Effect of phospholipids on the regulation of a soluble galactosyltransferase in aortic wall

A galactosyltransferase activity is located in the cell-sap of aortic intima-media cells. This enzymatic system calatyzes [14C]galactose transfer from UDP-[14C]galactose into endogenous and exogenous proteinic acceptors. Labelled products are isolated from the proteinic fraction obtained in 20% trichloroacetic acid pellet or from organic solvent extractions. Maximal [14C]galactose incorporation occurs at pH 7.8 in Tris-HCl buffer in the presence of 0.1 mM MnCl2 at 30 degrees C. The enzymatic activity is modified by phospholipids, particularly by phosphatidic acid and lysophosphatidylcholine, which behave as mixed inhibitors, while L-alpha-phosphatidylserine interacts as a competitive inhibitor. The effect of phospholipids is not stereospecific but appeared to be closely related to their polar headgroups, especially the acidic headgroups of phosphatidylcholine and phosphatidic acid. The chain length and the unsaturation degree of fatty acids involved in phospholipid structures are not a main factor of regulation. The lysophosphatidylcholine effect could be explained by its solubilization properties, as non-ionic detergents interact in the same way with galactosyltransferase activity. Exogenous phospholipids probably interact with the enzymatic environment by their own molecular arrangement and so could exert a control on galactosyltransferase activity or lead to a conformation change of this enzyme.     

Changes in Phospholipid Composition of a Winter Wheat Cultivar during Germination at 2 C and 24 C

Evaluation of various solvent systems for lipid extraction of wheat Triticum aestivum L. cv. Rideau seeds showed that boiling 2-propanol followed by the Bligh-Dyer procedure was the most efficient method, with respect to lipid yield and ability to inactivate lipolytic enzymes. Ten phospholipids were identified in dry seeds; the major components being phosphatidylcholine, lysophosphatidylcholine, N-acyl lysophosphatidyl-ethanolamine, N-acylphosphatidylethanolamine, and phosphatidylethanolamine. After growth for 1 week (2 C) or 31 hours (24 C), the proportions of phosphatidylethanolamine + lysophosphatidic acid and phosphatidic acid increased, lysophosphatidylcholine decreased, and the remaining phospholipids showed little change. At 5 weeks (2 C) or 72 hours (24 C), the seedlings showed 5-fold increases in the proportion of phosphatidic acid largely at the expense of phosphatidylcholine, small decreases in N-acyl lysophosphatidylethanolamine and N-acylphosphatidylethanolamine, and significant increases in lysophosphatidylcholine. The changes in phosphatidic acid and phosphatidylcholine are interpreted as being partially due to increasing phospholipase D activity during germination. In general, the phospholipid composition was similar in morphologically equivalent seedlings grown at 2 C or 24 C. The increased membrane content in seedlings grown at 2 C does not reflect any preferential synthesis of individual phospholipids.     

The effect of phosphate deficiency on membrane phospholipid composition of bean (Phaseolus vulgaris L.) roots

Plasma membranes were isolated from roots of bean (Phaseolus vulgaris L.) plants cultured on phosphate sufficient or phosphate deficient medium. The phospholipid composition of plasma membranes was analyzed and compared with that of the microsomal fraction. Phosphate deficiency had no influence on lipid/protein ratio in microsomal as well as plasma membrane fraction. In phosphate deficient roots phospholipid content was lower in the plasma membrane, but did not change in the microsomal fraction. Phosphatidylcholine and phosphatidylethanolamine were two major phospholipids in plasmalemma and microsomal membranes (80 % of the total). After two weeks of phosphate starvation a considerable decrease (about 50 %) in phosphatidylcholine and phosphatidylethanolamine in microsomal membranes was observed. The decline in two major phospholipids was accompanied by an increase in phosphatidic acid and lysophosphatidylcholine content. The effect of alterations in plasma membrane phospholipids on membrane function e.g. nitrate uptake is discussed.     

Modulation of solubilized brain fucosyltransferase activity by phospholipids

Phospholipids interact on Triton X-100 solubilized GDP-fucose: asialofetuin fucosyltransferase (EC 2.4.1.68) isolated from sheep brain. This enzymatic activity is modulated by charged phospholipids. In particular, phosphatidic acid and analogues markedly inhibit the transfer of fucose from GDP-[14C]fucose. Kinetic studies show that phosphatidic acid interacts as a mixed inhibitor: the velocity and affinity of fucosyltransferase for the GDP-fucose and asialofetuin substrates are strongly decreased. However, this inhibitory effect is not related to stereospecificity, and the different parameters involved in the enzymatic reaction of glycosylation are not modified. The nature of fatty acids and chemical bond (ester or ether) occurring in the carbohydrate chain does not modify the behaviour of phosphatidic acid with respect to fucosyltransferase activity. Further, the physical state of phosphatidic acid (gel phase or liquid crystalline phase) has no influence. However, as the inhibition is closely pH-dependent, these data suggest that phosphatidic acid might directly interact with the active site of the enzyme and induce a conformational change.     

Lysophosphatidic Acid Decreases Glutamate and Glucose Uptake by Astrocytes

Abstract: The brain is a rich source of the lipid biomediator lysophosphatidic acid, and lysophosphatidic acid levels can significantly increase following brain trauma. Responses of primary rat brain astrocytes to this novel lipid are defined in the current study. Treatment of cells with lysophosphatidic acid resulted in a time- and dose-dependent inhibition of glutamate uptake. Inhibition of glutamate uptake was specific because the related phospholipids, phosphatidic acid, lysophosphatidylcholine, and lysophosphatidylglycerol, did not inhibit this uptake under comparable conditions, i.e., treatment with 10 µ M lipid for 30 min. Lysophosphatidic acid treatment of cells resulted in an increase in lipid peroxidation, as measured by the thiobarbituric acid assay. This increase in content of thiobarbituric acid-reactive substances was largely inhibited by treatment with dithiothreitol or propyl gallate; however, such treatment did not affect the lysophosphatidic acid-induced inhibition of glutamate uptake. Lysophosphatidic acid also inhibited glucose uptake with a dose-response curve that paralleled the inhibition of glutamate uptake. By impairing uptake of glutamate by astrocytes, lysophosphatidic acid may exacerbate excitotoxic processes in various neurodegenerative conditions.     

Quantitative analysis of phospholipids by 31P-NMR

High-field 31P nuclear magnetic resonance spectroscopy was used to quantitate phospholipids in mixtures in organic solvents. The sample is dissolved in chloroform-methanol and analyzed at 161.7 MHz with decoupling of the protons. Signals were identified using authentic compounds, and their relative distribution was measured in mole percent. The method has good accuracy and reproducibility, and was used to analyze phosphatidylcholine, phosphatidylethanolamine, sphingomyelin, lysophosphatidylcholine, lysophosphatidylethanolamine, phosphatidylinositol, cardiolipin, and phosphatidic acid in egg lecithin. Four commercial egg phospholipids and the phospholipids from a total lipid extract of rat liver were analyzed. The method could be utilized to analyze phospholipids from other sources.     

A practical method to analyze the phospholipids in the amniotic fluid using HPTLC

An operative method using HPTLC which makes possible the determination of nine phospholipids in the amniotic fluid individually is described: lysophosphatidylcholine, sphingomyelin, phosphatidylcholine, phosphatidylinositol, phosphatidylserine, phosphatidylethanolamine, phosphatidylglycerol, phosphatidic acid and cardiolipine. Since a simple and reliable method for clinical practice was sought, the working techniques, materials, chromatographic solvents and staining reagents were chosen accordingly. The standardization method was made by using standard phospholipids. This method was tested with 34 samples of amniotic fluid. A discussion of the practical application in the determination of some ratios of fetal pulmonary maturity is made.     

Glucocorticoid induction of pulmonary maturation in the rabbit fetus. The effect of maternal injection of betamethasone on the activity of enzymes in fetal lung.

1. Maternal administration of betamethasone (0.2 mg/kg) on day 25 or 26 of gestation produced a significant decrease in the lung/body weight ratio of the rabbit fetuses within 24 h. 2. The incorporation of [14C]choline but not [14C]ethanolamine into the lipids of fetal lung slices was significantly increased, indicating that there was a specific effect on phosphatidylcholine synthesis. 3. The activities of a number of marker enzymes for subcellular organelles were elevated, especially in the lungs of fetuses delivered on day 26. The increases in monoamine oxidase (mitochondrial outer membrane), beta-glycerophosphatase and aqueously dispersed phosphatidic acid-dependent phosphatidic acid phosphohydrolase (lysosomal) activities were significant. 4. Although the activity of cholinephosphotransferase was not affected by glucocorticoid treatment, the activities of glycerol-3-phosphate phosphatidyltransferase and the activities of two enzymes in the auxiliary pathways for the production of disaturated phosphatidylcholine (lysophosphatidylcholine:lysophosphatidylcholine transacylase and lysophosphatidylcholine:acyl-CoA acyl-transferase) were significantly increased. 5. Membrane-bound phosphatidic acid-dependent phosphatidic acid phosphohydrolase activity was elevated to a lesser extent than the aqueously dispersed phosphatidate-dependent activity and this increase was not significant. 6. The incorporation of E135S]methionine into protein by slices of fetal lung was significantly reduced after maternal treatment with betamethosone. 7. These results are consistent with the general view that glucocorticoids can induce pulmonary maturation and surfactant production in the rabbit fetus but indicate that some of the former hypotheses regarding the mechanism by which lipid synthesis is accelerated must be reevaluated.     

Mannosylation of endogenous and exogenous phosphatidic acid by liver microsomal membranes. Formation of phosphatidylmannose

Hamster liver post-nuclear membranes catalyze the transfer of mannose from GDP-mannose to endogenous dolichyl phosphate and to a second major endogenous acidic lipid. This mannolipid was believed to be synthesized from endogenous retinyl phosphate and was tentatively identified as retinyl phosphate mannose (Ret-P-Man) (De Luca, L. M., Brugh, M. R. Silverman-Jones, C. S. and Shidoji, Y. (1982) Biochem. J. 208, 159-170). To characterize this endogenous mannolipid in more detail, we isolated and purified the mannolipid from incubations containing hamster liver membranes and GDP-[14C]mannose and compared its properties to those of authentic Ret-P-Man. We found that the endogenous mannolipid was separable from authentic Ret-P-Man on a Mono Q anion exchange column, did not exhibit the absorbance spectrum characteristic of a retinol moiety, and was stable to mild acid under conditions which cleave authentic Ret-P-Man. The endogenous mannolipid was sensitive to mild base hydrolysis and mannose was released from the mannolipid by snake venom phosphodiesterase digestion. These properties were consistent with the endogenous acceptor being phosphatidic acid. Addition of exogenous phosphatidic acid, but not phospholipids with a head group blocking the phosphate moiety, to incubations containing hamster liver membranes and GDP-[14C]mannose resulted in the synthesis of a mannolipid with chromatographic and physical properties identical to the endogenous mannolipid. A double-labeled mannolipid was synthesized in incubations containing hamster liver membranes, GDP-[14C]mannose, and [3H]phosphatidic acid. Mannosyl transfer to exogenous phosphatidic acid was saturable with increasing concentrations of phosphatidic acid and GDP-mannose and specific for glycosyl transfer from GDP-mannose. Class E Thy-1-negative mutant mouse lymphoma cell membranes, which are defective in dolichyl phosphate mannose synthesis, also fail to transfer mannose from GDP-mannose to exogenous phosphatidic acid or retinyl phosphate. Amphomycin, an inhibitor of dolichyl phosphate mannose synthesis, blocked mannosyl transfer to the endogenous lipid, and to exogenous retinyl phosphate and phosphatidic acid. We conclude that the same mannosyltransferase responsible for dolichyl phosphate mannose synthesis can also utilize in vitro exogenous retinyl phosphate and phosphatidic acid as well as endogenous phosphatidic acid as mannosyl acceptors.     

冬虫夏草及杜仲磷脂成分的研究

本文对不同产地的冬虫夏草及杜仲磷脂成分进行了分析研究。以钼蓝比色法测定了它们的总磷脂含量,采用薄层色谱扫描和吸光度比例系数校正法测定了其磷脂组成及相对百分含量。冬虫夏草约含8种磷脂组分,主要成分为磷脂酰胆碱、磷脂酰叽醇、磷脂酰丝氨酸、磷脂酰乙醇胺和磷脂酸。杜仲约含6种磷脂组分,其中以溶血磷脂酰胆碱和磷脂酰胆碱为主。     

Phospholipid content in "late" posttraumatic epileptic focus in rabbits

The phospholipids content in the cortex, hippocamp and stem tissues in rabbits with "late" posttraumatic epileptic focus (1 year after the light brain injury) was estimated. There was established an increase of the content of phosphatidylethanolamine, sphingomyeline, phosphatidylinosite, phosphatidic acid, non-identified phospholipids, decrease of the content of phosphatidylcholine and cardiolipine and appearance of lysophosphatidylcholine in the cortical epileptic focus tissues. The conclusion is made that the changes within the phospholipids pool may cause the epileptogenic disturbance in the neurons, i.e. the changes of functional properties of the excitable membranes and the activity of the mytochondrial phospholipid-dependent enzyme complex.     

Increased secretion of lignolytic enzymes by the Lentinus tigrinus fungus after addition of butanol and toluene in submerged cultivation

We studied the effects of butanol and toluene on secretion of lignolytic enzymes by the Lentinus tigrinus fungus during submerged cultivation. Addition of butanol and toluene during the trophophase was followed by an increase in laccase and peroxidase activity of the culture and change in the composition of phospholipids and fatty acids. The ratio of phosphatidylcholine and phosphatidic acid decreased, while the amount of lysophosphatidylcholine, phosphatidylethanolamine, phosphoinositides, phosphatidylserine, and unsaturated fatty acids decreased. These changes resulted in an increase in the unsaturation index.     

De novo biosynthesis of docosahexaenoyl-phosphatidic acid in bovine retinal microsomes

Lysophosphatidic acid stimulated several-fold the formation of docosahexaenoyl-phosphatidic acid from 14C-labeled docosahexaenoic acid (22:6 (n-3] in the bovine retina. 1-Palmitoyl- and 1-oleoyl-sn-glycerol 3-phosphate were the preferred acceptors. Most of the activity was localized in the 105 000 X g microsomal fraction. Despite the very high content of 22:6 in the phospholipids of photoreceptor membranes, only about 1% of the microsomal activity was found in discs isolated from rod outer segments. The newly synthesized docosahexaenoyl-phosphatidic acid was further metabolized to diacylglycerols, triacylglycerols, phosphatidylcholine and phosphatidylserine. The de novo synthesis of docosahexaenoyl-phosphatidylcholine was stimulated by 1 mM CDPcholine. Lysophosphatidic acid and lysophosphatidylcholine up to 50 microM do not compete with each other for 22:6 in the formation of their respective diacylated lipids. This suggests that this fatty acid is introduced into phosphatidic acid and phosphatidylcholine via different acylation systems. We conclude that, in addition to the deacylation-acylation cycle, there is also an active pathway for the acylation of 22:6 into glycerolipids during the de novo biosynthesis of phosphatidic acid.     

Alkylacetylglycerophosphocholine effects on the metabolism of phospholipids in rabbit platelets: effects of extracellular Ca2+ and prostacyclin

Alkylacetylglycerophosphocholine (AGEPC) stimulation of 32P-labeled lysophosphatidic acid formation in washed rabbit platelets was dependent on extracellular Ca2+. Its accumulation was slower and required a higher concentration of AGEPC in comparison to the degradation of inositol phospholipids and production of phosphatidic acid induced by the same agonist. These results suggest that the formation of lysophosphatidic acid is not directly related to the primary activation of rabbit platelets by AGEPC. AGEPC elicited a preferential degradation of inositol phospholipids in the following order: phosphatidylinositol 4,5-bisphosphate greater than phosphatidylinositol 4-phosphate greater than phosphatidylinositol. The degradation of inositol phospholipids and subsequent production of phosphatidic acid were affected by pretreatment of platelets with prostacyclin or ethylene glycol bis (beta-aminoethyl ether) N,N'-tetraacetic acid (EGTA). Synergistic inhibitions of these metabolic changes were observed in the platelets pretreated with both prostacyclin and EGTA. These results were compared with effects of prostacyclin and EGTA on serotonin release induced by AGEPC, and the possible roles of metabolic changes in phospholipids induced by AGEPC are discussed with respect to the mechanism of platelet activation.     

Prostacyclin inhibition of phosphatidic acid synthesis in human platelets is not mediated by protein kinase C

The activation of protein kinase C in human platelets by phorbol-12, 13- dibutyrate (PDBu) results in the phosphorylation of a 40,000 dalton protein. This phosphorylation is time- and concentration-dependent. Maximal phosphorylation is rapid and is not affected by indomethacin or prostacyclin. PDBu does not promote activation of the phosphodiesteratic cleavage (phospholipase C) of the inositol phospholipids and the subsequent formation of 1,2-diacylglycerol or its phosphorylated product, phosphatidic acid. If platelets exposed to PDBu are subsequently stimulated with thrombin, this stimulus does not initiate further 40,000 dalton protein phosphorylation but will promote the formation of phosphatidic acid and also the phosphorylation of a 20,000 dalton protein (myosin light chain). However, prostacyclin will prevent the subsequent stimulation of phosphatidic acid synthesis by thrombin in a concentration-dependent manner. The fact that prostacyclin can affect the response to thrombin, even in the presence of phorbol ester, supports the idea that the enzymes related to the formation of phosphatidic acid or inhibition of its synthesis are not related to the phosphorylated 40K protein.     

Changes in conformation of spin-labeled calmodulin by phospholipids

Spin-labeled calmodulin was synthesized and the effects of phospholipids on its conformation were examined by ESR spectroscopy. Phosphatidylserine (0.1-1.0 mM) increased the signal intensity of the ESR spectrum of spin-labeled calmodulin and decreased the apparent rotational correlation time in the presence of 0.1 mM CaCl2. This change was reversed by addition of excess calcium, and in the absence of calcium phosphatidylserine did not change the spectrum, suggesting that the change in spin-labeled calmodulin brought about by phosphatidylserine was not induced by a hydrophobic interaction of the two, but by inhibition of the binding of calcium to calmodulin. L-Serine and O-phospho-L-serine had no effect on the ESR signals of spin-labeled calmodulin. The effects of various other phospholipids were also examined. Their inhibitory activities were in the order phosphatidic acid greater than phosphatidylserine greater than phosphatidylglycerol = phosphatidylinositol; phosphatidylethanolamine and phosphatidylcholine had no effect on the spectra. The effects of these phospholipids were dependent on their binding activities toward calcium. Furthermore, phosphatidic acid and phosphatidylserine at 1 mM reduced the activity of calmodulin-dependent phosphodiesterase by 16.4 and 8.7%, respectively. These findings indicate that spin-labeled calmodulin did not interact with the phospholipids by a hydrophobic interaction, but that calcium binding to spin-labeled calmodulin interfered with phosphatidic acid, phosphatidylserine, phosphatidylglycerol and phosphatidylinositol, and some of these phospholipids inactivated calmodulin. Thus the activity of calmodulin may be regulated in part by some phospholipids.     

Acidic phospholipids and lysophospholipids stimulate cAMP phosphodiesterase of rat liver microsomal membranes

Acidic phospholipids and lysophospholipids modify cAMP phosphodiesterase activity of rat liver microsomal membranes to different extents, depending on the cAMP concentrations employed. At low concentrations, they activate the hormone-sensitive low-Km form of the enzyme through an increase of Vmax (diphosphatidylglycerol greater than phosphatidylglycerol greater than phosphatidic acid = lysophosphatidylserine greater than phosphatidylserine greater than lysophosphatidylcholine). At high concentrations, only lysophospholipids activate the high-Km form of phosphodiesterase through a marked increase in both Vmax and apparent Km for the cAMP.     

Modulation of 32Pi incorporation into phospholipids of polymorphonuclear leukocytes by ionophore A23187.

The effects of ionophore A23187 on the incorporation of 32Pi into phospholipids and on 45Ca2+ uptake and release by polymorphonuclear leukocytes were examined. A23187 increased 32Pi incorporation into phosphatidic acid, phosphatidylglycerol, phosphatidylserine, and the phosphoinositides. It also promoted a rapid burst uptake and release of 45Ca2+ by leukocytes. External Ca2+, but not Mg2+, was required for full stimulation of 32Pi incorporation into phosphatidic acid and the phosphoinositides. In the absence of external Ca2+, the increased radiophosphorus activity of phosphatidic acid, phosphatidylserine and the phosphoinositides was grossly reduced but not eliminated, and the decreased radiophosphorus activity of phosphatidylcholine became pronounced. In addition, the ionophore effect on 32Pi incorporation into leukocyte phospholipids was not abolished by ethyleneglycol bis(beta-amino-ethylether)-N,N'-tetraacetic acid. ATP radiophosphorus activity was also enhanced by the presence of A23187, but the enhancement was much less than that of the acidic phospholipids. Based on these findings, it is suggested that the increased 32Pi incorporation into the acidic phospholipids of leukocytes induced by A23187 was not solely derived from the higher radioactivity of ATP, increased Ca2+ fluxes and perturbation of cellular Ca2+ distribution of leukocytes exposed to A 23187 may trigger part of the altered 32Pi incorporation into phospholipids.     

Effect of retinoic acid on the acylation of phospholipids in granulocytes in vitro

The influence of retinoic acid on the incorporation of [1-14C]palmitic acid and [1-14C]arachidonic acid into phospholipids was examined in guinea pig peritoneal granulocytes. All-trans-retinoic acid inhibited the incorporation of both fatty acids into phosphatidic acid and phosphatidylinositol. However, it stimulated the incorporation of both fatty acids into phosphatidylcholine but not other phospholipids. All-trans-retinoic acid was more effective than 13-cis-retinoic acid. The influence of all-trans-retinoic acid on the acylation of phospholipids was concentration-dependent with significant effect occurring at 2.1 microM. The loss of labeled fatty acids from prelabeled phospholipids and the transport of labeled fatty acids into granulocytes were not responsive to the presence of retinoic acid in the incubation media. These results suggest that retinoic acid may affect the activities of acyltransferases involved in the synthesis of phosphatidic acid, phosphatidylinositol and phosphatidylcholine.     

Increased secretion of lignolytic enzymes by the <Emphasis Type="Italic">Lentinus tigrinus</Emphasis> fungus after addition of butanol and toluene in submerged cultivation

We studied the effects of butanol and toluene on secretion of lignolytic enzymes by the Lentinus tigrinus fungus during submerged cultivation. Addition of butanol and toluene during the trophophase was followed by an increase in laccase and peroxidase activity of the culture and change in the composition of phospholipids and fatty acids. The ratio of phosphatidylcholine and phosphatidic acid decreased, while the amount of lysophosphatidylcholine, phosphatidylethanolamine, phosphoinositides, phosphatidylserine, and unsaturated fatty acids decreased. These changes resulted in an increase in the unsaturation index.