 mM CaCl₂ was pulsed as the alternative growth medium. Specimens with intact biofilms were taken out on days 5, 12 and 20. The model was assessed by viable counts of the biofilm, mineral loss and lesion depth in the dentin specimens (by transversal microradiography) and pH measurements in the groove (by pH microelectrode). The results showed that biofilms formed on the dentin surface specimens were constant in viable counts for the low SPF, while this parameter tended to increase with time under the high SPF. Lesions with intact surfaces were formed and the lesion size increased significantly over time and increased significantly with increasing SPF. Typical Stephan curves were found after sucrose pulsing. The pH inside the groove returned to neutral under low SPF, but remained below 6.5 under high SPF. With the CDFF S. mutans biofilm model, lesions can be created in dentin within reasonable experimental time periods, as a result of the presence of a biofilm and in response to carbohydrate challenges.

Purification of brain tubulin through two cycles of polymerization–depolymerization in a high-molarity buffer

Microtubules can be assembled in vitro from purified α/β tubulin heterodimers in the presence of GTP. Tubulin is routinely obtained from animal brain tissue through repetitive cycles of polymerization–depolymerization, followed by ion-exchange chromatography to remove any contaminating microtubule-associated proteins and motors. Here, we show that only two cycles of polymerization–depolymerization of pig brain tubulin in the presence of a high-molarity PIPES buffer allow the efficient removal of contaminating proteins and production of a high-concentration tubulin solution. The proposed protocol is rapid and yields more active tubulin than the traditional ion-exchange chromatography-based procedures.

Investigation of electrochemical properties of FMN and FAD adsorbed on titanium electrode

The electrochemical properties (such as the values of the formal potentials, the dependence of the formal potentials on solution pH, the reversibility of the electrochemical process) of flavin mononucleotide (FMN) and flavin adenine dinucleotide (FAD) adsorbed on a titanium electrode were dependent on the electrolyte. The formal potentials of adsorbed FMN and FAD in phosphate, HEPES and PIPES buffers at pH 7 were similar to those for dissolved flavins (−460 to −480 mV vs. SCE) and changed linearly with a slope of about 52 mV per pH unit in the pH region 3 to 8. In TRIS buffer, the formal potentials of adsorbed FMN and FAD were also pH-dependent, however, with invariance in the pH range 4.5 to 5.5. In non-buffered solutions (KCl, LiCl, NaCl, CsCl, CaCl 2, Na 2SO 4 at different concentrations), the electrochemical behavior of adsorbed FMN and FAD differed from that of dissolved flavins and was dependent on the electrolyte (especially at pH 4.5 and pH 5). Under certain conditions (electrolyte, concentration, pH), a two-step oxidation of FMN could be observed.

Degradation of copper-NTA by Mesorhizobium sp. NCIMB 13524

Abstract In a series of enrichment isolations, a mixed culture was obtained from soil which was able to degrade copper nitrilotriacetate. In phosphate buffered (metal-complexing) medium this mixed culture grew on and completely degraded Mn-, Fe-, Co-, Cu- and Zn-NTA after 14 d incubation at 30°C. In PIPES-buffered (non-complexing) medium, the mixed culture completely degraded free NTA and Mn-, Fe- and Zn-NTA; Cu-NTA was partially degraded (54%) and Co-NTA was not degraded. A pure culture of Mesorhizobium sp. (NCIMB 13524) isolated from the mixed culture completely degraded free NTA and Mn-, Fe-, Co-, Ni- Cu-, and Zn-NTA in phosphate buffer after 2– 24 d incubation at 30°C. In PIPES buffer, growth and NTA degradation by Mesorhizobium sp. was inhibited stoichiometrically by copper, suggesting that only the free NTA was degraded. In addition, fully complexed Ni-NTA was not degraded in PIPES buffer, and Co-NTA was only partially degraded (18.1%). The importance of speciation, and the effect of the buffer on NTA degradation is discussed.

Tris (2,2′-bipyridil) copper (II) chloride complex: a biomimetic tyrosinase catalyst in the amperometric sensor construction

The use of tris (2,2′-bipyridil) copper (II) chloride complex, [Cu(bipy) 3]Cl 2·6H 2O, as a biomimetic catalyst, is reported in the construction of an amperometric sensor for dopamine. The sensor was prepared modifying a glassy carbon electrode with a Nafion® membrane doped with the complex. The optimized conditions for the sensor response were obtained in 0.25 mol dm −3 Pipes buffer (pH 7.0) containing 150 μmol dm −3 H 2O 2, with an applied potential of −50 mV versus saturated calomel electrode (SCE). In these conditions, a linear response range between 9 and 230 μmol dm −3, with a sensitivity of 1.43±0.01 nA dm 3 μmol −1 cm −2 and a detection limit of 4.8 μmol dm −3 were observed for dopamine. The response time for this sensor was about 1 s, presenting the same response for at least 150 successive measurements, with a good repeatability (4.8%) expressed as relative standard deviation for n=13. After its construction, this sensor can be used after 180 days without loss of sensitivity, kept at room temperature. The difference of the sensor response between four preparations was 4.2%. A detailed investigation about the sensor response for other eighteen phenolic compounds and five interfering species was performed. The sensor was applied for dopamine determination in pharmaceutical preparation with success.

Thermochemistry of the biochemical reaction: { pyrophosphate(aq) + H2O(l) = 2 phosphate(aq)}

Calorimetric enthalpies of reaction have been measured for the overall biochemical reaction{pyrophosphate(aq) + H2O(l) = 2phosphate (aq)} . The reaction was catalyzed by alkaline phosphatase and, to simplify the thermochemistry, was carried out in the absence of Mg 2+(aq). Measurements were performed with phosphate buffer ( pH = 7.19 and 7.94), PIPES buffer ( pH = 7.13), and HEPES buffer ( pH = 7.86). The results of these measurements were analyzed by using an equilibrium model. These calculations lead to the standard molar enthalpy changeΔrHmo=−(17.3 ± 0.6)kJ·mol−1 (temperature T= 298.15 K and ionic strengthI= 0) for the reference reaction{HP2O73−(aq) + H2O(l) = 2HPO42−(aq) + H+(aq)} . Values of the apparent equilibrium constantK′ for the overall biochemical reaction from the literature were also analyzed by using the equilibrium model in order to obtain what is believed to be a reliable value for the equilibrium constantK= 4.7 · 10−4 for the reference reaction. The values ofK and ΔrHmo for the reference reaction have been used together with values from the CODATA tables to calculate standard molar formation properties for the pyrophosphate species.

Modulation of Mouse Paneth Cell α-Defensin Secretion by mIKCa1, a Ca2+-activated, Intermediate Conductance Potassium Channel

Paneth cells in small intestinal crypts secrete microbicidal alpha-defensins in response to bacteria and bacterial antigens (Ayabe, T., Satchell, D. P., Wilson, C. L., Parks, W. C., Selsted, M. E., and Ouellette, A. J. (2000) Nat. Immunol. 1, 113- 38). We now report that the Ca(2+)-activated K(+) channel mIKCa1 modulates mouse Paneth cell secretion. mIKCa1 cDNA clones identified in a mouse small intestinal crypt library by hybridization to human IKCa1 cDNA probes were isolated, and DNA sequence analysis showed that they were identical to mIKCa1 cDNAs isolated from erythroid cells and liver. The genomic organization was found to be conserved between mouse and human IKCa1 as shown by comparisons of the respective cDNA and genomic sequences. Reverse transcriptase-PCR experiments using nested primers amplified mIKCa1 from the lower half of bisected crypts and from single Paneth cells, but not from the upper half of bisected crypts, villus epithelium, or undifferentiated crypt epithelial cells, suggesting a lineage-specific role for mIKCa1 in mouse small bowel epithelium. The cloned mIKCa1 channel was calcium-activated and was blocked by ten structurally diverse peptide and nonpeptide inhibitors with potencies spanning 9 orders of magnitude and indistinguishable from that of the human homologue. Consistent with channel blockade, charybdotoxin, clotrimazole, and the highly selective IKCa1 inhibitors, TRAM-34 and TRAM-39, inhibited (approximately 50%) Paneth cell secretion stimulated by bacteria or bacterial lipopolysaccharide, measured both as bactericidal activity and secreted cryptdin protein, but the inactive analog, TRAM-7, did not block secretion. These results demonstrate that mIKCa1 is modulator of Paneth cell alpha-defensin secretion and disclose an involvement in mucosal defense of the intestinal epithelium against ingested bacterial pathogens.

Electrochemical behavior of the bis(2,2'-bipyridyl)copper(II) complex immobilized on a self-assembled monolayer modified electrode for L-ascorbic acid detection.

Modification of a gold electrode has been achieved by immobilizing a bis(2,2'-bipyridyl)copper(II) complex in a self-assembled monolayer (SAM) of 3-mercaptopropionic acid. The electrostatic interaction of the negatively charged SAM with a di-positive copper complex allowed the attachment. The modified electrode exhibited excellent redox behavior. The dependence of the modified electrode response was investigated in terms of pH, supporting electrolyte and ionic strength. Moreover, it showed good electrocatalytic activity for ascorbic acid oxidation, allowing convenient quantification at levels down to 8.1 x 10(-8) mol l(-1). The [Cu(bipy)2]/SAM modified electrode under optimized operational conditions (PIPES buffer 0.01 mol l(-1) at pH 6.8 and 200 mV vs. SCE) presented a linear response range between 1.0 micromol l(-1) and 100.0 micromol l(-1) for ascorbic acid. This modified electrode also presented an excellent repeatability, showing a relative standard deviation of 2.1% for a series of 12 successive measurements of a 5.0 micromol l(-1) ascorbic acid solution. Furthermore, the electroactivity was maintained over a long period (e.g., 92% after 100 determinations).

In vitro measurement of available iron in fortified foods

The objective of this study was to investigate the influence of base systems used to regulate pH values of digests on iron dialysability as an indicator of bioavailable iron. We studied the pertinence of measuring titratable acidity to pH 7.5 with KOH to calculate mEq of NaHCO 3 to be used for pancreatic digestion/dialysis. The pH achieved using the same mEq of each base was lower when using NaHCO 3 than when using KOH. The discrepancy between achieved and attempted pH was uneven for several iron sources. Differences in pH regulation procedure, including type and concentration of base or buffer added to the pepsin digest rendered different final digest/dialysate pH values, thus affecting dialysable iron. A modification of in vitro equilibrium dialysis method is proposed using PIPES buffer of sufficient molarity to obtain a uniform final pH of 6.5 in digest/dialysate systems. The main factors taken into account to calculate buffer concentration were buffer capacity of food matrix (HCl mEq required to reach pH 2), HCl mEq included in the aliquot of pepsin suspension, acid or base mEq generated through enzymatic hydrolysis during in vitro digestion and intrinsic food pH (HCl mEq to adjust food matrix pH to 6.5). With these data buffer molarity for each food matrix can be calculated. Modifications suggested for the equilibrium dialysis method allowed development of a uniform final pH of the digest/dialysate system in a variety of foods assayed.

La 3+ stabilizes the hexagonal II (H II) phase in phosphatidylethanolamine membranes

The mechanism of the effects of the lanthanum ion (La 3+) and the gadolinium ion (Gd 3+), which are lanthanides, on the function of membrane proteins and the stability of the membrane structure is not well understood. We investigated the effects of La 3+ on the stability of the hexagonal II (H II) phase of the phosphatidylethanolamine (PE) membrane at 20°C by small-angle X-ray scattering. As PE membrane we used DPOPE (dipalmitoleoylphosphatidylethanolamine) membrane, which was in the L α phase in 10 mM PIPES buffer (pH 7.4) at 20°C. An L α to H II phase transition occurred in the DPOPE membrane at 1.4 mM La 3+ in 0 M KCl, and at 0.4 mM La 3+ in 0.5 M KCl and above the critical concentrations the membranes were in the H II phase, indicating that La 3+ stabilizes the H II phase rather than the L α phase. The basis vector length, d, of DPOPE and DOPE (dioleoylphosphatidylethanolamine) membranes containing 16 wt% tetradecane in excess water condition did not change with an increase in La 3+ concentration, suggesting that La 3+ did not change the spontaneous curvature of these PE monolayer membranes. The chain-melting transition temperature of the dielaidoylphosphatidylethanolamine membrane increased with an increase in La 3+ concentration, indicating that the lateral compression pressure increased. To elucidate the effects of a small percentage of ‘guest’ lipids with longer acyl chains than the average length of ‘host’ lipids on the stability of the H II phase, we investigated the effects of the concentration of a guest lipid (DOPE) in a host lipid (DPOPE) membrane on their phase behavior and structure. 12 mol% DOPE induced an L α to H II phase transition in DOPE/DPOPE membrane, without changing the spontaneous curvature of the monolayer membrane. We found that Ca 2+ also induced an L α to H II phase transition in the DPOPE membrane, and compared the effects of Ca 2+ on PE membranes with those of La 3+. Based on these results, we have proposed a new model for the mechanism of the L α to H II phase transition and the stabilization of the H II phase by La 3+.

Mitochondrial glycerol phosphate acyltransferase directs the incorporation of exogenous fatty acids into triacylglycerol.

The mitochondrial isoform of glycerol-3-phosphate acyltransferase (GPAT), the first step in glycerolipid synthesis, is up-regulated by insulin and by high carbohydrate feeding via SREBP-1c, suggesting that it plays a role in triacylglycerol synthesis. To test this hypothesis, we overexpressed mitochondrial GPAT in Chinese hamster ovary (CHO) cells. When GPAT was overexpressed 3.8-fold, triacylglycerol mass was 2.7-fold higher than in control cells. After incubation with trace [(14)C]oleate ( approximately 3 microm), control cells incorporated 4.7-fold more label into phospholipid than triacylglycerol, but GPAT-overexpressing cells incorporated equal amounts of label into phospholipid and triacylglycerol. In GPAT-overexpressing cells, the incorporation of label into phospholipid, particularly phosphatidylcholine, decreased 30%, despite normal growth rate and phospholipid content, suggesting that exogenous oleate was directed primarily toward triacylglycerol synthesis. Transiently transfected HEK293 cells that expressed a 4.4-fold increase in GPAT activity incorporated 9.7-fold more [(14)C]oleate into triacylglycerol compared with control cells, showing that the effect of GPAT overexpression was similar in two different cell types that had been transfected by different methods. When the stable, GPAT-overexpressing CHO cells were incubated with 100 microm oleate to stimulate triacylglycerol synthesis, they incorporated 1.9-fold more fatty acid into triacylglycerol than did the control cells. Confocal microscopy of CHO and HEK293 cells transfected with the GPAT-FLAG construct showed that GPAT was located correctly in mitochondria and was not present elsewhere in the cell. These studies indicate that overexpressed mitochondrial GPAT directs incorporation of exogenous fatty acid into triacylglycerol rather than phospholipid and imply that (a) mitochondrial GPAT and lysophosphatidic acid acyltransferase produce a separate pool of lysophosphatidic acid and phosphatidic acid that must be transported to the endoplasmic reticulum where the terminal enzymes of triacylglycerol synthesis are located, and (b) this pool remains relatively separate from the pool of lysophosphatidic acid and phosphatidic acid that contributes to the synthesis of the major phospholipid species.