Lipids of ripening tomato fruit and its mitochondrial fraction

The lipid composition of tomato fruit and its mitochondrial fraction were examined at various stages of fruit ripeness. Phosphatidyl choline, phosphatidyl ethanolamine, monogalactosyl diglyceride, digalactosyl diglyceride and phosphatidyl inositol were found to be the major lipids of tomato pericarp at all stages of ripeness. Mitochondrial lipids resembled those of the parent tissue except for the absence of monogalactosyl diglyceride and a greater percentage of diphosphatidyl glycerol and phosphatidic acid. Changes in the lipid-protein ratio of mitochondria were noted with ripening.     

Intracellular transport of phosphatidic acid and phosphatidylcholine into lipid bodies: use of fluorescent lipids to study lipid-body formation in an oleaginous fungus

Fluorescent phosphatidic acid and phosphatidylcholine were used to characterize lipid-transport pathways into lipid bodies in an oleaginous fungus, Mortierella ramanniana var. angulispora. Several characteristics of the lipid transport such as temperature dependence and ATP dependence were evaluated. The transport depicted by these fluorescent lipids was consistent with metabolism of radiolabelled lipids, indicating that fluorescent lipids are useful to study lipid-body formation in this fungus. The results dissect lipid transport of phosphatidic acid and phosphatidylcholine into lipid bodies and reveal regulatory steps for lipid-body formation in this fungus.     

The Fusion of Liposomes to Rat Brain Microsomal Membranes Regulates Phosphatidylserine Synthesis

Rat brain microsomal membranes were fused to liposomes prepared with several pure lipids, namely, phosphatidylserine, phosphatidylinositol, phosphatidic acid, and mixtures of phosphatidic acid and phosphatidylcholine or phosphatidylethanolamine. The fusion between liposomes and microsomes was measured by the octadecyl rhodamine B chloride method. The extent and other properties of fusion largely depend on the lipid used to prepare liposomes; phosphatidic acid and phosphatidylinositol fuse more extensively than other lipid classes. The activity of serine base exchange is affected by the fusion between rat brain microsomes and lipids. It is strongly inhibited by phosphatidylserine, but it is activated by phosphatidic acid. The inhibition produced by phosphatidylserine on its own synthesis is proposed as a mechanism for controlling the formation of phosphatidylserine in rat brain microsomes.     

成串采收对番茄果实采后乙烯合成及贮藏品质的影响

为研究成串采收对番茄果实采后乙烯合成及贮藏品质的影响,对广西田阳县两个栽培区两种嫁接砧木的串收番茄的采后生理指标进行测定,探讨了该采收方式对番茄果实采后保鲜的作用机制。结果表明:整个贮藏期,不同栽培区不同嫁接砧木的番茄成串采收的果实,乙烯生成量明显低于对照的常规单果采收。其中,砧木1号Ⅰ区的串收番茄的乙烯生成量,采后5 d即下降至最低点(0.35 nL·g~(-1)·h~(-1)),显著低于其对照(1.36 nL·g~(-1)·h~(-1)),其他栽培区和砧木组合的串收番茄,在采后15 d乙烯生成量达到最低值。串收番茄的类胡萝卜素、番茄红素和抗坏血酸等果实内天然抗氧化物质的含量,在贮藏前期快速升高,且峰值显著高于对照。此外,成串采收处理还一定程度抑制了果实后熟阶段可溶性糖的积累和可滴定酸的分解。因此,番茄成串采收处理,可能通过抑制果实采后乙烯的合成,同时提高类胡萝卜素、番茄红素和抗坏血酸的水平,并且推迟糖和酸等营养物质的后熟变化进程,实现其延长果实货架期,提高商品品质的作用。     

Lipid-protein interactions in ADP-ATP carrier/egg phosphatidylcholine recombinants studied by spin-label ESR spectroscopy

The stoichiometry and specificity of lipid-protein interaction, as well as the lipid exchange rates at the protein interface, have been determined from the electron spin resonance spectra of spin-labeled lipids in reconstituted complexes of the mitochondrial ADP-ATP carrier with egg phosphatidylcholine. With the exception of cardiolipin and phosphatidic acid, the lipids studied are found to compete for approximately 50 sites at the intramembranous surface of the protein dimer. This number of first-shell lipid sites is unusually large for a protein of this size. The specificity for the protein is in the order stearic acid approximately phosphatidic acid approximately cardiolipin greater than phosphatidylserine greater than phosphatidylglycerol approximately phosphatidylcholine, with the maximum association constant relative to phosphatidylcholine being approximately 4. The selectivity for anionic lipids was partially screened with increasing ionic strength, but to a lesser extent for cardiolipin and phosphatidic acid than for stearic acid. Only in the case of phosphatidylserine was the selectivity reduced at high ionic strength to a level close to that for phosphatidylcholine. The off rates for lipid exchange at the protein surface were independent of lipid/protein ratio and correlated in a reciprocal fashion with the different lipid selectivities, varying from 5 x 10(6) s-1 for stearic acid at low ionic strength to 2 x 10(7) s-1 for phosphatidylcholine and phosphatidylglycerol. The off rates for cardiolipin were unusually low in comparison with the observed selectivity, and indicated the existence of a special population of sites (ca. 30% of the total) for cardiolipin, at which the exchange rate was very low.(ABSTRACT TRUNCATED AT 250 WORDS)     

Hydrolysis of lipid mixtures by rat hepatic lipase

The hydrolysis of phospholipid mixtures by purified rat hepatic lipase, also known as hepatic triglyceride lipase, was studied in a Triton X-100/lipid mixed micellar system. Column chromatography of the mixed micelles showed elution of Triton X-100 and binary lipid mixtures of phosphatidic acid, phosphatidylcholine, phosphatidylethanolamine and phosphatidylserine as a single peak. This indicated that the mixed micelles were homogenous and contained all components in the designated molar ratios. The molar ratio of Triton X-100 to lipid was kept constant at 4 to 1. Labeling one lipid with 3H and the other lipid with 14C enabled us to determine the hydrolysis of both components of these binary lipid mixed micelles. We found that the hydrolysis of phosphatidylcholine was activated by the inclusion of small amounts of phosphatidic acid (2.5-fold), phosphatidylethanolamine (1.5-fold) or phosphatidylserine (1.4-fold). The maximal activation of phosphatidylcholine hydrolysis was observed when 5 mol% of phosphatidylethanolamine, 7.5 mol% phosphatidic acid or 5 mol% phosphatidylserine was added to Triton X-100 mixed micelles. The hydrolysis of phosphatidic acid was activated 30%, and that of phosphatidylserine was inhibited 30% when the molar proportion of phosphatidylcholine was less than 50 mol%. The hydrolysis of phosphatidylethanolamine was slightly activated when the mol% of phosphatidylcholine was below 5. The hydrolysis of phosphatidylserine was inhibited by phosphatidylethanolamine when the mol% of the latter was 50 or less whereas phosphatidylethanolamine hydrolysis was not affected by phosphatidylserine. Under the conditions used sphingomyelin and cholesterol did not have a significant effect on the hydrolysis of the phospholipids studied. In agreement with our previous study (Kucera et al. (1988) J. Biol. Chem. 263, 1920-1928) these studies show that the phospholipid polar head group is an important factor which influences the action of hepatic lipase and that the interfacial properties of the substrate play a role in the expression of the activity of this enzyme. The molar ratios of phosphatidic acid, phosphatidylethanolamine and phosphatidylserine which activated phosphatidylcholine hydrolysis correspond closely to the molar ratios of these lipids found in the surface lipid film of lipoproteins e.g., high density lipoproteins.(ABSTRACT TRUNCATED AT 400 WORDS)     

Lipid modulation of nicotinic acetylcholine receptor function: the role of neutral and negatively charged lipids.

The effects of negatively charged and neutral lipids on the function of the reconstituted nicotinic acetylcholine receptor from Torpedo californica were determined with two assays using acetylcholine receptor-containing vesicles: the ion flux response and the affinity-state transition. The receptor was reconstituted into three different lipid environments, with and without neutral lipids: (1) phosphatidylcholine/phosphatidylserine; (2) phosphatidylcholine/phosphatidic acid; and (3) phosphatidylcholine/cardiolipin. Analysis of the ion flux responses showed that: (1) all three negatively charged lipid environments gave fully functional acetylcholine receptor ion channels, provided neutral lipids were added; (2) in each lipid environment, the neutral lipids tested were functionally equivalent to cholesterol; and (3) the rate of receptor desensitization depends upon the type of neutral lipid and negatively charged phospholipid reconstituted with the receptor. The functional effects of neutral and negatively charged lipids on the acetylcholine receptor are discussed in terms of protein-lipid interactions and stabilization of protein structure by lipids.     

Segregation of phosphatidic acid-rich domains in reconstituted acetylcholine receptor membranes

Purified Acetylcholine Receptor (AcChR) from Torpedo has been reconstituted at low (approximately 1:3500) and high (approximately 1:560) protein to phospholipid molar ratios into vesicles containing egg phosphatidylcholine, cholesterol, and different dimyristoyl phospholipids (dimyristoyl phosphatidylcholine, phosphatidylserine, phosphatidylglycerol and phosphatidic acid) as probes to explore the effects of the protein on phospholipid organization by differential scanning calorimetry, infrared, and fluorescence spectroscopy. All the experimental results indicate that the presence of the AcChR protein, even at the lower protein to phospholipid molar ratio, directs lateral phase separation of the monoanionic phosphoryl form of the phosphatidic acid probe, causing the formation of specific phosphatidic acid-rich lipid domains that become segregated from the bulk lipids and whose extent (phosphatidic acid sequestered into the domain, out of the total population in the vesicle) is protein-dependent. Furthermore, fluorescence energy transfer using the protein tryptophan residues as energy donors and the fluorescence probes trans-parinaric acid or diphenylhexatriene as acceptors, establishes that the AcChR is included in the domain. Other dimyristoyl phospholipid probes (phosphatidylcholine, phosphatidylserine, phosphatidylglycerol) under identical conditions could not mimic the protein-induced domain formation observed with the phosphatidic acid probe and result in ideal mixing of all lipid components in the reconstituted vesicles. Likewise, in the absence of protein, all the phospholipid probes, including phosphatidic acid, exhibit ideal mixing behavior. Since phosphatidic acid and cholesterol have been implicated in functional modulation of the reconstituted AcChR, it is suggested that such a specific modulatory role could be mediated by domain segregation of the relevant lipid classes.     

Characteristics of a membrane-associated lipoxygenase in tomato fruit

Microsomal membranes isolated from the pericarp of maturegreen tomato (Lycopersicon esculentum) fruit rapidly metabolize exogenous radiolabeled linoleic acid into fatty acid oxidation products at 22°C. The reaction is strongly inhibited by n-propyl gallate, an inhibitor of lipoxygenase. The membranes also rapidly metabolize 16:0/18:2^* phosphatidylcholine into radiolabeled oxidation products that comigrate on TLC plates with those formed from free linoleic acid. At 30°C, the formation of fatty acid oxidation products from 16:0/18:2^* phosphatidylcholine is slower, and there is an initial accumulation of radiolabeled linoleic acid that is not evident at 22°C, which can be attributed to the action of lipolytic acyl hydrolase. Radiolabeled phosphatidic acid and diacylglycerol are also formed during metabolism of 16:0/18:2^* phosphatidylcholine by the microsomal membranes, and there is no breakdown of either linoleic acid or phosphatidylcholine by heat-denatured membranes. When Triton X-100 treated membranes were used, the same patterns of metabolite formation from radiolabeled linoleic acid and 16:0/18:2^* phosphatidylcholine were observed. Thus, the enzymes mediating the breakdown of these radiolabeled compounds appear to be tightly associated with the membranes. Collectively, the data indicate that there is a lipoxygenase associated with microsomal membranes from tomato fruit that utilizes free fatty acid substrate released from phospholipids. The microsomal lipoxygenase is strongly active over a pH range of 4.5 to 8.0, comprises approximately 38% of the total (microsomal plus soluble) lipoxygenase activity in the tissue, has an apparent K_m of 0.52 millimolar and an apparent V_max of 0.186 millimoles per minute per milligram of protein. The membranous enzyme also cross-reacts with polyclonal antibodies raised against soybean lipoxygenase-1 and has an apparent molecular mass of 100 kilodaltons.     

Influence of lipid headgroup on the specificity and exchange dynamics in lipid-protein interactions. A spin-label study of myelin proteolipid apoprotein-phospholipid complexes

The pH and salt dependences of the interaction of phosphatidic acid, phosphatidylserine, and stearic acid with myelin proteolipid apoprotein (PLP) in dimyristoylphosphatidylcholine (DMPC) recombinants have been studied by electron spin resonance spectroscopy, using spin-labeled lipids. The two-component spin-label spectra have been analyzed both by spectral subtraction and by simulation using the exchange-coupled Bloch equations to give the fraction of lipids motionally restricted by the protein and the rate of lipid exchange between the fluid and motionally restricted lipid populations. For stearic acid, phosphatidic acid, and phosphatidylserine, the fraction of motionally restricted spin-label increases with increasing pH, with pKa's of 7.7, 7.6, and ca. 9.4, respectively. The corresponding pKa's for the bulk lipid regions of the bilayer are estimated, from changes in the ESR spectra, to be 6.7, 7.4, and 11, respectively. In the dissociated state at pH 9.0, the fraction of motionally restricted component decreases with increasing salt concentration, reaching an approximately constant value at [NaCl] = 0.5-1.0 M for all three negatively charged lipids. The net decreases for stearic acid and phosphatidic acid are considerably smaller (by ca. 30%) than those obtained on protonating the two lipids, whereas for phosphatidylserine the fraction of motionally restricted lipid in high salt is reduced to that corresponding to phosphatidylcholine. For a fixed lipid/protein ratio, the on-rate for exchange at the lipid-protein interface is independent of the degree of selectivity and has a shallow temperature dependence, as expected for a diffusion-controlled process.(ABSTRACT TRUNCATED AT 250 WORDS)     

Intracellular translocation and metabolism of a fluorescent phosphatidic acid analogue in cultured fibroblasts

We have investigated the metabolism and intracellular translocation of a fluorescent derivative of phosphatidic acid, 1-acyl-2-[(N-4-nitrobenzo-2-oxa-1,3-diazole)aminocaproyl] phosphatidic acid (C6-NBD-PA), and its metabolites, in Chinese hamster fibroblasts. This derivative is rapidly transferred from phospholipid vesicles to cells at 2 degrees C, and results in fluorescent labeling of the mitochondria, endoplasmic reticulum, and nuclear membrane of intact cells during its metabolism predominantly to fluorescent diglyceride (Pagano, R. E., Longmuir, K. J., Martin, O. C., and Struck, D. K. (1981) J. Cell Biol. 91, 872-877). In the present study, we show that, upon warming to 37 degrees C, the fluorescence associated with the endoplasmic reticulum was greatly reduced, while cytoplasmic lipid droplets, which were initially nonfluorescent, became intensely labeled. This altered intracellular distribution of fluorescence was accompanied by further metabolism of the fluorescent lipids to NBD-triglyceride and NBD-phosphatidylcholine. Although NBD-fatty acid was also produced, it was not re-utilized in the synthesis of other cellular lipids. Subcellular fractionation experiments demonstrated that primarily NBD-labeled triglyceride was associated with the intracellular lipid droplets, although substantial amounts of NBD-labeled phosphatidic acid, phosphatidylcholine, and diglyceride were also present in the whole cell extracts. This finding was confirmed in a separate experiment in which the fluorescent lipids associated with the intracellular lipid droplets were selectively and irreversibly photobleached in situ. Extraction and analysis of the fluorescent lipids revealed that NBD-triglyceride was preferentially photobleached. These results indicate that "sorting" of the NBD-labeled lipids into various cytoplasmic compartments accompanied their metabolism.     

Synthesis and properties of radioiodinated phospholipid analogues that spontaneously undergo vesicle-vesicle and vesicle-cell transfer

An efficient method for the synthesis and purification of a variety of iodinated phospholipid analogues is described. 1-Acyl-2-[[[3-(3-[125I]iodo-4-hydroxyphenyl)- propionyl]amino]caproyl]phosphatidylcholine (125I-PC) was prepared by alkylation of 1-acyl-2-(aminocaproyl)phosphatidylcholine with monoiodinated Bolton-Hunter reagent. 125I-Labeled phosphatidic acid, phosphatidylethanolamine, and phosphatidylserine were produced from 125I-PC by phospholipase D catalyzed base exchange in the presence of ethanol-amine or L-serine. All of these lipid analogues transferred readily from donor vesicles into recipient membranes. When an excess of acceptor vesicles was mixed with a population of donor vesicles containing the iodinated analogues, approximately 50% of the 125I-labeled lipids transferred to the acceptor vesicle population. In addition, under appropriate incubation conditions, these lipids were observed to transfer from vesicles to mammalian cells. Autoradiographic analysis of 125I-labeled lipids extracted from the cells after incubation with vesicles at 2 degrees C for 60 min revealed that a large proportion of the 125I-labeled phosphatidic acid was metabolized to 125I-labeled diglyceride and 125I-labeled phosphatidylcholine, whereas no metabolism of exogenously supplied 125I-labeled phosphatidylethanolamine or 125I-labeled phosphatidylcholine could be detected.     

Stoichiometry and specificity of lipid-protein interaction with myelin proteolipid protein studied by spin-label electron spin resonance

The interaction of spin-labeled lipids with the myelin proteolipid apoprotein in complexes with dimyristoylphosphatidylcholine of varying lipid/protein ratios has been studied with electron spin resonance spectroscopy. A first shell of approximately 10 lipids per 25 000-dalton protein is found to be motionally restricted by the protein interface. This stoichiometry is consistent with a hexameric arrangement of the protein in the membrane. A selectivity of the various spin-labeled lipids for the motionally restricted component at the protein interface is found in the order stearic acid greater than phosphatidic acid greater than cardiolipin approximately greater than phosphatidylserine greater than phosphatidylglycerol approximately equal to phosphatidylcholine greater than phosphatidylethanolamine greater than androstanol approximately greater than cholestane.     

The rat retina is a useful in vivo model to study membrane lipid synthesis: Rates of biosynthesis of neutral glycerides and phospholipids

The phospholipid composition was studied in the whole rat retina, as well as in its subcellular fractions. A relative enrichment of phosphatidic acid, phosphatidylethanolamine, and phosphatidylserine was observed in rod outer segments (ROS) in comparison with entire retina: nuclear-photoreceptor inner segmentssynaptic bodies (P₁) and synaptosomal-mitochondrial (P₂) fractions. Phosphatidylcholine was the predominant phospholipid class found in all subcellular fractions analyzed. The microsomal fraction was relatively enriched in phosphatidic acid and in phosphatidylinositol. In addition, the rat eye has been used as an in vivo system to study membrane lipid synthesis. After intravitreal injections of [2-³H]glycerol a rapid labeling of retinal glycerolipids took place. Up to 120 min after injection only the glycerol backbone of lipids was labeled. Phosphatidic acid and diacylglycerol displayed rapid rates of synthesis and breakdown. Fastest rates of labeling were attained by phosphatidylcholine followed by phosphatidylinositol. Differences were found when in vitro labeling by [2-³H]glycerol was compared with intravitreal injections. Labeling of phospholipids of subcellular fractions by intravitreally injected [2-³H]glycerol showed that most of the label accumulated in microsomal phosphatidylcholine and phosphatidylinositol. Diacylglycerols and phosphatidylethanolamine also took up 10 and 20% respectively of the precursor. It is concluded that the rat eye is a useful experimental model to study synthesis and metabolism of membrane lipids in the retina.     

Calcium- and calmodulin-regulated breakdown of phospholipid by microsomal membranes from bean cotyledons

Evidence for the involvement of Ca²⁺ and calmodulin in the regulation of phospholipid breakdown by microsomal membranes from bean cotyledons has been obtained by following the formation of radiolabeled degradation products from [U-¹⁴C]phosphatidylcholine. Three membrane-associated enzymes were found to mediate the breakdown of [U-¹⁴C] phosphatidylcholine, viz. phospholipase D (EC 3.1.4.4), phosphatidic acid phosphatase (EC 3.1.3.4), and lipolytic acyl hydrolase. Phospholipase D and phosphatidic acid phosphatase were both stimulated by physiological levels of free Ca²⁺, whereas lipolytic acyl hydrolase proved to be insensitive to Ca²⁺. Phospholipase D was unaffected by calmodulin, but the activity of phosphatidic acid phosphatase was additionally stimulated by nanomolar levels of calmodulin in the presence of 15 micromolar free Ca²⁺. Calmidazolium, a calmodulin antagonist, inhibited phosphatidic acid phosphatase activity at IC₅₀ values ranging from 10 to 15 micromolar. Thus the Ca²⁺-induced stimulation of phosphatidic acid phosphatase appears to be mediated through calmodulin, whereas the effect of Ca²⁺ on phospholipase D is independent of calmodulin. The role of Ca²⁺ as a second messenger in the initiation of membrane lipid degradation is discussed.     

Changes in the lipid composition of Paecilomyces persicinus P-10 M1 during growth and cephalosporin C production

The lipid composition of the fungus Paecilomyces persicinus P-10 M1 was monitored daily for 6 days to detect any changes during growth and cephalosporin C production. Total lipid yields and cephalosporin C production were maximal after 72 h of incubation. Analysis of the total lipids revealed that the neutral lipid fraction was elevated at this time, whereas polar lipids were depressed. Phosphatidylethanolamine and phosphatidylcholine represented the major phospholipids detected. Phosphatidylethanolamine levels were descending when cephalosporin C was detected at its highest concentration. Increases in phosphatidylcholine levels paralleled those of cephalosporin C but reached a maximum at 48 h after the latter. Diphosphatidylglycerol, phosphatidic acid, and phosphatidylserine were also detected. Fatty acids present in the total lipid fraction ranged in carbon length from C12 to C24. The major acids were C16 (palmitic), C18:1 (oleic), and C18:2 (linoleic). All fatty acids exhibited minor variations in concentration during the 6-day period, and none displayed a direct correlation with cephalosporin C yields.     

Changes in the lipid composition of Paecilomyces persicinus P-10 M1 during growth and cephalosporin C production.

The lipid composition of the fungus Paecilomyces persicinus P-10 M1 was monitored daily for 6 days to detect any changes during growth and cephalosporin C production. Total lipid yields and cephalosporin C production were maximal after 72 h of incubation. Analysis of the total lipids revealed that the neutral lipid fraction was elevated at this time, whereas polar lipids were depressed. Phosphatidylethanolamine and phosphatidylcholine represented the major phospholipids detected. Phosphatidylethanolamine levels were descending when cephalosporin C was detected at its highest concentration. Increases in phosphatidylcholine levels paralleled those of cephalosporin C but reached a maximum at 48 h after the latter. Diphosphatidylglycerol, phosphatidic acid, and phosphatidylserine were also detected. Fatty acids present in the total lipid fraction ranged in carbon length from C12 to C24. The major acids were C16 (palmitic), C18:1 (oleic), and C18:2 (linoleic). All fatty acids exhibited minor variations in concentration during the 6-day period, and none displayed a direct correlation with cephalosporin C yields.     

Lipid composition of the electron transport membrane of Haemophilus parainfluenzae

The principal lipids associated with the electron transport membrane of Haemophilus parainfluenzae are phosphatidylethanolamine (78%), phosphatidylmonomethylethanolamine (0.4%), phosphatidylglycerol (18%), phosphatidylcholine (0.4%), phosphatidylserine (0.4%), phosphatidic acid (0.2%), and cardiolipin (3.0%). Phospholipids account for 98.4% of the extractible fatty acids. There are no glycolipids, plasmalogens, alkyl ethers, or lipo amino acid esters in the membrane lipids. Glycerol phosphate esters derived from the phospholipids by mild alkaline methanolysis were identified by their staining reactions, mobility on paper and ion-exchange column chromatography, and by the molar glycerol to phosphate ratios. Eleven diacyl phospholipids can be separated by two-dimensional thin-layer chromatography. Each lipid served as a substrate for phospholipase D, and had a fatty acid to phosphate ratio of 2:1. Each separated diacyl phospholipid was deacylated and the glycerol phosphate ester was identified by paper chromatography in four solvent systems. Of the 11 separated phospholipids, 3 were phosphatidylethanolamines, 2 were phosphatidylserines, and 2 were phosphatidylglycerols. Phosphatidylcholine, cardiolipin, and phosphatidic acid were found at a single location. Phosphatidylmonomethylethanolamine was found with the major phosphatidylethanolamine. Three distinct classes of phospholipids are separable according to their relative fatty acid compositions. (i) The trace lipids consist of two phosphatidylethanolamines, two phosphatidylserines, phosphatidylcholine, phosphatidic acid, and a phosphatidylglycerol. Each lipid represents less than 0.3% of the total lipid phosphate. These lipids are characterized by high proportions of the short (C(10) to C(14)) and long (C(19) to C(22)) fatty acids with practically no palmitoleic acid. (ii) The major phospholipids (93% of the lipid phosphate) are phosphatidylethanolamine, phosphatidylmonomethylethanolamine, and phosphatidylglycerol. These lipids contain a low proportion of the short (C(19)) fatty acids. Palmitic and palmitoleic acids represent over 80% of the total fatty acids. (iii) The fatty acid composition of the cardiolipin is intermediate between the other two classes. Both palmitoleic and the longer fatty acids represent a significant proportion of the total fatty acid.     

Muscarinic Acetylcholine Receptor Enhances Phosphatidylcholine Hydrolysis via Phospholipase D in Bovine Chromaffin Cells in Culture

Although it is well-established that inositol-containing lipids serve as precursors of intracellular second messenger molecules in chromaffin cells, we describe some findings that show the formation of diacylglycerol from phosphatidylcholine in response to agonist-mediated stimulation. Stimulation of chromaffin cells by acetylcholine produced a high turnover of phosphatidylcholine, as suggested by the release of [3H]choline derived from [3H]-phosphatidylcholine in experiments performed with [3H]choline chloride-prelabeled cells. An enhanced breakdown of phosphatidylcholine was also inferred from the finding of an increased formation of [3H]diacylglycerol in chromaffin cells prelabeled with [3H]glycerol. The diacylglycerol mass that accumulated after stimulation showed a distinct temporal course and seemed to exceed the mass that has been reported to be derived from phosphatidylinositol. In keeping with the purported origin from phosphatidylcholine, diacylglycerol showed a high content in [3H]oleate molecular species. Phospholipase D activity measurements and experiments performed in the presence of propranolol (an inhibitor of phosphatidic acid:phosphohydrolase) suggested that phosphatidylcholine is hydrolyzed by a phospholipase D activity, producing phosphatidic acid, which is subsequently degraded to diacylglycerol, rather than by a phospholipase C. Incubation of chromaffin cells in the presence of atropine before addition of acetylcholine showed complete inhibition of the increased formation of [3H]-diacylglycerol, whereas d-tubocurarine failed to do so. Taken together, these results suggest that acetylcholine activates phosphatidylcholine breakdown and diacylglycerol formation in chromaffin cells via a muscarinic-type receptor.     

Correlation of phospholipid structure with functional effects on the nicotinic acetylcholine receptor. A modulatory role for phosphatidic acid.

Fourier transform infrared spectroscopy is used to characterize specific interactions between negatively charged lipids, such as phosphatidic acid, and the purified nicotinic acetylcholine receptor from Torpedo californica. The specific interaction of phosphatidic acid with acetylcholine receptor is demonstrated by the receptor-induced perturbation of the lipid ionization state, which is monitored using Fourier transform infrared bands arising from the phosphate head group. The acetylcholine receptor shifts the pKa of phosphatidic acid molecules adjacent to the receptor to a lower value by almost 2 pH units from 8.5 to 6.6. Decreased pH also leads to changes in ion channel function and to changes in the secondary structure of the acetylcholine receptor in membranes containing ionizable phospholipids. Phospholipase D restores functional activity of acetylcholine receptor reconstituted in an unfavorable environment containing phosphatidylcholine by generating phosphatidic acid. Lipids such as phosphatidic acid may serve as allosteric effectors for membrane protein function and the lipid-protein interface could be a site for activity-dependent changes that lead to modulation of synaptic efficacy.