Relationships between molecular structure and kinetic and thermodynamic controls in lipid systems. Part III. Crystallization and phase behavior of 1-palmitoyl-2,3-stearoyl-sn-glycerol (PSS) and tristearoylglycerol (SSS) binary system

The phase behavior of 1-palmitoyl-2,3-distearoyl-sn-glycerol (PSS)/tristearoylglycerol (SSS) binary system was investigated in terms of polymorphism, crystallization and melting behavior, microstructure and solid fat content (SFC) using widely different constant cooling rates. Kinetic phase diagrams were experimentally determined from the DSC heating thermograms and analyzed using a thermodynamic model to account for non-ideality of mixing. The kinetic phase diagram presented a typical eutectic behavior with a eutectic point at the 0.5(PSS) mixture with a probable precipitation line from 0.5(PSS) to 1.0(PSS), regardless of the rate at which the sample was cooled. The eutectic temperature decreased only slightly with increasing cooling rate. PSS has a strong effect on the physical properties of the PSS-SSS mixtures. In fact, the overall phase behavior of the PSS-SSS binary system was determined, for a very large part, by the asymmetrical TAG. Moreover, PSS is a key driver of the high stability observed in crystal growth, polymorphism and phase development. Levels as low as 10% PSS, when cooled slowly, and 30% when cooled rapidly, were found to be sufficient to suppress the effect of thermal processing.     

An ultraviolet spectrophotometric assay for the screening of sn-2-specific lipases using 1,3-O-dioleoyl-2-O-α-eleostearoyl-sn-glycerol as substrate

In the present study, we propose a continuous assay for the screening of sn-2 lipases by using triacylglycerols (TAGs) from Aleurites fordii seed (tung oil) and a synthetic TAG containing the α-eleostearic acid at the sn-2 position and the oleic acid (OA) at the sn-1 and sn-3 positions [1,3-O-dioleoyl-2-O-α-eleostearoyl-sn-glycerol (sn-OEO)]. Each TAG was coated into a microplate well, and the lipase activity was measured by optical density increase at 272 nm due to transition of α-eleostearic acid from the adsorbed to the soluble state. The sn-1,3-regioselective lipases human pancreatic lipase (HPL), LIP2 lipase from Yarrowia lipolytica (YLLIP2), and a known sn-2 lipase, Candida antarctica lipase A (CALA) were used to validate this method. TLC analysis of lipolysis products showed that the lipases tested were able to hydrolyze the sn-OEO and the tung oil TAGs, but only CALA hydrolyzed the sn-2 position. The ratio of initial velocities on sn-OEO and tung oil TAGs was used to estimate the sn-2 preference of lipases. CALA was the enzyme with the highest ratio (0.22 ± 0.015), whereas HPL and YLLIP2 showed much lower ratios (0.072 ± 0.026 and 0.038 ± 0.016, respectively). This continuous sn-2 lipase assay is compatible with a high sample throughput and thus can be applied to the screening of sn-2 lipases.     

Production of aromatic green gasoline additives via catalytic pyrolysis of acidulated peanut oil soap stock

Catalytic pyrolysis was used to generate gasoline-compatible fuel from peanut oil soap stock (PSS), a high free fatty acid feedstock, using a fixed-bed reactor at temperatures between 450 and 550°C with a zeolite catalyst (HZSM-5). PSS fed at 81 gh(-1) along with 100 mL min(-1) inert gas was passed across a 15 g catalyst bed (WHSV=5.4h(-1), gas phase residence time=34s). Results indicate that fuel properties of PSS including viscosity, heating value, and O:C ratio were improved significantly. For PSS processed at 500°C, viscosity was reduced from 59.6 to 0.9 mm(2)s(-1), heating value was increased from 35.8 to 39.3 MJL(-1), and the O:C ratio was reduced from 0.07 to 0.02. Aromatic gasoline components (e.g., BTEX), were formed in concentrations as high as 94% (v/v) in catalytically-cracked PSS with yields ranging from 22% to 35% (v/v of PSS feed).     

Modification of seed oil content and acyl composition in the brassicaceae by expression of a yeast sn-2 acyltransferase gene.

A putative yeast sn-2 acyltransferase gene (SLC1-1), reportedly a variant acyltransferase that suppresses a genetic defect in sphingolipid long-chain base biosynthesis, has been expressed in a yeast SLC deletion strain. The SLC1-1 gene product was shown in vitro to encode an sn-2 acyltransferase capable of acylating sn-1 oleoyl-lysophosphatidic acid, using a range of acyl-CoA thioesters, including 18:1-, 22:1-, and 24:0-CoAs. The SLC1-1 gene was introduced into Arabidopsis and a high erucic acid-containing Brassica napus cv Hero under the control of a constitutive (tandem cauliflower mosaic virus 35S) promoter. The resulting transgenic plants showed substantial increases of 8 to 48% in seed oil content (expressed on the basis of seed dry weight) and increases in both overall proportions and amounts of very-long-chain fatty acids in seed triacylglycerols (TAGs). Furthermore, the proportion of very-long-chain fatty acids found at the sn-2 position of TAGs was increased, and homogenates prepared from developing seeds of transformed plants exhibited elevated lysophosphatidic acid acyltransferase (EC 2.3.1.51) activity. Thus, the yeast sn-2 acyltransferase has been shown to encode a protein that can exhibit lysophosphatidic acid acyltransferase activity and that can be used to change total fatty acid content and composition as well as to alter the stereospecific acyl distribution of fatty acids in seed TAGs.     

Microsomal Lyso-Phosphatidic Acid Acyltransferase from a Brassica oleracea Cultivar Incorporates Erucic Acid into the sn-2 Position of Seed Triacylglycerols

Developing seeds from Brassica oleracea (L.) var botrytis cv Sesam were examined for the ability to biosynthesize and incorporate erucic acid into triacylglycerols (TAGs). Seed embryos at mid-development contained a high concentration of erucic acid in diacylglycerols and TAGs, and substantial levels were also detected in free fatty acids, acyl-coenzyme A (CoA), phosphatidic acid, and phosphatidylcholine. Homogenates and microsomal fractions from seeds at mid-development produced [14C]eicosenoyl- and [14C]erucoyl-CoAs from [14C]oleoyl-CoA in the presence of malonyl-CoA and reducing equivalents in vitro. These fatty acids were incorporated into TAGs via the Kennedy pathway. However, unlike most Brassicaceae, the B. oleracea was able to insert significant erucic acid into the sn-2 position of TAGs. It was shown that the lyso-phosphatidic acid acyltransferase (LPAT) incorporated erucic acid into the sn-2 position of lyso-phosphatidic acid. The erucoyl-CoA:LPAT activity during seed development and the sn-2 erucic acid content of the TAG fraction in mature seed were compared to those in B. napus, Tropaeolum majus, and Limnanthes douglasii. There was a correlation between the in vitro erucoyl-CoA:LPAT activity and the sn-2 erucic acid content in seed TAGs. To our knowledge, this is the first member of the Brassicaceae reported to have an LPAT able to use erucoyl-CoA. This observation has important implications for efforts being made to increase the erucic acid content in B. napus, to supply strategic industrial feedstocks.     

The determination of the asymmetrical stereochemical distribution of fatty acids in triacylglycerols

The fatty acid distribution in triacylglycerols (TAGs) is a factor that contributes to the intrinsic properties of oils from different species variants. Many hypotheses have been proposed to explain the specific distribution of fatty acids in the different naturally occurring oils. Currently, the 1,3-random-2-random theory is more or less accepted, but it has been widely shown that most vegetable oils do not behave randomly in the sn-1 and sn-3 stereochemical positions. For this reason, complex methodologies have been developed to analyze the fatty acid composition of the three stereochemical positions in TAGs. In this article, we propose that by calculating the asymmetric alpha coefficient, the stereochemical asymmetry of fatty acids in TAG molecular species can be defined. This coefficient reflects the relative content of fatty acids at the sn-1 and sn-3 positions and may overcome the problems found mainly with complex sn-1, sn-2, and sn-3 stereochemical analysis of fatty acids in TAG. The alpha coefficient is calculated from the fatty acid, sn-2 fatty acid, and TAG composition of the oil. Indeed, through this coefficient, it has been possible to show that, despite having the same overall content, the stearic acid distribution in the sn-1 and sn-3 positions is not random in some oils.     

Phosphatidylserine synthase 2: high efficiency for synthesizing phosphatidylserine containing docosahexaenoic acid

Phosphatidylserine (PS), the major anionic phospholipid in eukaryotic cell membranes, is synthesized by the integral membrane enzymes PS synthase 1 (PSS1) and 2 (PSS2). PSS2 is highly expressed in specific tissues, such as brain and testis, where docosahexaenoic acid (DHA, 22:6n-3) is also highly enriched. The purpose of this work was to characterize the hydrocarbon-chain preference of PSS2 to gain insight on the specialized role of PSS2 in PS accumulation in the DHA-abundant tissues. Flag-tagged PSS2 was expressed in HEK cells and immunopurified in a functionally active form. Purified PSS2 utilized both PE plasmalogen and diacyl PE as substrates. Nevertheless, the latter was six times better utilized, indicating the importance of an ester linkage at the sn-1 position. Although no sn-1 fatty acyl preference was noted, PSS2 exhibited significant preference toward DHA compared with 18:1 or 20:4 at the sn-2 position. Preferential production of DHA-containing PS (DHA-PS) was consistently observed with PSS2 purified from a variety of cell lines as well as with microsomes from mutant cells in which PS synthesis relies primarily on PSS2. These findings suggest that PSS2 may play a key role in PS accumulation in brain and testis through high activity toward DHA-containing substrates that are abundant in these tissues.     

Effects of palm oil and transesterified palm oil on chylomicron and VLDL triacylglycerol structures and postprandial lipid response

The effects of positional distribution of triacylglycerol (TAG) fatty acids to TAG structures in chylomicrons and VLDL, and to postprandial lipemia, were studied in 10 healthy premenopausal women using a 6-h oral fat load test and a randomized, double-blind cross-over design. Molecular level information of TAG regioisomerism was obtained with a tandem mass spectrometric method. The positional distribution of fatty acids in chylomicron TAGs was similar to the respective dietary fat; 79% of the analyzed regioisomers in palm oil and 84% of the analyzed regioisomers in transesterified oil were found in chylomicron TAGs 3 h after the oral fat loads. VLDL TAGs were equal after the two fat loads in all but one regioisomer. Similarities in the fatty acid compositions of chylomicron TAGs suggest that palmitic acid was absorbed equally from both test fats. The proportion of palmitoleic acid in the chylomicrons was increased. Fat with palmitic acid predominantly in the sn-1 and sn-3 positions caused a larger incremental area of total TAGs in plasma and reduced plasma insulin values at the beginning of the postprandial response (0-90 min) compared with fat with palmitic acid randomly distributed. The relationship between TAG molecular structures in dietary fats and in lipoproteins provides new means for understanding the effects of fatty acid positional distribution on human lipid metabolism.     

The binary phase behavior of 1, 3-dipalmitoyl-2-stearoyl-sn-glycerol and 1, 2-dipalmitoyl-3-stearoyl-sn-glycerol

The binary phase behavior of purified 1, 3-dipalmitoyl-2-stearoyl-sn-glycerol (PSP) and 1, 2-dipalmitoyl-3-stearoyl-sn-glycerol (PPS) was investigated at a very slow (0.1 °C/min) and a relatively fast (3.0 °C/min) cooling rate. Mixtures with molar fractions of 0.1 increments were studied in terms of melting and crystallization, polymorphism, solid fat content (SFC), hardness and microstructure. Only the α-form of a double chain length (DCL) structure was detected for all mixtures in both experiments. The kinetic phase diagram, constructed using heating DSC thermograms, displayed two distinct behaviors separated by a singularity at the 0.5_PSP composition: a eutectic in the X_PSP ≤ 0.5 and a monotectic in the X_PSP ≤ 0.5 concentration region. The singularity was attributed to the formation of a 1:1 (mol:mol) molecular compound. Apart from the segment from 0.0_PSP to the eutectic point, X_E, the simulation of the liquidus line using a model based on the Hildebrand equation suggested that the molecular interactions are strong and tend to favor the formation of unlike pairs in the liquid state and that the miscibility is not significantly dependent on cooling rate. The kinetic effects are manifest in all measured properties, particularly dramatically in the X_PSP ≤ X_E concentration region. An analysis of induction time as measured by pulse nuclear magnetic resonance (pNMR) showed that PPS retards crystal growth, an effect which can explain the peculiarity of this concentration region. At both cooling rates, fit of the SFC (%) versus time curves to a modified form of the Avrami model revealed two common growth modes for all the mixtures. The polarized light microscope (PLM) of the PSP-PPS mixtures revealed networks made of spherulitic crystallites of size, growth direction and boundaries that are varied and sensitive to composition and cooling rate. The change in the microstructure and final SFC (%), particularly noticeable at compositions close to the eutectic, explain in part the differences seen in relative hardness.     

Evidence for stereospecific phospholipid-cholesterol interaction in lipid bilayers

The CH2 proton NMR linewidths of sn-3 and sn-1 dipalmitoyl phosphatidylcholine respond differently to the addition of cholesterol to the lipid vesicles. This result points to a stereospecific phospholipid-cholesterol interaction in the "hydrogen belt" region.     

Simulation of gel phase formation and melting in lipid bilayers using a coarse grained model

The transformation between a gel and a fluid phase in dipalmitoyl-phosphatidylcholine (DPPC) bilayers has been simulated using a coarse grained (CG) model by cooling bilayer patches composed of up to 8000 lipids. The critical step in the transformation process is the nucleation of a gel cluster consisting of 20-80 lipids, spanning both monolayers. After the formation of the critical cluster, a fast growth regime is entered. Growth slows when multiple gel domains start interacting, forming a percolating network. Long-lived fluid domains remain trapped and can be metastable on a microsecond time scale. From the temperature dependence of the rate of cluster growth, the line tension of the fluid-gel interface was estimated to be 3+/-2 pN. The reverse process is observed when heating the gel phase. No evidence is found for a hexatic phase as an intermediate stage of melting. The hysteresis observed in the freezing and melting transformation is found to depend both on the system size and on the time scale of the simulation. Extrapolating to macroscopic length and time scales, the transition temperature for heating and cooling converges to 295+/-5 K, in semi-quantitative agreement with the experimental value for DPPC (315 K). The phase transformation is associated with a drop in lateral mobility of the lipids by two orders of magnitude, and an increase in the rotational correlation time of the same order of magnitude. The lipid headgroups, however, remain fluid. These observations are in agreement with experimental findings, and show that the nature of the ordered phase obtained with the CG model is indeed a gel rather than a crystalline phase. Simulations performed at different levels of hydration furthermore show that the gel phase is stabilized at low hydration. A simulation of a small DPPC vesicle reveals that curvature has the opposite effect.     

The effect of pH, of ATP and of modification with pyridoxal 5-phosphate on the conformational transition between the Na+-form and the K+-form of the (Na+ +K+)-ATPase

An increase in pH decreases the Na+ concentration (Na+ +K+ = 150 mM) necessary for half-maximum activation of the (Na+ +K+)-ATPase at non-saturating concentrations of ATP just as an increase in the concentration of ATP at a given pH. It also decreases the concentration of Na+ necessary for transformation from the K+-form to the Na+-form at equilibrium conditions (Na+ +K+ = 150 mM). An increase in pH increases the rate of the transformation from the K+-form to the Na+-form of the system and decreases the rate of the reverse reaction. The pH effect on the conformation suggests that the K+-form is a protonated form and the Na+-form a deprotonated one. The similarity between the effect of an increase in pH with non-saturating concentrations of ATP and that of an increase in ATP at a given pH suggests that ATP exerts its effect on the transformation from the K+ - to the Na+-form by a decrease in pK values of the system, i.e., by releasing protons, a Bohr effect. Enzyme modified by reaction with pyridoxal 5-phosphate terminated by NaBH4 behaves at a given pH as if it were non-modified enzyme but at a higher pH. The 'pH effect' is seen after modification by pyridoxal 5-phosphate in the presence of ATP, of Na+ without and with ATP, of K+ with ATP but not in the presence of K+ alone. The modification has also a 'pH effect' on the rate of the transformation from the K+ -form to the Na+ -form and on the reverse reaction. There are at least two different pyridoxal 5-phosphate-reactive groups (amino groups), one which can be protected by ATP and which is of importance for activity and another which is not protected by ATP and which is of importance for the pH effect on the conformation. The effect of a protonation-deprotonation of amino groups on the conformation is explained by an involvement of the amino groups in salt bridge formation in between and inside the polypeptide chains, a hemoglobin-like situation. The protonated K+ -form is then a tense T-structure with a high K+, low Na+ affinity and the deprotonated Na+ -form a relaxed, R-structure with high Na+, low K+ affinity. ATP facilitates deprotonation by decreasing pK values. Oligomycin has 'pH effect' on the K0.5 for Na+ under equilibrium and steady-state conditions, but oligomycin has no effect on the rate of the transformation from the K+ -form to the Na+ -form, but gives a pronounced decrease of the rate of the reverse reaction, indicating that oligomycin does not react with the K+ -form but with the Na+ -form of the system and prevents the protonation, the E1 to E2 transformation.     

Fourier transform infrared study on the thermotropic behaviour of fully hydrated 1-palmitoyl-2-[10-(pyren-1-y) decanoyl]-sn-glycero-3-phosphatidylcholine

Fourier transform infrared (FTIR) spectroscopy was used to study the thermotropic behaviour of fully hydrated 1-palmitoyl-2-[10-(pyren-1-yl)-decanoyl]-sn-glycero-3-phosphatidyl choline (PPDPC) in the temperature range of 3-30 degrees C. Several changes in the spectral features of PPDPC were observed. Major alterations analogous to the gel-to-liquid crystalline phase transition of saturated phosphatidylcholines were evident at approximately 16 degrees C in both the wavenumbers and the halfbandwidths of five different vibrational modes of PPDPC, viz. asymmetric and symmetric CH2 stretching, C = O stretching, and CH2 bending. Also the pyrene ring deformation mode changed at this temperature. Using Fourier self-deconvolution technique we resolved the carbonyl stretching mode into two bands at approx. 1741 and 1726 cm-1. These bands are due to conformational differences in the ester linkages of the two acyl chains, and are further assigned on the basis of literature data to the sn-1 and sn-2 carbonyl groups, respectively. The ratio of the relative intensities of these two bands is shown to depend on the phase state of the phospholipid.     

Effect of hydrostatic pressure on the bilayer phase behavior of symmetric and asymmetric phospholipids with the same total chain length

The bilayer phase transitions of palmitoylstearoyl-phosphatidylcholine (PSPC), diheptadecanoyl-PC (C17PC) and stearoylpalmitoyl-PC (SPPC) which have the same total carbon numbers in the two acyl chains were observed by differential scanning calorimetry and high-pressure optical method. As the temperature increased, these bilayers exhibited four phases of the subgel (Lc), lamellar gel (L beta'), ripple gel (P beta') and liquid crystal (L alpha), in turn. The Lc phase was observed only in the first heating scan after cold storage. The temperatures of the phase transitions were almost linearly elevated by applying pressure. The temperature-pressure phase diagrams and the thermodynamic quantities associated with the phase transitions were compared among the lipid bilayers. For all the bilayers studied, the pressure-induced interdigitated gel (L beta I) phase appeared above the critical interdigitation pressure (CIP) between the L beta' and P beta' phases. The CIPs for the PSPC, C17PC and SPPC bilayers were found to be 50.6, 79.1 and 93.0 MPa, respectively. Contribution of two acyl chains to thermodynamic properties for the phase transitions of asymmetric PSPC and SPPC bilayers was not even. The sn-2 acyl chain lengths of asymmetric PCs governed primarily the bilayer properties. The fluorescence spectra of Prodan in lipid bilayers showed the emission maxima characteristic of bilayer phases, which were dependent on the location of Prodan in the bilayers. Second derivative of fluorescent spectrum exhibited the original emission spectrum of Prodan to be composed of the distribution of Prodan into multiple locations in the lipid bilayer. The F'497/F'430 value, a ratio of second derivative of fluorescence intensity at 497 nm to that at 430 nm, is decisive evidence whether bilayer interdigitation will occur. With respect to the L beta'/L beta I phase transition in the SPPC bilayer, the emission maximum of Prodan exhibited the narrow-range red-shift from 441 to 449 nm, indicating that the L beta I phase in the SPPC bilayer has a less polar "pocket" formed by a space between uneven terminal methyl ends of the sn-1 and sn-2 chains, in which the Prodan molecule remains stably.     

Specific volumes of unsaturated phosphatidylcholines in the liquid crystalline lamellar phase

The specific volumes of seven 1,2-diacyl-sn-glycero-3-phosphocholines with symmetric, unbranched acyl chains containing one, four, or six cis double bonds per chain, or with a saturated sn-1 chain and one, four, or six cis double bonds in the sn-2 chain were determined by the neutral buoyancy method. Experiments were conducted in the liquid crystalline lamellar phase over the temperature range from 5 to 35 degrees C. It is demonstrated that the molecular volume of phosphatidylcholines can be well approximated as the sum of a constant volume of the polar lipid head region and the temperature-dependent volumes of hydrocarbon chain CH2, CH, and terminal CH3 groups. A linear dependence of chain segment volumes on temperature was observed. A self-consistent set of partially temperature-dependent volumes is obtained that allows prediction of phosphatidylcholine molecular volumes within very tight error margins.     

Differential scanning calorimetric study of a homologous series of fully hydrated saturated mixed-chain C(X):C(X + 6) phosphatidylcholines

The successive high-resolution differential scanning calorimetric (DSC) thermograms for aqueous dispersions of a homologous series of mixed-chain phosphatidylcholines, C(X):C(X + 6)PC, have been recorded and analyzed. In this series of saturated mixed-chain phosphatidylcholines, the total number of carbon atoms in the sn-1 acyl chain increases from 11 to 20, and the sn-2 acyl chain is always 6 methylene units longer than the sn-1 acyl chain. In the initial heating DSC thermograms, two prominent endothermic transitions are detected for all the samples prepared from the various C(X):C(X + 6)PCs except C(12):C(18)PC. In contrast, a single exothermic transition is observed on cooling for all the samples except C(13):C(19)PC. The temperature difference between the two endothermic transitions increases linearly as the acyl chain length of C(X):C(X + 6)PC becomes progressively longer. Interestingly, the main phase transition occurs before the subtransition for C(11):C(17)PC dispersions. Our DSC data further demonstrate that the thermodynamic parameters (Tm, delta H, and delta S) associated with the main phase transition for fully hydrated C(13):C(19)PC and other identical MW phosphatidylcholines are inversely related to the corresponding values of the chain-length inequivalence (delta C/CL) for these lipids. This linear relationship can be employed to map the Tm values for aqueous dispersions prepared from a large number of mixed-chain phosphatidylcholines whose values of delta C/CL are within the range of 0.1-0.4.     

Mixed-chain phosphatidylcholine bilayers: structure and properties

Calorimetric and X-ray diffraction data are reported for two series of saturated mixed-chain phosphatidylcholines (PCs), 18:0/n:0-PC and n:0/18:0-PC, where the sn-1 and sn-2 fatty acyl chains on the glycerol backbone are systematically varied by two methylene groups from 18:0 to 10:0 (n = 18, 16, 14, 12, or 10). Fully hydrated PCs were annealed at -4 degrees C and their multilamellar dispersions characterized by differential scanning calorimetry and X-ray diffraction. All mixed-chain PCs form low-temperature "crystalline" bilayer phases following low-temperature incubation, except 18:0/10:0-PC. The subtransition temperature (Ts) shifts toward the main (chain melting) transition temperature (Tm) as the sn-1 or sn-2 fatty acyl chain is reduced in length; for the shorter chain PCs (18:0/12:0-PC, 12:0/18:0-PC, and 10:0/18:0-PC), Ts is 1-2 degrees C greater than Tm, and the subtransition enthalpy (delta Hs) is much greater than for the longer acyl chain PCs. Tm decreases with acyl chain length for both series of PCs except 18:0/10:0-PC, while for the positional isomers, n:0/18:0-PC and 18:0/n:0-PC, Tm is higher for the isomer with the longer acyl chain in the sn-2 position of the glycerol backbone. The conversion from the crystalline bilayer Lc phase to the liquid-crystalline L alpha phase with melted hydrocarbon chains occurs through a series of phase changes which are chain length dependent. For example, 18:0/18:0-PC undergoes the phase changes Lc----L beta'----P beta'----L alpha, while the shorter chain PC, 10:0/18:0-PC, is directly transformed from the Lc phase to the L alpha phase. However, normalized enthalpy and entropy data suggest that the overall thermodynamic change, Lc----L alpha, is essentially chain length independent. On cooling, the conversion to the Lc phases occurs via bilayer gel phases, L beta', for the longer chain PCs or through triple-chain interdigitated bilayer gel phases, L beta, for the shorter chain PC 18:0/12:0-PC and possibly 10:0/18:0-PC. Molecular models indicate that the bilayer gel phases for the more asymmetric PC series, 18:0/n:0-PC, must undergo progressive interdigitation with chain length reduction to maintain maximum chain-chain interaction. The L beta phase of 18:0/10:0-PC is the most stable structure for this PC below Tm. The formation and stability of the triple-chain structures can be rationalized from molecular models.     

Occlusion of Na+ by the Na,K-ATPase in the presence of oligomycin

Oligomycin occludes Na+ in an E1-form of the Na,K-ATPase. The rate constants for the release of Na+ from the E1-form and for the transition to the E2-form are about 0.5 s-1. The effect of oligomycin is not seen using other cations which also have a Na+-like effect on the enzyme conformation. The inhibitory effect of oligomycin on the ADP-ATP dependent Na:Na exchange but not on the accompanying ADP-ATP exchange can be explained from a decrease in the rate of release of Na+ from an E1 approximately phosphoform with Na+ occluded, E'1 approximately P (Na3), i.e. with Na+ in the membrane phase, to an E"1 approximately PNa3 form with Na+ not occluded. E"1 approximately PNa3 is at a step before formation of E2-P, and disappears at a high rate when ADP reacts with E"1 approximately P (Na3).     

Novel chromatographic resolution of chiral diacylglycerols and analysis of the stereoselective hydrolysis of triacylglycerols by lipases

In the present study, we propose a general and accessible method for the resolution of enantiomeric 1,2-sn- and 2,3-sn-diacylglycerols based on derivatization by isocyanates, which can be easily used routinely by biochemists to evaluate the stereopreferences of lipases in a time course of triacylglycerol (TAG) hydrolysis. Diacylglycerol (DAG) enantiomers were transformed into carbamates using achiral and commercially available reagents. Excellent separation and resolution factors were obtained for diacylglycerols present in lipolysis reaction mixtures. This analytical method was then applied to investigate the stereoselectivity of three model lipases (porcine pancreatic lipase, PPL; lipase from Rhizomucor miehei, MML; and recombinant dog gastric lipase, rDGL) in the time course of hydrolysis of prochiral triolein as a substrate. From the measurements of the diglyceride enantiomeric excess it was confirmed that PPL was not stereospecific (position sn-1 vs sn-3 of triolein), whereas MML and rDGL preferentially hydrolyzed the ester bond at position sn-1 and sn-3, respectively. The enantiomeric excess of DAGs was not constant with time, decreasing with the course of hydrolysis. This was due to the fact that DAGs can be products of the stereospecific hydrolysis of TAGs and substrates for stereospecific hydrolysis into monoacylglycerols.     

Physical properties of the transmembrane signal molecule, sn-1-stearoyl 2-arachidonoylglycerol. Acyl chain segregation and its biochemical implications

sn-1,2-diacylglycerol (DAG), a key intermediate in lipid metabolism, activates protein kinase C and is a fusogen. Phosphoinositides, the main sources of DAG in cell signaling, contain mostly stearoyl and arachidonoyl in the sn-1 and -2 positions, respectively. The polymorphic behavior of sn-1-stearoyl-2-arachidonoylglycerol (SAG) was studied by differential scanning calorimetry, x-ray powder diffraction, and solid state magic angle spinning (MAS) (13)C NMR. Three alpha phases were found in the dry state. X-ray diffraction indicated that the acyl chains packed in a hexagonal array in the alpha phase, and the two sub-alpha phases packed with pseudo-hexagonal symmetry. In the narrow angle range strong diffractions of approximately 31 and approximately 62 A were present. High power proton-decoupled MAS (13)C NMR of isotropic SAG gave 16 distinct resonances of the 20 arachidonoyl carbons and 5 distinct resonances of the 18 stearoyl carbons. Upon cooling, all resonances of stearoyl weakened and vanished in the sub-alpha(2) phase, whereas arachidonoyl carbons from 8/9 to 20 gave distinct resonances in the frozen phases. Remarkably, the omega-carbon of the two acyl chains had different chemical shifts in alpha, sub-alpha(1), and sub-alpha(2) phases. Large differences in spin lattice relaxation of the stearoyl and arachidonoyl methene and methyl groups were demonstrated by contact time (cross-polarization) MAS (13)C NMR experiments in the solid phases alpha, sub-alpha(1), and sub-alpha(2). This shows that stearoyl and arachidonoyl in SAG have different environments in the solid states (alpha, sub-alpha(1), and sub-alpha(2) phases) and may segregate during cooling. The NMR and long spacing x-ray diffraction results suggest that SAG does not pack in a conventional double layer with the two acyls in a hairpin fashion. Our findings thus provide a physicochemical basis for DAG hexagonal phase domain separation within membrane bilayers.