The lung lamellar body as a functioning membrane in protein-catalyzed phosphatidylcholine transfer

Lung lamellar bodies and liver mitochondria were used to demonstrate that soluble phospholipid transfer proteins from lung transfer phosphatidylcholine to both of these acceptors. The initial rate of transfer to lung lamellar bodies is about half that of the rate of transfer to the liver mitochondria when both acceptor membranes are present at saturating concentrations. Phosphatidylcholine unilamellar vesicles were used to demonstrate that the fatty acyl composition of the membrane phosphatidylcholine is a significant determinant of the rate of phosphatidylcholine transfer catalyzed by these proteins. The lamellar bodies have a unique phosphatidylcholine composition, and these studies suggest that this is an important factor in determining the lower initial rate of transfer to lamellar bodies. The studies have also characterized two phospholipid transfer proteins in rat lung in terms of isoelectric point. Isoelectric points for the two proteins which transfer phosphatidylcholine were found to be 5.6 ± 0.08 and 6.2 ± 0.03.     

Stimulation of potato microsomal NADH-cytochrome c reductase by acidic phospholipids

Potato microsomal membranes were solubilized by 0.5% sodium cholate solutions. Separation of lipids from proteins was realized by two successive gel filtrations on two different Sephadex columns. Lipid-free microsomal proteins maintained a high NADH-ferricyanide reductase activity but had a lowered (20%) NADH-cytochrome c reductase activity. The latter activity was strongly stimulated when lipid-free proteins were integrated, by sonication, into phosphatidylserine or phosphatidylinositol liposomes. Some stimulation was obtained also with phosphatidylcholine-lysophosphatidylcholine (7:3) mixtures. Other phospholipids were far less active or even inhibitory. Acidic phospholipids stimulate NADH-cytochrome c reductase activity by increasing noticeably the apparent affinities of enzymatic proteins for NADH or cytochrome c.     

Stimulation of plant plasma membrane Ca2+-ATPase activity by acidic phospholipids

The effect of phospholipids on the activity of the plasma membrane (PM) Ca²⁺-ATPase was evaluated in PM isolated from germinating radish ( Raphanus sativus L. cv. Tondo Rosso Quarantino) seeds after removal of endogenous calmodulin (CaM) by washing the PM vesicles with EDTA. Acidic phospholipids stimulated the basal Ca²⁺-ATPase activity in the following order of efficiency: phosphatidylinositol 4,5-diphosphate (PIP2)≈phosphatidylinositol 4-monophosphate>phosphatidylinositol≈phosphatidylserine≈phosphatidic acid. Neutral phospholipids as phosphatidylcholine and phosphatidylethanolamine were essentially ineffective. When the assays were performed in the presence of optimal free Ca²⁺ concentrations (10 μ M ) acidic phospholipids did not affect the Ca²⁺-ATPase activated by CaM or by a controlled trypsin treatment of the PM, which cleaved the CaM-binding domain of the enzyme. Analysis of the dependence of Ca²⁺-ATPase activity on free Ca²⁺ concentration showed that acidic phospholipids increased V_max and lowered the apparent K_m for free Ca²⁺ below the value measured upon tryptic cleavage of the CaM-binding domain; in particular, PIP2 was shown to lower the apparent K_m for free Ca²⁺ of the Ca²⁺-ATPase also in trypsin-treated PM. These results indicate that acidic phospholipids activate the plant PM Ca²⁺-ATPase through a mechanism only partially overlapping that of CaM, and thus involving a phospholipid-binding site in the Ca²⁺-ATPase distinct from the CaM-binding domain. The physiological implications of these results are discussed.     

Stimulation by local anesthetics of the metabolism of acidic phospholipids in the rat pineal gland

The efficacy of five local anesthetics in causing stimulation of phospholipid metabolism in rat pineal gland $\text{in vitro}$ paralleled their anesthetic potency and decreased in the order: dibucaine, tetracaine, cocaine, procaine, lidocaine. When stimulation occurred, the patterns of labeling resembled that produced by propranolol, a β-adrenergic receptor blocking agent with local anesthetic activity. Isotope incorporation into phosphatidylglycerol and CDP-diglyceride was markedly enhanced and increases of labeling of phosphatidic acid and phosphatidylinositol were also seen. At concentrations of 1–10 mM, propranolol and local anesthetics inhibited labeling of phosphatidylcholine and phosphatidylethanolamine by more than 90% and incorporation of ³²P_i into other phospholipids to a smaller extent.     

Arachidonyl transfer from diacyl phosphatidylcholine to ether phospholipids in rat platelets.

High levels of ether phospholipids were found in rat platelets. Alkylacyl compounds constituted 18 and 29% of glycerophosphocholine (GPC) and glycerophosphoethanolamine (GPE). Alkenylacyl compounds, not detected in GPC, represented 40% of GPE. Arachidonate comprised 60%, 42% and 26% of the acyl residues in the sn-2 position of alkenylacyl-GPE, alkylacyl-GPE and alkylacyl-GPC respectively. Based on all arachidonate being linked to the sn-2 position of the diacyl species, the arachidonate level was 47% in diacyl-GPE and 30% in diacyl-GPC. The incorporation and metabolic fate of arachidonate in various phospholipid classes of resting platelets was examined. Arachidonate was essentially recovered in the diacyl phospholipids and very poorly in alkylacyl- and alkenylacyl-GPE and GPC after 30 min incubation in the presence of [14C]arachidonic acid. Upon reincubation of the platelets after removal of free arachidonate, the radioactivity was gradually lost from diacyl-GPC. Concomitantly, the radioactivities in alkylacyl-GPC, alkylacyl-GPE, alkenylacyl-GPE and to a lower extent in diacyl-GPE were increased. Labelling of glycerophosphoinositol was not changed. This labelling transfer was linear up to 5-6 h, except for alkylacyl-GPC; then labelling remained constant. These data strongly suggest that free arachidonate incorporation through the Lands pathway occurs only for diacyl species and that arachidonate incorporation into the ether phospholipids is achieved by exchange from diacyl-GPC. Based on specific activities related to phosphorus content, the arachidonate incorporation rates into diacyl-GPE and diacyl-GPC were approximately equivalent. The very large differences between specific radioactivities related to arachidonate observed at the starting reincubation time were strongly attenuated when labelling equilibrium was reached. The turnover rate by this exchange pathway was higher in alkylacyl-GPC than in alkyl- and alkenylacyl-GPE. This finding agrees with the selectivity for arachidonate observed in the acylation of PAF-acether in human neutrophils [Chilton, O'Flaherty, Ellis, Swendsen & Wykle (1983) J. Biol. Chem. 258, 7268-7271].     

Evidence for protein-catalyzed transfer of platelet activating factor by macrophage cytosol

Platelet activating factor (PAF) is a potent, proinflammatory lipid. PAF is synthesized in response to stimuli and is rapidly destroyed by specific acetylhydrolases. In order to express its biological activity, PAF and its metabolites are transported among subcellular membranes by as yet unexplained mechanisms. We report here an assay system using methylcarbamyl-PAF (CPAF, 1-O-hexadecyl-2-O-(N-methylcarbamyl)-sn-glycero-3-phosphocholine) and a vesicle-extrusion technique for examining protein-catalyzed intermembrane transfer of CPAF, and demonstrate the presence of proteins catalyzing the separate transfer of CPAF and diacyl phosphatidylcholine in macrophage cytosol. The CPAF transfer activity is heat- and trypsin-sensitive and elutes from gel-filtration columns well separated from proteins catalyzing the transfer of phosphatidylcholine.     

Stimulation of glycogen phosphorylase kinase by phospholipids

The acidic phospholipids phosphatidylinositol (PI), phosphatidylserine (PS), phosphatidylinositol 4-phosphate (PIP), phosphatidylinositol 4,5-biphosphate (PIP2) and the neutral phospholipid lysophosphatidylcholine (LPC) were found to stimulate (3 to 8-fold) the activity of nonactivated rabbit skeletal muscle phosphorylase kinase at pH 6.8, without significantly affecting the activity at pH 8.2. In this respect, phosphatidylcholine and phosphatidylethanolamine were ineffective, while the anionic detergent sodium dodecyl sulfate (SDS) and the anionic steroid dehydroisoandrosterone sulfate (DIAS) were able to mimic the action of phospholipids. SDS was also found to be a very efficient activator of the autophosphorylation of phosphorylase kinase (20-fold activation at 200 microM). The activating effect of phospholipids largely depends on the size of lipid vesicles, which is connected with the procedure of their preparation. These results suggest that phosphorylase kinase belongs to the class of Ca2+-dependent enzymes, which are sensitive to stimulation by calmodulin, limited proteolysis and anionic amphiphiles.     

An in vitro ESR study of uncatalyzed rat liver protein-catalyzed spin-labeled phosphatidylcholine exchange

ESR spectrometry has been used to study fatty acid spin-labeled phosphatidylcholine exchange from single bilayer donor vesicles to various acceptor systems, such as intact or differently treated mitochondria, phospholipid multilamellar vesicles or single bilayer vesicles. This exchange is catalyzed by soluble non-specific rat liver protein, first investigated by Bloj and Zilversmit in 1977 (J. Biol. Chem. 252, 1613--1619). Non-catalyzed phosphatidylcholine exchange has also been studied. Full inhibition of both mechanisms occurs with lipid-depleted acceptor mitochondria, while N-ethylmaleimide-treated mitochondria behave as good acceptors during catalyzed exchange but are in no way effective during spontaneous exchange. Non-catalyzed exchange does not take place with phospholipase D-treated mitochondria as acceptors, while the pure catalyzed mechanism is inhibited by 28%. Neither multilamellar nor single bilayer phospholipid vesicles exchange spin-labeled phosphatidylcholine in the absence of protein, the former being a poorer acceptor system than the latter during catalyzed exchange, when this activity is 31 and 80%, respectively, of that of intact mitochondria. The hypothesis is made that the spontaneous mechanism is active among intact natural membranes and could be of some importance in vivo. Furthermore, the biomembrane protein moiety is assumed to be involved in the catalyzed exchange more as a phospholipid spacer than as a binder between the exchange protein and the membrane involved. Phospholipids, on the contrary, appear to be important for both functions.     

Regulation of liver base-exchange activity by acidic phospholipids

Liver microsomes were enriched in liposomal acidic lipids by Ca²⁺-dependent fusion of liposomes at pH 7.0. The extent of fusion was monitored by the transfer of radioactive cholesteryl oleate. The enrichment of membranes in phosphatidylserine inhibited ethanolamine base-exchange, whereas the fusion with phosphatidylinositol inhibited both ethanolamine and serine base-exchange reactions. In contrast, these two phospholipids had scarce effects on choline base-exchange. Phosphatidic acid did not suppress any of the three base-exchange activities. Possible functional implications are discussed.Abbreviations DTT dithiothreitol - HEPES 4-(2-hydroxyethyl)-1-piperazineethansulfonic acid - SHB suerose-HEPES buffer (0.25M sucrose, 3mM HEPES, pH 7.4)     

Stimulation of cholinephosphotransferase activity by phosphatidylcholine transfer protein. Regulation of membrane phospholipid synthesis by a cytosolic protein

The effect of rat liver phosphatidylcholine transfer protein on the incorporation of CDP-choline and dioleoylglycerol into phosphatidylcholine catalyzed by rat liver microsomal CDP-choline: 1,2-diacyl-sn-glycerol cholinephosphotransferase was studied. In the presence of phosphatidylcholine transfer protein, the incorporation of CDP-choline into phosphatidylcholine was markedly stimulated. Phosphatidylcholine transfer protein isolated from either rat or bovine liver was capable of this stimulatory effect; in contrast, phosphatidylinositol transfer protein from rat liver had no effect on phosphatidylcholine synthesis. Kinetic analysis showed that microsomal phosphatidylcholine synthesis increased 2.4-fold after 1 min and reached a maximum of approximately 10-fold within 10 min in the presence of phosphatidylcholine transfer protein; in the absence of this protein phosphatidylcholine synthesis stopped after 2-4 min. These results suggest that phosphatidylcholine transfer protein permits phosphatidylcholine synthesis to proceed further. With the addition of phospholipid vesicles, as an acceptor membrane in the reaction mixture, there was a significant amount of protein-mediated transfer of synthesized phosphatidylcholine to the vesicles. Measurable transfer of synthesized phosphatidylcholine to vesicles could only be detected after a lag of 2-4 min. The stimulation of cholinephosphotransferase could be nearly abolished by increasing the amount of added phospholipid vesicles; concurrently, a greater transfer to the vesicles was observed. These results describe a new property of phosphatidylcholine transfer protein which may be of physiological significance in the regulation of phosphatidylcholine synthesis in mammalian tissues.     

An in vitro ESR study of uncatalyzed and rat liver protein-catalyzed spin-labeled phosphatidylcholine exchange

ESR spectrometry has been used to study fatty acid spin-labeled phosphatidylcholine exchange from single bilayer donor vesicles to various acceptor systems, such as intact or differently treated mitochondria, phospholipid multilamellar vesicles or single bilayer vesicles. This exchange is catalyzed by soluble non-specific rat liver protein, first investigated by Bloj and Zilversmit in 1977 (J. Biol. Chem. 252, 1613–1619). Non-catalyzed phosphatidylcholine exchange has also been studied. Full inhibition of both mechanisms occurs with lipid-depleted acceptor mitochondria, while $\text{N-}\text{ethylmaleimide-treated}$ mitochondria behave as good acceptors during catalyzed exchange but are in no way effective during spontaneous exchange. Non-catalyzed exchange does not take place with phospholipase D-treated mitochondria as acceptors, while the pure catalyzed mechanism is inhibited by 28%. Neither multilamellar nor single bilayer phospholipid vesicles exchange spin-labeled phosphatidylcholine in the absence of protein, the former being a poorer acceptor system than the latter during catalyzed exchange, when this activity is 31 and 80%, respectively, of that of intact mitochondria. The hypothesis is made that the spontaneous mechanism is active among intact natural membranes and could be of some importance in vivo. Furthermore, the biomembrane protein moiety is assumed to be involved in the catalyzed exchange more as a phospholipid spacer than as a binder between the exchange protein and the membrane involved. Phospholipids, on the contrary, appear to be important for both functions.     

Modulation of the phospholipid transfer protein-mediated transfer of phospholipids by diacylglycerols

Previous studies have shown that diacylglycerols (DAG) are formed during triglyceride hydrolysis in very low density lipoproteins (VLDL), a process that is accompanied by an elevated phospholipid transfer protein (PLTP)-mediated transfer of phospholipids (PL) from VLDL to high density lipoprotein. Because PLTP has been also shown to transfer DAG, we hypothesized that DAG might modulate PL transfer through a mechanism of competition with respect to PLTP. To address this question we performed in vitro PL transfer assays using specifically designed PL donor particles. These were single bilayer vesicles (SBV) and large (EM-L) or small (EM-S) lipid emulsions, containing various proportions of DAG. The PLTP-mediated transfers of PL decreased as the volumes of the particle cores increased (SBV > EM-S > EM-L). In all cases, these transfers were inhibited by DAG in a concentration-dependent manner. We determined the core-to-surface distribution of DAG and we measured their relative affinity for PLTP by comparison with that of PL. From these parameters, we calculated the theoretical effects of DAG on PL transfers that would result from a competition mechanism. The experimental data showed that the inhibiting effects of DAG on PL transfers were much more important than those predicted from our calculations. Additional data showed that a large part of DAG effects was in fact due to their ability to increase the viscosity of the particle PL surfaces, as calculated from electron spin resonance experiments.These results show that DAG can modulate the PLTP-dependent PL transfers, both by competition with PL and by increasing the viscosity of the particle surfaces. These findings might be physiopathologically relevant in situations where elevated plasma concentrations of DAG might result from hypertriglyceridemia.-Lalanne, F., C. Motta, Y. Pafumi, D. Lairon, and G. Ponsin. Modulation of the phospholipid transfer protein-mediated transfer of phospholipids by diacylglycerols. J. Lipid Res. 2001. 42: 142;-149.     

Synergistic stimulation of EpsE ATP hydrolysis by EpsL and acidic phospholipids

EpsE is a cytoplasmic component of the type II secretion system in Vibrio cholerae. Through ATP hydrolysis and an interaction with the cytoplasmic membrane protein EpsL, EpsE supports secretion of cholera toxin across the outer membrane. In this study, we have determined the effect of the cytoplasmic domain of EpsL (cyto-EpsL) and purified phospholipids on the ATPase activity of EpsE. Acidic phospholipids, specifically cardiolipin, bound the copurified EpsE/cyto-EpsL complex and stimulated its ATPase activity 30-130-fold, whereas the activity of EpsE alone was unaffected. Removal of the last 11 residues (residues 243-253) from cyto-EpsL prevented cardiolipin binding as well as stimulation of the ATPase activity of EpsE. Further mutagenesis of the C-terminal region of the EpsL cytoplasmic domain adjacent to the predicted transmembrane helix suggested that this region participates in fine tuning the interaction of EpsE with the cytoplasmic membrane and influences the oligomerization state of EpsE thereby stimulating its ATPase activity and promoting extracellular secretion in V. cholerae.     

Effect of acidic phospholipids on sphingosine kinase

Sphingosine-1-phosphate (SPP) is a unique sphingolipid metabolite involved in cell growth regulation and signal transduction. SPP is formed from sphingosine in cells by the action of sphingosine kinase, an enzyme whose activity can be stimulated by growth factors. Little is known of the mechanisms by which sphingosine kinase is regulated. We found that acidic phospholipids, particularly phosphatidylserine, induced a dose-dependent increase in sphingosine kinase activity due to an increase in the apparent Vmax of the enzyme. Other acidic phospholipids, such as phosphatidylinositol, phosphatidic acid, phosphatidylinositol bisphosphate, and cardiolipin stimulated sphingosine kinase activity to a lesser extent than phosphatidylserine, whereas neutral phospholipids had no effect. Diacylglycerol, a structurally similar molecule which differs from phosphatidic acid in the absence of the phosphate group, failed to induce any changes in sphingosine kinase activity. Our results suggest that the presence of negative charges on the lipid molecules is important for the potentiation of sphingosine kinase activity, but the effect does not directly correlate with the number of negative charges. These results also support the notion that the polar group confers specificity in the stimulation of sphingosine kinase by acidic glycerophospholipids. The presence of a fatty acid chain in position 2 of the glycerol backbone was not critical since lysophosphatidylserine also stimulated sphingosine kinase, although it was somewhat less potent. Dioleoylphosphatidylserine was the most potent species, including a fourfold stimulation, whereas distearoyl phosphatidylserine was completely inactive. Thus, the degree of saturation of the fatty acid chain of the phospholipids may also play a role in the activation of sphingosine kinase. © 1996 Wiley-Liss, Inc.     

Ubiquitin carrier protein-catalyzed ubiquitin transfer to histones. Mechanism and specificity

Ubiquitinated derivatives of histones H2A and H2B, in which the carboxyl terminus of ubiquitin is joined to epsilon-amino groups of specific lysine residues of each histone, occur in vivo. Certain ubiquitin carrier proteins (E2s) catalyze ubiquitin transfer to histones (Pickart, C. M., and Rose, I. A. (1985) J. Biol. Chem. 260, 1573-1581). The catalytic activities of these purified ubiquitin carrier proteins have been quantitatively characterized with purified histones, in order to determine if one or more of them exhibits specificity for H2A over other histones (H3,H4) which are not known to be ubiquitinated in vivo. The results show the following. 1) No E2 exhibits strong specificity for H2A over the other histones. 2) For a given histone, kinetics of formation of its monoubiquitinated adduct do not differ strongly among the E2s; sigmoid kinetics (nH = 2) are generally observed, with values of K 0.5 ranging from 2-6 microM. 3) E214K catalyzes primarily monoubiquitination. 4) E220K catalyzes multiple ubiquitination (up to three ubiquitin/histone) by a processive mechanism that involves joining of ubiquitin carboxyl termini to multiple histone lysine residues. 5) E235K also catalyzes processive ubiquitination, with formation of polyubiquitinated products exhibiting a lag phase. Many of the polyubiquitinated adducts produced at low histone concentration are larger than expected for monoubiquitination of every histone-lysine residue, and polyubiquitination is selectively inhibited by substitution of reductively methylated ubiquitin for ubiquitin. These results suggest that E235K uniquely catalyzes ubiquitin transfer to lysine residues of previously conjugated ubiquitin molecule(s). The implications of these results for biological mechanisms of histone ubiquitination are discussed.     

Single plate separation of lung phospholipids including disaturated phosphatidylcholine

We have developed an improved thin-layer chromatographic method for separation of lung phospholipids. Individual phospholipids are completely separated in the first dimension. All phospholipids, except phosphatidylcholine, are then removed. The phosphatidylcholine-containing area is reacted with osmium tetroxide and saturated phosphatidylcholine species are separated from the unsaturated oxidation products by subsequent chromatography in the second dimension. This method should prove useful in studies on lung surfactant metabolism and secretion, in prediction of fetal lung maturity by analysis of amniotic fluid phospholipids, and in studies on surfactant obtained by bronchoscopy in adult lung diseases.     

Rapidly-labelled, acidic phospholipids of the goldfish brain

Homogenates and particulate fractions of goldfish brain incorporated radioactivity from γ-[³²P]ATP selectively into acidic phospholipids during brief periods of incubation. Phosphatidate and lysophosphatidate became strongly labelled and activity was also found in phosphatidyl inositol phosphate and in phosphatidyl inositol diphosphate. When tetraphenylborate (a K⁺-complexing agent) was added, a selective stimulation of incorporation of ³²P into phosphatidate occurred. The addition of perchlorate (also known to bind K⁺) did not produce a similar stimulation, nor did the addition of K⁺ block the stimulation by tetraphenylborate. The stimulation of the labelling of phospholipids by tetraphenylborate appeared to be the result of multiple actions. Besides the evidence that it acted by stimulating the phosphoinositide phosphodiesterase of brain, data were obtained suggesting that it stimulated diglyceride kinase and blocked endogenous destruction of ATP as well. The stimulation by tetraphenylborate was blocked by addition of atropine but not of arecoline.