Studies on the endogenous phospholipids of mammalian kidney and their in vitro hydrolysis by endogenous phospholipases: a thin layer chromatographic and densitometric study

The phosphoglycerides profile of six species of mammalian kidney (guinea pig, pig, cat, dog, mouse and rat) and their in vitro response to the endogenous phospholipases were determined by TLC technology in conjunction with densitometric measurements. Changes in their phospholipids profile subsequent to in vitro incubation of whole tissue homogenate of these kidneys for 60 min, at pH 7.4, 38 degrees C, and prior to phospholipids extraction have shown that the deacylation of the endogenous cardiolipin (CL) is the most prevalent lipolytic event of all mammalian kidneys studied. Concurrent with the deacylation of CL, there was also formation of monolysocardiolipin (MLCL) and a reduction in CL level. To a much lesser extent, lyso alkenyl phosphatidyl ethanolamine (LPE) was also produced concomitant with a decrease of the endogenous alkenyl phosphatidyl ethanolamine (PE) level. The deacylation of PE plasmalogen to its lyso form confirms the action of endogenous PLA(2) releasing sn-2 fatty acids.     

Phospholipid remodeling/generation in Giardia: the role of the Lands cycle.

Recent results suggest that Giardia is able to carry out deacylation/reacylation reactions (the Lands cycle) to generate new phospholipids, effectively bypassing the de novo synthesis of the entire phospholipid molecule. The successful operation of this deacylation/reacylation cycle is important for Giardia because this protozoan parasite possesses limited lipid synthesis ability. This article discusses how Giardia might use the Lands cycle to alter phospholipids acquired from the host during its colonization in the human small intestine.     

Characterization of phospholipase activities in chromaffin granule ghosts isolated from the bovine adrenal medulla

Highly purified chromaffin granule membranes contain high levels (100 nmol/mg protein) of long-chain free fatty acids (Husebye, E.S. and Flatmark, T. (1984) J. Biol. Chem. 259, 15272-15276), as well as lysophosphatidylcholine (268 nmol/mg protein) and lysophosphatidylethanolamine (92 nmol/mg protein). The release of saturated and unsaturated long-chain fatty acids from endogenous phospholipids was 38 and 28 nmol/mg protein per h, respectively, at 37 degrees C and pH 7.5 (alkaline pH optimum). p-Bromophenacyl bromide inhibited the release of palmitate and oleate by 88 and 65%, respectively. The deacylation of membrane phospholipids was not significantly affected by micromolar free Ca2+. Based on experiments with pancreatic phospholipase A2, stearate and arachidonate were found to be suitable markers for deacylation at the sn-1 and sn-2 positions, respectively. Experiments with exogenously added labeled phosphatidylcholines confirmed that chromaffin granule ghosts contain a phospholipase A2 activity (alkaline pH optimum). The preparations also revealed a phospholipase A1 activity (acid pH optimum). Finally, the ghosts contain a lysophospholipase activity (alkaline pH optimum), that accounts for the major part of the deacylation of membrane phospholipids, notably the release of saturated fatty acids (stearate and palmitate). It is unlikely that the high content of lysophospholipids is an artifact of the procedure by which the granule ghosts are isolated.     

Differential activation of phosphatidylinositol deacylation and a pathway via diphosphoinositide in macrophages responding to zymosan and ionophore A23187

The inositol phospholipids of peritoneal macrophages were prelabeled with [3H]inositol to enable studies on the enzymatic mechanisms of stimulus-induced phosphatidylinositol breakdown. Ionophore A23187 induced a rapid breakdown of phosphatidylinositol in the presence of Ca2+ with 25% loss occurring within 5 min. The main water-soluble product of this breakdown was identified as inositol diphosphate. Since the accumulation of inositol diphosphate far exceeded the concomitant decrease in polyphosphoinositides, an increased phosphorylation of phosphatidylinositol must have preceded, or accompanied, the degradation of diphosphoinositide. The degradation of phosphatidylinositol induced by A23187 was shown to be strictly dependent on Ca2+. The monovalent cation ionophore monensin and platelet-activating factor increased the level of diphosphoinositide but caused no net degradation of inositol phospholipids. The same effect was seen with ionophore A23187 in the absence of Ca2+. Zymosan particles also induced extensive degradation of phosphatidylinositol. Products of phosphodiesterase-catalyzed cleavage of inositol lipids were observed, but the pathway of deacylation dominated as evidenced by the accumulation of lysophosphatidylinositol and glycerophosphoinositol. Deacylation was also enhanced in response to concanavalin A. Thus, in mouse peritoneal macrophages phosphatidylinositol breakdown occurred primarily by deacylation or via diphosphoinositide, depending on the stimulus, rather than through a phosphatidylinositol phosphodiesterase reaction.     

Stimulation of deacylation in Madin-Darby canine kidney cells. Specificity of deacylation and prostaglandin production in 12-O-tetradecanoylphorbol-13-acetate-treated cells

Madin-Darby canine kidney cells deacylate arachidonic acid from cellular phospholipid in response to 12-O-tetradecanoyl-phorbol-13-acetate (TPA) and convert the free arachidonic acid to prostaglandins. We have used this system to characterize the acyl specificity of deacylation. Cells were labeled with either [14C]linoleic, [14C]eicosatrienoic (delta 8,11,14 or delta 5,8,11), or [14C]arachidonic acid and stimulated with 10 nM TPA. We found that TPA stimulated the deacylation of all four acids, primarily from phosphatidylethanolamine and phosphatidylcholine.l Only products from linoleic (presumably through chain elongation and desaturation), eicosatrienoic (delta 8,11,14), and arachidonic acids produced prostaglandins. Those produced from linoleic and eicosatrienoic acid (delta 8,11,14)-labeled cells were determined to be primarily of the 1-series, while arachidonic acid-labeled cells produced prostaglandins of the 2-series. Together these results indicate that the stimulated deacylation of phospholipids is not specific for arachidonic acid and that the membrane acyl composition controls the particular series of prostaglandin which is produced.     

Diacylglycerol induces deacylation of phosphatidylinositol and mobilization of arachidonic acid in mouse macrophages. Comparison with induction by phorbol diester.

1,2-Dioctanoyl-sn-glycerol (2-50 microM) was found, like phorbol myristate acetate (greater than or equal to 3 nM) to stimulate phospholipase A-type cleavage of phosphatidylinositol and the release of arachidonic acid from macrophage phospholipids. The 1,3 isomer of dioctanoylglycerol was inactive, whereas racemic 1,2-dioctanoylglycerol was half as potent as the 1,2-sn enantiomer. Dioctanoylglycerol-induced deacylation of phosphatidylinositol was only partly dependent on extracellular calcium but was more severely inhibited by depletion of intracellular calcium. Chlorpromazine inhibited the deacylation of phosphatidylinositol, whereas inhibitors of cyclo-oxygenase and lipoxygenase were ineffective. Since both phorbol myristate acetate and 1,2-dioctanoyl-sn-glycerol are known to activate protein kinase C, the results suggest that this kinase is involved in the sequence of events leading to release of arachidonic acid in macrophages.     

Phospholipid synthesis by extracellular phospholipase A2 in organic solvents

The catalytic activity of extracellular phospholipase A2 was studied in low polarity solvents where hydrolytic enzymes have been demonstrated to catalyze synthesis reactions. It was demonstrated that extracellular phospholipase A2 can catalyze the esterification of lysophosphatidylcholine with oleic acid. Up to 6.5% of lysophosphatidylcholine can be esterified into phosphatidylcholine. This activity requires a preincubation of the enzyme in a pH 9 aqueous solution containing calcium, before the incubation in the non-aqueous solvent. No transfer of fatty acid between a phospholipid and a lysophospholipid or between two phospholipids was observed. These results may be useful in understanding the function of the membrane phospholipase A2 which may catalyze acylation or deacylation depending on the local physico-chemical environment.     

The phospholipids of Pneumococcus I-192R, A.T.C.C. 12213: Some structural rearrangements occurring under mild conditions

1. The phospholipids from the non-capsulated strain of Pneumococcus I-192R, A.T.C.C. 12213, were separated into three fractions by chromatography on columns of silicic acid and DEAE-cellulose (acetate form). 2. The water-soluble phosphate esters produced by deacylation of each fraction were separated by chromatography on columns of DEAE-cellulose (HCO(3) (-) form). 3. Three deacylated products, diglycerol phosphate, glycerylphosphorylglycerol phosphate and bis(glycerylphosphoryl)glycerol, were identified by analysis, by chemical degradations and by comparison with synthetic materials. 4. From a study of freshly isolated lipids prepared and worked up under conditions where exposure to acid was minimal, it was concluded that the Pneumococcus contains phosphatidylglycerol and bisphosphatidylglycerol, in the molar proportion 1:2.5-3.0, and that the deacylation product glycerylphosphorylglycerol phosphate was probably an artifact of the isolation procedure. 5. Acid-catalysed isomerization (phosphodiester migration) of diglycerol phosphate and bis(glycerylphosphoryl)glycerol and transesterification (glycerol phosphate transfer) of diglycerol phosphate were observed. The structures of the products were established by degradation. 6. A novel mechanism for the biosynthesis of bisphosphatidylglycerol is presented.     

Gas-liquid chromatography of plant galactolipids and their deacylation and methanolysis products.

The gas-liquid chromatography of monogalactosyl diglyceride (MGDG) and digalactosyl diglyceride (DGDG) and their deacylation and methanolysis products is reported. MGDG and DGDG and their galactosyl monoglycerides were chromatographed as their trimethylsilyl derivatives. Galactosyl monoglycerides were produced by partial deacylation of the diglycerides with Grignard's reagent and pancreatic lipase. The products of complete deacylation, mono- and digalactosyl glycerols, were separated as O-methyl, O-acetyl, O-trimethylsilyl and O-trifluoroacetyl derivatives. Gas-liquid chromatography of derivatives of the methanolysis products of MGDG and DGDG and the methylated galactosyl glycerols allowed the separation and quantitative recovery of the galactose and glycerol of both lipids and the two galactoses of DGDG.     

Two distinct pathways of platelet-activating factor-induced hydrolysis of phosphoinositides in primary cultures of rat Kupffer cells

Stimulation of rat Kupffer cells in primary culture with platelet-activating factor (PAF) caused a rapid hydrolysis of phosphatidylinositol 4,5-bisphosphate and phosphatidylinositol 4-phosphate with a concomitant increase in the levels of myo-inositol 1,4,5-trisphosphate and myo-inositol 1,4-bisphosphate. This phospholipase C-mediated hydrolysis of polyphosphoinositides was independent of extracellular Ca2+ but was inhibited by the intracellular Ca2+ antagonist TMB-8. A second slower response to PAF was characterized by deacylation of PI leading to the accumulation of glycerophosphoinositol (GPI). PAF-induced GPI synthesis was not inhibited by TMB-8. These effects of PAF were accompanied by initial transient mobilization of Ca2+ from intracellular stores followed by a rather slow influx of Ca2+ from the extracellular medium. PAF-stimulated deacylation and phosphodiesteric hydrolysis of inositol lipids were differentially affected by cholera toxin and pertussis toxin. Pretreatment of the Kupffer cells with either of these toxins caused inhibition of phospholipase C activity. Pertussis toxin also inhibited PAF-stimulated deacylation. However, cholera toxin itself stimulated GPI release and addition of PAF to the cholera toxin-treated cells caused a further increase in GPI release. Phorbol ester inhibited PAF-induced phosphodiesteric hydrolysis of phosphoinositides, but not deacylation. PAF-induced metabolism of phosphoinositides was inhibited by the PAF antagonist, U66985. These results suggest that PAF-induced phosphodiesteric hydrolysis and deacylation of inositol phospholipids are regulated via distinct mechanisms involving activation of separate G-proteins in rat Kupffer cells. Also the regulation of phosphoinositide metabolism by Ca2+ mobilization from two separate Ca2+ pools is indicated by this study.     

Kinetic aspects of the role of phospholipids in D-beta-hydroxybutyrate dehydrogenase activity

Interactions of phospholipids with D-beta-hydroxybutyrate dehydrogenase (BDH), a lecithin-requiring enzyme, have been studied by a kinetic approach. The process of reactivation of BDH by phospholipids, which follows a second-order mechanism, reveals that (1) at least 2 mol of lecithins is essential for the reactivation of the enzyme, and (2) the enzyme contains two dependent binding sites for lecithins. The graphic representation of the time course of reactivation shows a latent phase which decreases when there is an increase in the amount of phospholipids. A Scatchard plot treatment of the reactivation kinetic data reveals the presence of two classes of phospholipid binding sites, which exhibit high and low affinities related to the binding of four and two lecithin molecules, respectively. The effect of temperature on BDH activity and on the inactivation of the apoenzyme with N,N'-dicyclohexylcarbodiimide (a specific carboxyl reagent) or with phenylglyoxal (a specific arginine reagent) shows a break at 22-24 degrees C, indicating a slight structural change in the enzyme-active site around this temperature. In addition, the variations in enzyme kinetic parameters, according to the nature of phospholipids, are in agreement with conformational changes related to the nature and to the fluidity state of phospholipids. However, the apparent NAD+ binding constant does not depend on the phospholipid's fluidity.     

IL-1 alpha increases arachidonyl-CoA: lysophospholipid acyltransferase activity and stimulates [3H]arachidonate incorporation into phospholipids in rat mesangial cells.

The proinflammatory cytokine interleukin-1 alpha is a potent stimulus of prostaglandin synthesis. We have previously shown that IL-1 amplifies mesangial cell prostaglandin synthesis by inducing synthesis of a non-pancreatic phospholipase A2. Phospholipase A2 activation results in the formation of lysophospholipids and free fatty acids. We now investigate the effects of IL-1 alpha on reacylation of lysophospholipids. Incubations with IL-1 alpha for 24 hours significantly stimulated mesangial cell [3H]arachidonic acid incorporation but not [3H]oleic acid incorporation into phosphatidylinositol and phosphatidylethanolamine. Lysophospholipid acyltransferase activity was measured in vitro. Cytokine treatment increased enzyme activity when lysophosphatidylcholine, lysophosphatidylethanolamine and lysophosphatidylinositol were used as exogenous substrates. We conclude that IL-1 promotes cellular phospholipid remodeling by stimulating the deacylation and reacylation of phospholipids.     

Rapid solid phase isolation of 20 specific tRNAs from Escherichia coli.

A solid phase procedure has been developed for the rapid isolation of all 20 species of tRNA from Escherichia coli. The overall yields for a single preparation cycle ranged from 62 to 96%, the average being 80%. The values for the amino acid acceptor activities of the tRNA species equaled those reported in the literature for highly purified tRNAs. Starting from crude tRNA, a given tRNA species can easily be isolated in less than 2 h. One milliliter of the resin, which is reusable, is sufficient for the isolation of 200 mg of a specific tRNA. The procedure requires a bifunctional reagent, one moiety of which (--SO2Cl) reacts with the amino acid on the aminoacylated tRNA, the other, with the --SH group on the resin. Thus, only the desired tRNA species is bound to the resin; any of the other tRNAs in the filtrate can be isolated in another cycle. Raising the pH results in deacylation and release from the resin of the desired tRNA species. For tRNA Cys, it is necessary to block the --SH of cysteine prior to reaction with the bifunctional reagent. Side reactions involving the bifunctional reagent. Side reactions involving the bifunctional reagent and tRNA are either easily reversible or negligible (less than 0.01%).     

High resolution 31P NMR of extracted phospholipids

The common phospholipids from biological sources were quantitated using phosphorus-31 nuclear magnetic resonance (NMR) spectroscopy in conjunction with an analytical reagent composed of two parts: 1) 2 ml of reagent chloroform in which was dissolved 0.01-100 mg of crude tissue lipid extracted from tissue sources using chloroform-methanol 2:1, the extract having been washed with 0.2 vol. of 0.1 M KCl; 2) 1 ml of an aqueous methanol reagent composed of one part 0.2 M (ethylenedinitrilo)-tetraacetic acid in D2O titrated to pH 6 with CsOH and four parts of reagent methanol. In a magnetic field of 11.75 Tesla, the extracted phospholipids yield narrow signals (1.8-3.2 Hz at half-height), corresponding to each generic species, e.g., phosphatidylcholines, phosphatidylethanolamines, etc., permitting resolution among the various phospholipid families and their lyso and plasmalogen derivatives. The reagent permits assays of high precision and accuracy using a modest amount of NMR spectrometer time (ca. 15 min/assay). The procedures described, which are compared to high-performance liquid chromatography, are convenient for the routine analysis of phospholipids from biological sources.     

Blockade of arachidonic acid incorporation into phospholipids induces apoptosis in U937 promonocytic cells

Arachidonic acid (AA) participates in a reacylation/deacylation cycle of membrane phospholipids, the so-called Lands cycle, that serves to keep the concentration of this free fatty acid in cells at a very low level. To manipulate the intracellular AA level in U937 phagocytes, we have used several pharmacological strategies to interfere with the Lands cycle. We used inhibitors of the AA reacylation pathway, namely thimerosal and triacsin C, which block the conversion of AA into arachidonoyl-CoA, and a CoA-independent transacylase inhibitor that blocks the movement of AA within phospholipids. In addition, we used cells overexpressing group VIA phospholipase A(2), an enzyme with key roles in controlling basal fatty acid deacylation reactions in phagocytic cells. All of these different strategies resulted in the expected increase of cellular free AA but also in the induction of cell death by apoptosis. Moreover, when used in combination with any of the aforementioned drugs, AA itself was able to induce apoptosis at doses as low as 10 muM. Blocking cyclooxygenase or lipoxygenases had no effect on the induction of apoptosis by AA. Collectively, these results indicate that free AA levels within the cells may provide an important cellular signal for the onset of apoptosis and that perturbations of the mechanisms controlling AA reacylation, and hence free AA availability, may decisively affect cell survival.     

Role of carnitine and carnitine palmitoyltransferase as integral components of the pathway for membrane phospholipid fatty acid turnover in intact human erythrocytes.

The deacylation and reacylation process of phospholipids is the major pathway of turnover and repair in erythrocyte membranes. In this paper, we have investigated the role of carnitine palmitoyltransferase in erythrocyte membrane phospholipid fatty acid turnover. The role of acyl-L-carnitine as a reservoir of activated acyl groups, the buffer function of carnitine, and the importance of the acyl-CoA/free CoA ratio in the reacylation process of erythrocyte membrane phospholipids have also been addressed. In intact erythrocytes, the incorporation of [1-14C]palmitic acid into acyl-L-carnitine, phosphatidylcholine, and phosphatidylethanolamine was linear with time for at least 3 h. The greatest proportion of the radioactivity was found in acyl-L-carnitine. Competition experiments using [1-14C]palmitic and [9,10-3H]oleic acid demonstrated that [9,10-3H]oleic acid was incorporated preferentially into the phospholipids and less into acyl-L-carnitine. When an erythrocyte suspension was incubated with [1-14C]palmitoyl-L-carnitine, radiolabeled palmitate was recovered in the phospholipid fraction, and the carnitine palmitoyltransferase inhibitor, 2-tetradecylglycidic acid, completely abolished the incorporation. ATP depletion decreased incorporation of [1-14C]palmitic and/or [9,10-3H]oleic acid into acyl-L-carnitine, but the incorporation into phosphatidylcholine and phosphatidylethanolamine was unaffected. In contrast, ATP depletion enhanced the incorporation into phosphatidylcholine and phosphatidylethanolamine of the radiolabeled fatty acid from [1-14C]palmitoyl-L-carnitine. These data are suggestive of the existence of an acyl-L-carnitine pool, in equilibrium with the acyl-CoA pool, which serves as a reservoir of activated acyl groups. The carnitine palmitoyltransferase inhibition by 2-tetradecylglycidic acid or palmitoyl-D-carnitine caused a significant reduction of radiolabeled fatty acid incorporation into membrane phospholipids, only when intact erythrocytes were incubated with [9,10-3H]oleic acid. These latter data may be explained by the differences in rates and substrates specificities between acyl-CoA synthetase and the reacylating enzymes for palmitate and oleate, which support the importance of carnitine palmitoyltransferase in modulating the optimal acyl-CoA/free CoA ratio for the physiological expression of the membrane phospholipids fatty acid turnover.     

Inositol lipids, phosphatidate and diacylglycerol share stearoylarachidonoylglycerol as a common backbone in thrombin-stimulated human platelets.

Gel-filtered human platelets were stimulated with 5i.u. of thrombin/ml for times up to 1 min. The fatty acid composition of inositol-containing phospholipids, phosphatidic acid and diacylglycerol was determined by g.l.c. in control and thrombin-stimulated platelet suspensions. Inositol phospholipids were found to have similar proportions of stearic and arachidonic acids, the sum of these representing 86.6% of the total fatty acids in phosphatidylinositol (PtdIns), 76.9% in phosphatidylinositol 4-phosphate (PtdIns4P) and 85.4% in phosphatidylinositol 4,5-bisphosphate [PtdIns(4,5)P2]. However, arachidonic and stearic acids were less abundant in phosphatidic acid (PtdA) and diacylglycerols in non-stimulated platelets. A transient decrease in the mass of PtdIns(4,5)P2 was observed after 5-10s of thrombin stimulation, followed by an increase after 30s. The amounts of PtdIns4P and PtdIns decreased throughout the experiment. A transient accumulation of stearoylarachidonoylglycerol was observed at 5s, whereas stearoylarachidonoylglycerol 3-phosphate (PtdA) was produced in increasing amounts throughout the experiment. The decrease in inositol-containing phospholipids was not fully compensated for by the production of diacylglycerol or PtdA [or PtdIns(4,5)P2] at 1 min. All the changes in inositol phospholipids, as well as those observed in diacylglycerols and PtdA, were due to a parallel reduction or increase in the contents of stearic and arachidonic acids, with a stoichiometry equal to 1. Taken together, this suggests an interconversion of all these lipids with the utilization of a common backbone, stearoylarachidonoylglycerol. The deacylation of this diacylglycerol could account for up to 4-5nmol of arachidonate/10(9) platelets after 1 min stimulation by thrombin.     

Kinetic behaviour of alpha-chymotrypsin in reverse micelles. A stopped-flow study.

At the aim of investigating whether the early rapid phase of enzyme turnover is different in reverse micelles compared with bulk water, the kinetic properties of alpha-chymotrypsin have been studied in reverse micelles formed by sodium bis(2-ethylhexyl)sulfosuccinate in isooctane. Pre-steady state and steady-state kinetic constants, in water and in reverse micelles, have been determined by stopped-flow spectrophotometry for the hydrolysis of two substrates, namely acetyl-L-tryptophan-p-nitrophenyl ester and p-nitrophenyl acetate. It has been shown that, for both substrates, the acylation rate constant (k2) is very much lower in reverse micelles than in water. However, the deacylation rate constant (k3) and the turnover number (kcat) are not significantly changed in reverse micelles with respect to bulk water. Therefore, despite considerable rate changes in the acylation step, deacylation is rate limiting both in water as well as in reverse micelles, under the experimental conditions used.     

Reversible activation/inactivation of the deacylation/acylation cycle in rat liver microsomes

Rat liver microsomes incorporate [¹⁴C]palmitoyl CoA into membrane phospholipids via the deacylation/acylation cycle. This activity is reversibly inactivated/activated by treatment of the microsomes with ATP, MgCl₂, and 105,000g supernatant or with 105,000g supernatant alone. These observations suggest that the acylation cycle is controlled by a mechanism involving phosphorylation/dephosphorylation. As the pool of lysolecithin in the membranes is not altered by conditions increasing incorporation of palmitoyl CoA into phospholipid, it is probable that the site of regulation of deacylation/acylation is at the acyltransferase rather than the phospholipase.     

Platelet-activating factor promotes arachidonate incorporation into phosphatidylinositol and phosphatidylcholine in neutrophils

Platelet-activating factor (1-0-hexadecyl-2-acetyl-sn-glycero-3-phosphocholine, PAF) promotes the incorporation of [1-14C]arachidonic acid most significantly into phosphatidylinositol (PI) and phosphatidylcholine (PC) during the early stages of guinea pig neutrophil-PAF interaction. The stimulation reached a maximum at 10(-7) M and started to decline at 10(-6) M. No changes in the mass of each phospholipid were detected in neutrophils challenged by PAF for 1 to 5 minutes. The stimulation by PAF on the formation of [14C]arachidonoyl-PC but not [14C]arachidonoyl-PI was dependent on the presence of external Ca2+. These results suggest that the increased acylation of PI and PC elicited by PAF is secondary to an increased deacylation of these phospholipids and the mechanisms by which PAF stimulates the deacylation of PI and PC may be different.