Species Of Phosphatidylglycerol Research Articles

Wounding stimulates the accumulation of glycerolipids containing oxophytodienoic acid and dinor-oxophytodienoic acid in Arabidopsis leaves.

Although oxylipins can be synthesized from free fatty acids, recent evidence suggests that oxylipins are components of plastid-localized polar complex lipids in Arabidopsis (Arabidopsis thaliana). Using a combination of electrospray ionization (ESI) collisionally induced dissociation time-of-flight mass spectrometry (MS) to identify acyl chains, ESI triple-quadrupole (Q) MS in the precursor mode to identify the nominal masses of complex polar lipids containing each acyl chain, and ESI Q-time-of-flight MS to confirm the identifications of the complex polar lipid species, 17 species of oxylipin-containing phosphatidylglycerols, monogalactosyldiacylglycerols (MGDG), and digalactosyldiacylglycerols (DGDG) were identified. The oxylipins of these polar complex lipid species include oxophytodienoic acid (OPDA), dinor-OPDA (dnOPDA), 18-carbon ketol acids, and 16-carbon ketol acids. Using ESI triple-Q MS in the precursor mode, the accumulation of five OPDA- and/or dnOPDA-containing MGDG and two OPDA-containing DGDG species were monitored as a function of time in mechanically wounded leaves. In unwounded leaves, the levels of these oxylipin-containing complex lipid species were low, between 0.001 and 0.023 nmol/mg dry weight. However, within the first 15 min after wounding, the levels of OPDA-dnOPDA MGDG, OPDA-OPDA MGDG, and OPDA-OPDA DGDG, each containing two oxylipin chains, increased 200- to 1,000-fold. In contrast, levels of OPDA-hexadecatrienoic acid MGDG, linolenic acid (18:3)-dnOPDA MGDG, OPDA-18:3 MGDG, and OPDA-18:3 DGDG, each containing a single oxylipin chain, rose 2- to 9-fold. The rapid accumulation of high levels of galactolipid species containing OPDA-OPDA and OPDA-dnOPDA in wounded leaves is consistent with these lipids being the primary products of plastidic oxylipin biosynthesis.

Lipidomics of cellular and secreted phospholipids from differentiated human fetal type II alveolar epithelial cells

Maturation of fetal alveolar type II epithelial cells in utero is characterized by specific changes to lung surfactant phospholipids. Here, we quantified the effects of hormonal differentiation in vitro on the molecular specificity of cellular and secreted phospholipids from human fetal type II epithelial cells using electrospray ionization mass spectrometry. Differentiation, assessed by morphology and changes in gene expression, was accompanied by restricted and specific modifications to cell phospholipids, principally enrichments of shorter chain species of phosphatidylcholine (PC) and phosphatidylinositol, that were not observed in fetal lung fibroblasts. Treatment of differentiated epithelial cells with secretagogues stimulated the secretion of functional surfactant-containing surfactant proteins B and C (SP-B and SP-C). Secreted material was further enriched in this same set of phospholipid species but was characterized by increased contents of short-chain monounsaturated and disaturated species other than dipalmitoyl PC (PC16:0/16:0), principally palmitoylmyristoyl PC (PC16:0/14:0) and palmitoylpalmitoleoyl PC (PC16:0/16:1). Mixtures of these PC molecular species, phosphatidylglycerol, and SP-B and SP-C were functionally active and rapidly generated low surface tension on compression in a pulsating bubble surfactometer. These results suggest that hormonally differentiated human fetal type II cells do not select the molecular composition of surfactant phospholipid on the basis of saturation but, more likely, on the basis of acyl chain length.

Identification and characterization of human cardiolipin synthase

The mitochondrial phospholipid cardiolipin is synthesized from cytidinediphosphate-diacylglycerol and phosphatidylglycerol, a process catalyzed by the enzyme cardiolipin synthase. In this study, we identified a human candidate gene/cDNA for cardiolipin synthase, C20orf155. Expression of this candidate cDNA in the (cardiolipin synthase-deficient) crd1Δ yeast confirmed that it indeed encodes human cardiolipin synthase. Purified mitochondria of the crd1Δ expressing human cardiolipin synthase were used to characterize the enzyme. It has an alkaline pH optimum, requires divalent cations for activity and appears to have a different substrate preference for cytidinediphosphate-diacylglycerol species when compared to phosphatidylglycerol species. The possible implications for CL synthesis and remodeling are discussed.

Pulmonary surfactant proteins and lipids as modulators of inflammation and innate immunity

The pulmonary surfactant system of the human lung consists of unique lipids and proteins that contribute to the biophysical and innate immune properties of the organ. Surfactant protein A (SP-A) is an oligomeric protein consisting of 18 protomers with collagen and lectin-like domains that recognizes glycoconjugates, lipids and protein determinants on both host cells and invading microorganisms. The authors examined the interaction of SP-A with Mycoplasma pneumoniae and the influence of the protein upon the innate immune response to the bacteria. The authors quantified SP-A interaction with bacteria using ELISA, and identified the major surface ligand by thin layer chromatography, HPLC and mass spectrometry. The inflammatory response of human and rat macrophages was measured by quantifying tumour necrosis factor-alpha secretion using ELISA, and nitric oxide production. SP-A bound the bacteria with high affinity and enhanced the inflammatory response of human and rat macrophages to the organism and its membranes. Analysis of the interaction of SP-A with the bacteria revealed that the major ligand was a phospholipid. The lipid ligand was purified by a combination of thin layer and HPLC, and identified by mass spectrometry. The mass spectrometry demonstrated that the SP-A reactive lipid consisted of several disaturated molecular species of phosphatidylglycerol (PtdGro). Additional experiments were performed to determine if disaturated PtdGro was capable of interfering with the action of SP-A as an inhibitor of bacterial lipopolysaccharide-induced inflammatory mediator production by macrophages. The disaturated PtdGro failed to alter the anti-inflammatory action of SP-A but unexpectedly these same studies revealed that unsaturated PtdGro can modify the host response to lipopolysaccharide. These findings reveal that both the lipids and proteins of pulmonary surfactant play a role in regulating the host response to invading microorganisms.

Molecular Species Analysis of Phosphatidylglycerols in Escherichia coli by Reversed-Phase HPLC/ESI-MS

A simple and sensitive method for the determination of molecular species of bacterial phosphatidylglycerols (1,2-diacyl-sn-glycero-3-phospho-1'-sn-glycerol, PG) as an intact form is described. The PG fraction isolated from Escherichia coli IAM12119 was subjected to reversed-phase high-performance liquid chromatography (HPLC) in conjunction with ion-trap mass spectrometry with electrospray ionization (ESI-MS). The HPLC analysis was performed with an ODS column (25 cm × 4.6 mm i.d., 5 μm particle size) using methanol/water/aqueous ammonium hydroxide (93 : 7 : 0.1, v/v/v) as the mobile phase, which gave a clear resolution of individual molecular species containing saturated, monounsaturated and cyclopropane fatty acids. In addition to a prominent deprotonated [M-H]− molecule, the negative ESI-MS gave weak product ions, [M-RCO]−, [M-H-RCOOH]− and [RCOO]−, by which individual molecular species could be identified. These product ions, derived from a fatty acid at the sn-2 position, showed greater intensities than those from the sn-1 position.

Total structure characterization of unsaturated acidic phospholipids provided by vicinal di-hydroxylation of fatty acid double bonds and negative electrospray ionization mass spectrometry

In the present work, the unsaturated fatty acid substituents of some phosphatidic acid, phosphatidylserine, phosphatidylinositol, and phosphatidylglycerol species were converted to their 1,2-di-hydroxy derivatives by OsO(4). The subsequent electrospray ionization tandem low-energy mass spectrometry analysis of the deprotonated species allowed positional determination of the double bonds by the production of specific product-ions. The product-ions are formed by charge-remote and charge-proximate homolytic cleavages as well as charge-directed heterolytic cleavages and rearrangements. The commercial availability of pure species of the phospholipids in question was limited, and a number of species were therefore synthesized. The developed method was used to fully characterize the two isobaric phosphatidylglycerol species 16:0/16:1Delta(9) and 16:0/16:1Delta(10) extracted from the bacteria Methylococcus capsulatus. The presence of these fatty acids was supported by a gas-chromatography mass spectrometry investigation of the dimethyloxazoline derivatives of the species of the lipid extract. The present work demonstrates that a total structure characterization of acidic unsaturated phospholipids in isolate or in mixtures is accomplished by vicinal di-hydroxylation of olefinic sites and subsequent electrospray ionization mass spectrometry of the derivatized phospholipids.

Surfactant abnormalities after single lung transplantation in dogs: impact of bronchoscopic surfactant administration

Objective Disturbances of the alveolar surfactant system have been implicated in the pathogenesis of reperfusion injury. The aim of this study was to evaluate the influence of exogenous surfactant administration on surfactant properties in a model of single lung transplantation. Methods We performed heterologous, left lung transplantation (+4°C ischemia; 24 hours, Euro-Collins solution) in 6 foxhounds (untreated) and in 6 animals that received calf lung surfactant extract (Alveofact) prior to explantation (only donor lung; 50 mg/kg body weight) and immediately after onset of reperfusion (both lungs, 200 mg/kg body weight). Separate but synchronized ventilation of each lung was performed, in a volume-controlled, pressure-limited mode, with animals in prone position. Bronchoalveolar lavage fluids were collected in pretransplantation lungs (control), after 24 hours of ischemia prior to transplantation (0 hours) and 6 and 12 hours after reperfusion in both the grafts and the recipient native lungs. Results Ischemic storage per se did not provoke any changes of the surfactant system; however, severe alterations occurred within 6 hours of reperfusion, resulting in a severe loss of surface activity, including a decrease in the percentage of the large surfactant aggregate fraction, reduction of the surfactant apoproteins SP-B and SP-C, the dipalmitoyl molecular species of phosphatidylcholine and phosphatidylglycerol within the large surfactant aggregate fraction. These abnormalities were restricted to the graft, with virtually normal surfactant function and composition being found in the recipient native lung. Surfactant administration fully normalized the biochemical and largely improved the biophysical surfactant properties, alongside maintenance of lung gas exchange properties. Conclusions Severe surfactant abnormalities occur exclusively in the graft when performing separate, synchronized ventilation of each lung to attenuate ventilator-induced lung injury. Bronchoscopic surfactant administration provides protection against these abnormalities and may be a therapeutic strategy in lung transplantation.

Genetic modification of the fatty acid unsaturation of phosphatidylglycerol in chloroplasts alters the sensitivity of tobacco plants to cold stress

ABSTRACTThe cis‐unsaturated molecular species of phosphatidylglycerol (PG) in chloroplasts have been implicated in the chilling sensitivity of plants. Homozygous lines of transgenic tobacco (Nicotiana tabacum) that overexpressed the cDNA for glycerol‐3‐phosphate acyltransferase, a key enzyme in the determination of the extent of cis‐unsaturation of PG, were established from a chilling‐sensitive squash (Cucurbita moschata). In transgenic plants, the proportion of saturated plus trans‐monounsaturated molecular species of PG increased from 24 to 65%. However, this change did not affect the architecture of the chloroplasts. Chilling stress decreased the growth and biomass production of young seedlings of transgenic plants more severely than those of wild‐type plants, and this observation suggests that the changes in the proportion of cis‐unsaturated PG affected not only leaves but also developing plants. Chilling stress also damaged inflorescences. In particular, the abscission of flower buds and inflorescence meristems from transgenic plants occurred more frequently than that from wild‐type plants. Thus, it is likely that decreases in the proportion of cis‐unsaturated PG enhanced the sensitivity to chilling of reproductive organs.

Molecular species compositions of lung and pancreas phospholipids in the cftr(tm1HGU/tm1HGU) cystic fibrosis mouse.

Fatty acid analysis of phospholipid compositions of lung and pancreas cells from a cystic fibrosis transmembrane regulator (CFTR) negative mouse (cftr(-/-))suggested that a decreased concentration of docosahexaenoate (22:6(n-3)) and increased arachidonate (20:4(n-6)) may be related to the disease process in cystic fibrosis (CF). Consequently, we have determined compositions of the major phospholipids of lung, pancreas, liver, and plasma from a different mouse model of CF, the cftr(tm1HGU/tm1HGU) mouse, compared with ZTM:MF-1 control mice. Electrospray ionization mass spectrometry permitted the quantification of all of the individual molecular species of phosphatidylcholine (PtdCho), phosphatidylethanolamine (PtdEtn), phosphatidylglycerol (PtdGly), phosphatidylserine (PtdSer), and phosphatidylinositol (PtdIns). There was no deficiency of 22:6(n-3) in any phospholipid class from lung, pancreas, or liver from mice with the cftr(tm1HGU/tm1HGU). Instead, the concentration of 20:4(n-6) was significantly decreased in plasma PtdCho species and in pancreas and lung species of PtdEtn, PtdSer, and PtdIns. These results demonstrate the variability of membrane phospholipid compositions in different mouse models of CF and suggest that in cftr(tm1HGU/tm1HGU) mice, the apparent deficiency was of 20:4n-6- rather than of 22:6n-3-containing phospholipid species. They highlight a need for detailed phospholipid molecular species analysis of cells expressing mutant CFTR from children with CF before the therapeutic effects of administering high doses of 22:6(n-3)-containing oils to children with CF can be fully evaluated.

Membranes of class IIa bacteriocin-resistant Listeria monocytogenes cells contain increased levels of desaturated and short-acyl-chain phosphatidylglycerols.

A major concern in the use of class IIa bacteriocins as food preservatives is the well-documented resistance development in target Listeria strains. We studied the relationship between leucocin A, a class IIa bacteriocin, and the composition of the major phospholipid, phosphatidylglycerol (PG), in membranes of both sensitive and resistant L. monocytogenes strains. Two wild-type strains, L. monocytogenes B73 and 412, two spontaneous mutants of L. monocytogenes B73 with intermediate resistance to leucocin A (+/-2.4 and +/-4 times the 50% inhibitory concentrations [IC50] for sensitive strains), and two highly resistant mutants of each of the wild-type strains (>500 times the IC50 for sensitive strains) were analyzed. Electrospray mass spectrometry analysis showed an increase in the ratios of unsaturated to saturated and short- to long-acyl-chain species of PG in all the resistant L. monocytogenes strains in our study, although their sensitivities to leucocin A were significantly different. This alteration in membrane phospholipids toward PGs containing shorter, unsaturated acyl chains suggests that resistant strains have cells with a more fluid membrane. The presence of this phenomenon in a strain (L. monocytogenes 412P) which is resistant to both leucocin A and pediocin PA-1 may indicate a link between membrane composition and class IIa bacteriocin resistance in some L. monocytogenes strains. Treatment of strains with sterculic acid methyl ester (SME), a desaturase inhibitor, resulted in significant changes in the leucocin A sensitivity of the intermediate-resistance strains but no changes in the sensitivity of highly resistant strains. There was, however, a decrease in the amount of unsaturated and short-acyl-chain PGs after treatment with SME in one of the intermediate and both of the highly resistant strains, but the opposite effect was observed for the sensitive strains. It appears, therefore, that membrane adaptation may be part of a resistance mechanism but that several resistance mechanisms may contribute to a resistance phenotype and that levels of resistance vary according to the type of mechanisms present.

Cyclodextrins: A potential tool for studying the role of glycerolipids in photosynthetic membranes

A novel cyclodextrin derivative, i.e., permethylated-alpha-cyclodextrin (PM-alpha-CD), was used for removing glycerolipids from spinach thylakoid membranes and investigating their role in photosynthetic activities. A three-step selective removal of each lipid class was observed in treated membranes. Up to a concentration of 4 mM, PM-alpha-CD (in the presence of 75 microg chlorophyll a+b/mL), PM-alpha-CD displayed a marked selectivity for anionic lipids [sulfoquinovosyldiacylglycerol (SQDG) and phosphatidylglycerol (PG)] in comparison with galactolipids. At this concentration, half of PG and SQDG were removed. Within this range of concentration, the volume response of treated thylakoids to variation of osmolarity, an indirect mean of verifying the structural integrity of the membrane, was not altered. Similarly, neither photosystem II (PSII) nor photosystem I (PSI) activity was affected. In contrast, the low-temperature fluorescence ratio F695/F740 drastically diminished from 1.45 to about 0.7, essentially due to the decrease of PSII fluorescence. The results derived from the fast fluorescence rise expressed in the form of a spider suggest that the fraction of inactivated (non-Q(A) reducing) reaction centers (RC) increases while the active (Q(A) reducing) RC remained intact. Raising the concentration of PM-alpha-CD from 4 to 7 mM resulted in a progressive but greater diminution of the galactolipid level than that of SQDG and PG. Within this concentration range, the integrity of the membrane was not altered, nor was either PSII or PSI activity, whereas the F695/F740 ratio decreased to about 0.45 as well as the fraction of inactivated RC. At concentrations above 7 mM of PM-alpha-CD, the integrity of the membrane was impaired, resulting in a decrease of both electron transport activities. At all concentrations, PM-alpha-CD did not show any selectivity toward either the acyl chains of the lipid molecules or the molecular species of PG. The results are discussed in terms of the role of glycerolipids in thylakoid membrane function and the relationship of the chemical structure of PM-alpha-CD and its lipid removal capacity.

Outer membrane composition of a lipopolysaccharide-deficient Neisseria meningitidis mutant.

In the pathogen Neisseria meningitidis, a completely lipopolysaccharide (LPS)-deficient but viable mutant can be obtained by insertional inactivation of the lpxA gene, encoding UDP-GlcNAc acyltransferase required for the first step of lipid A biosynthesis. To study how outer membrane structure and biogenesis are affected by the absence of this normally major component, inner and outer membranes were separated and their composition analysed. The expression and assembly of integral outer membrane proteins appeared largely unaffected. However, the expression of iron limitation-inducible, cell surface-exposed lipoproteins was greatly reduced. Major changes were seen in the phospholipid composition, with a shift towards phosphatidylethanolamine and phosphatidylglycerol species containing mostly shorter chain, saturated fatty acids, one of which was unique to the LPS-deficient outer membrane. The presence of the capsular polysaccharide turned out to be essential for viability without LPS, as demonstrated by using a strain in which LPS biosynthesis could be switched on or off through a tac promoter-controlled lpxA gene. Taken together, these results can help to explain why meningococci have the unique ability to survive without LPS.

Mitochondrial Lipid Alterations during Fas- and Radiation-Induced Apoptosis

In the present study, we investigated the dynamic alterations in mitochondrial lipids occurring during Fas- and radiation-induced cell death. Cross-linking of CD-95 on Fas-sensitive Jurkat cells produced rapid increases in two species of mitochondrial phosphatidylglycerol. By 2.5 h, phosphatidylglycerol decreases below basal levels, concomitant with an increase in mitochondrial ceramide. In addition, between 1.5 and 3.0 h after anti-Fas crosslinking, there is a continued loss of mitochondrial cardiolipin. When gamma irradiation was used to induce apoptosis, similar lipid changes occurred, although with somewhat slower kinetics. Fas-resistant Jurkat cells exhibited phosphatidylglycerol as the dominant lipid species in their mitochondria. Following Fas ligation, there is a transient decrease in phosphatidylglycerol, but cardiolipin and ceramide remained unchanged. The high basal levels of PG in Fas-resistant cells and the increase in PG levels in Fas-sensitive cells undergoing apoptosis was determined to be due to increased PGP synthase activity. Thus, critical mitochondrial lipids could potentially serve as novel targets in regulating the apoptotic process.

Analysis of phospholipid species in human blood using normal-phase liquid chromatography coupled with electrospray ionization ion-trap tandem mass spectrometry

A narrow-bore normal-phase high-performance liquid chromatography (HPLC) method was developed for separation of phospholipid classes in human blood. The separation was obtained using an HPLC diol column and a gradient of chloroform and methanol with 0.1% formic acid, titrated to pH 5.3 with ammonia and added 0.05% triethylamine. The HPLC system was coupled on-line with an electrospray ionisation ion-trap mass spectrometer. Chromatographic baseline separation was obtained between phosphatidylglycerol, phosphatidylcholine, phosphatidylethanolamine, lyso-phosphatidylcholine, phosphatidylinositol and phosphatidylserine, eluting in that order. The total run time was 30 min. Plasmalogen phosphatidylethanolamine and sphingomyelin, which both are substances with structural similarities to the glycerophospholipids, had similar retention time as phosphatidylethanolamine, but were well separated from the other glycerophospholipid classes. The species from each class were identified using MS 2 or MS 3, which forms characteristic lyso-fragments. The combination of lyso-fragment mass, molecular ion and chromatographic retention time was used to identify each species, including 20 species of phosphatidylglycerol. The mass spectra obtained for the phospholipid classes are presented. Using this system 17 disaturated phospholipid species not earlier described to be present in blood were identified. The limit of detection varied between different phospholipid classes and was in the range 0.1–5 ng of injected substance.

Bacterial cell membrane hydrolysis by secreted phospholipases A 2: a major physiological role of human group IIa sPLA 2 involving both bacterial cell wall penetration and interfacial catalysis

The ability of human group IIa secreted phospholipase A 2 (human sPLA 2) to hydrolyse the phospholipid membrane of whole cell suspensions of Gram-positive bacteria is demonstrated in real time using a continuous fluorescence displacement assay. Micrococcus luteus is used as a model system and demonstrates an almost absolute specificity for this human enzyme compared with porcine pancreatic and Naja naja venom sPLA 2s. This specificity is due to selective penetration of the highly cationic human sPLA 2 through the highly anionic bacterial cell wall. Disruption of the peptidoglycan cell wall by treatment with lysozyme allows all three enzymes to express similar hydrolytic activity against the anionic bacterial cell membrane. Extensive (>50%) phospholipid hydrolysis was observed and this was confirmed by electrospray mass spectrometry that allowed the identification of several molecular species of phosphatidylglycerol as the targets for hydrolysis. However, the bactericidal activity of the human enzyme under these assay conditions was low, highlighting the capacity of the organism to survive a major phospholipid insult. In addition to pure enzyme, the human sPLA 2 activity in tears was demonstrated using M. luteus as substrate. In comparison to M. luteus, cell suspensions of Staphylococcus aureus were highly resistant to hydrolysis by human sPLA 2 as well as to the pancreatic and venom enzymes. Treatment of this organism with the specific cell wall protease lysostaphin resulted in a dramatic enhancement in cell membrane phospholipid hydrolysis by all three sPLA 2s. Overall, the results highlight the potential of the human sPLA 2 as a selective antimicrobial agent against Gram-positive bacteria in vivo because this enzyme is essentially inactive against mammalian plasma membranes. However, the enzyme will be most effective in combination with other antimicrobial agents that enhance the permeability of the bacterial cell wall and where potentiation of the effectiveness of other antibiotics would be expected.

Hydrocarbon chain packing and the effect of ethanol on the thermotropic phase behavior of mixed-chain phosphatidylglycerols

Previous studies in this laboratory have delineated the relationship between the acyl chain asymmetry of mixed-chain phosphatidylcholines and the effect of ethanol concentration ([EtOH]) on their melting behavior (Li et al., Biophys J., 70 (1996) 2784–2794). This present investigation extends these findings to another phospholipid family by using high-resolution differential scanning calorimetry (DSC) to characterize the effect of ethanol concentration on the main phase transition temperature ( T m) of five molecular species of mixed-chain phosphatidylglycerol (PG). For C(14):C(18)PG, C(15):C(17)PG, C(16):C(16)PG, and C(17):C(15)PG, a biphasic profile in the T m versus [EtOH] plot was observed, and the minimum in the plot for each PG occurred at 33, 15, 19, and 36 mg/ml, respectively. This biphasic behavior is typical of phospholipids whose acyl chain asymmetry is fairly small. For C(18):C(14)PG, only a linear decrease in the T m was observed as a function of ethanol concentration; this effect is characteristic of highly asymmetric phospholipids. Our DSC results obtained with mixed-chain PG in the presence of ethanol demonstrate that the acyl chain asymmetry of the five lipids studied can be ranked as follows: C(15):C(17)PG<C(16):C(16)PG<C(14):C(18)PG<C(17):C(15)PG<C(18):C(14)PG, confirming independent results from this laboratory that the overall acyl chain structure of PG in the hydrated gel phase is opposite to that detected in the single crystals. In fully hydrated bilayers, the acyl chain conformation of PG is most likely to be similar to that of phosphatidylcholine.

Phosphatidylglycerol Is a Physiologic Activator of Nuclear Protein Kinase C

A major mechanism by which protein kinase C (PKC) function is regulated is through the selective targeting and activation of individual PKC isotypes at distinct subcellular locations. PKC betaII is selectively activated at the nucleus during G2 phase of cell cycle where it is required for entry into mitosis. Selective nuclear activation of PKC betaII is conferred by molecular determinants within the carboxyl-terminal catalytic domain of the kinase (Walker, S. D., Murray, N. R., Burns, D. J., and Fields, A. P. (1995) Proc. Natl. Acad. Sci. U. S. A. 92, 9156-9160). We previously described a lipid-like PKC activator in nuclear membranes, termed nuclear membrane activation factor (NMAF), that potently stimulates PKC betaII activity through interactions involving this domain (Murray, N. R., Burns, D. J., and Fields, A. P. (1994) J. Biol. Chem. 269, 21385-21390). We have now identified NMAF as phosphatidylglycerol (PG), based on several lines of evidence. First, NMAF cofractionates with PG as a single peak of activity through multiple chromatographic separations and exhibits phospholipase sensitivity identical to that of PG. Second, purified PG, but not other phospholipids, exhibits dose-dependent NMAF activity. Third, defined molecular species of PG exhibit different abilities to stimulate PKC betaII activity. 1,2-Dioleoyl-PG possesses significantly higher activity than other PG species, suggesting that both fatty acid side chain composition and the glycerol head group are important determinants for activity. Fourth, in vitro binding studies demonstrate that PG binds to the carboxyl-terminal region of PKC betaII, the same region we previously implicated in NMAF-mediated activation of PKC betaII. Taken together, our results indicate that specific molecular species of nuclear PG function to physiologically regulate PKC betaII activity at the nucleus.

Phosphatidylglycerol molecular species of photosynthetic membranes analyzed by high-performance liquid chromatography: theoretical considerations.

A reversed-phase high-performance liquid chromatography technique was developed to separate, identify, and quantify individual phosphatidylglycerol (PG) molecular species in thylakoid membranes isolated from higher plant leaves. PG was first separated by thin-layer chromatography; then the dinitrobenzoyl derivatives of diacylglycerols produced after phospholipase C hydrolysis of PG were separated by a C18 reversed-phase column and detected at 254 nm. A linear response of the detector was observed in the range of 0.025 to 12 nmol of PG molecular species. It was established that there was an excellent correlation (r = 0.996) between the carbon and double-bond number in the aliphatic residues and the relative retention time of dinitrobenzoyl derivatives. A new equivalent carbon number value (ECN*) which takes into consideration the number of cis-(nc) and trans-(nt) double bonds per molecular species was defined as ECN* = CN - 2nc - nt, where CN is the number of carbon atoms in the aliphatic residues. The logarithm of the retention time increased linearily as a function of ECN* value. However, in this type of correlation, it may happen that two molecular species of PG having distinct relative retention times had the same ECN* value. In this case, the two molecular species can be identified by the linear correlation (r = 1) existing between the reciprocal of the relative retention time and the number of double bonds (0 < or = n < or = 3) in the separate 18:n/delta 3-trans-hexadecenoic acid -16:1(3t)- and 18:n/16:0 molecular species series. The advantages of this method are good separation, cohort elution time, quantitative precision, and predictable retention times of PG molecular species from chloroplast membranes. The method has been used routinely to identify the ten PG molecular species of thylakoid membranes in squash, potato, lettuce, and spinach leaf: 18:3/16:1(3t), 18:3/16:0, 18:2/16:1(3t), 18:2/16:0, 18:1/16:1(3t), 18:1/16:0, 18:0/16:1(3t), 18:0/16:0, 16:0/16:1(3t), and 16:0/16:0.

Elevated Levels of High-Melting-Point Phosphatidylglycerols Do Not Induce Chilling Sensitivity in an Arabidopsis Mutant.

Molecular species of phosphatidylglycerol that contain only 16:0, 18:0, and 16:1-trans fatty acids undergo the transition from liquid crystalline phase to gel phase at temperatures well above 20[deg]C. Several lines of evidence have been used to implicate elevated proportions of these high-melting-point molecular species as a major cause of plant chilling sensitivity. In the fatty acid biosynthesis 1 (fab1) mutant of Arabidopsis, leaf phosphatidylglycerol contained 43% high-melting-point molecular species[mdash]a higher percentage than is found in many chilling-sensitive plants. Nevertheless, the mutant was completely unaffected (when compared with wild-type controls) by a range of low-temperature treatments that quickly led to the death of cucumber and other chilling-sensitive plants. Our results clearly demonstrate that high-melting-point phosphatidylglycerols do not mediate classic chilling damage. However, growth of fab1 plants was compromised by long-term (>2 weeks) exposure to 2[deg]C. This finding and other observations are consistent with a proposition that plants native to tropical and subtropical regions have evolved many traits that are incompatible with long-term growth or development in cooler climates but that may confer selective advantages at high temperatures.

Biosynthesis of Cardiolipin in Plant Mitochondria.

The properties of cardiolipin synthase were investigated in mitochondria and submitochondrial fractions from etiolated mung bean (Vigna radiata L.) seedlings. Direct evidence is presented that the enzyme utilizes CDP-diacylglycerol in addition to phosphatidylglycerol for the synthesis of cardiolipin. Cardiolipin synthase had an alkaline pH optimum of about 9 and required divalent cations for activity. Maximal activity was obtained in the presence of 16 mM MnCl2. The apparent Km values for CDP-diacylglycerol and phosphatidylglycerol were 0.8 and 50 [mu]M, respectively. Cardiolipin synthase was localized predominantly in the inner membrane of mung bean mitochondria and displayed a substrate species specificity. Highest activities were measured with the dioleoyl species of both CDP-diacylglycerol and phosphatidylglycerol, and somewhat lower activities were measured with mixed species of the two substrates containing a palmitoyl and an oleoyl group. On the other hand, the cardiolipin synthase hardly used the dipalmitoyl species and strongly discriminated against CDP-dipalmitoylglycerol from a mixture with CDP-dioleoylglycerol.