Symmetric transcription of simian virus 40 DNA in the nuclei of transformed mouse cells.

Na⁺ channels from lobster nerve membranes stored frozen in sucrose were incorporated into artificial liposomes. Crude soybean phospholipids or mixtures of purified phospholipids were suitable for reconstitution provided the latter included phosphatidylserine or another acidic phospholipid. The ²²Na flux into the reconstituted vesicles was increased (2 to 3-fold) by veratridine (0.25 – 1 mM) or grayanotoxin I (50 –150 μM) and the increment was abolished by 10 nM tetrodotoxin (K_i = 2 nM). The reconstituted vesicles were inactivated after incubation for 15 min at 40° and exposure to 20 μM dicyclohexylcardobiimide inhibited by 80% the response to the drugs.     

Endogenous phospholipid metabolism in stimulated neutrophils differential activation by FMLP and PMA

Endogenous phospholipid metabolism was examined during the initial 0–120 seconds of neutrophil (PMN) stimulation. When PMN were exposed to the chemotactic peptide FMLP (10^?7 M) or the tumor promotor, phorbol myristate acetate (PMA, 1 μg/ml) extensive changes in specific phospholipid (PL) classes were evident within 15 seconds. The profile and kinetics of stimulus-induced PL changes were stimulus-dependent. Five seconds after the addition of FMLP, PMN content of PC, PS and PA increased, while the level of PI decreased. Kinetic studies revealed that only PA levels remained elevated (0–120 s) while other PL decreased. In contrast, when cells were exposed to PMA (1 μg/ml), the levels of PC and PS rapidly increased (< 15 s). With PMA as stimulus, changes in PI and PA were not observed until > 60 s. Results indicate that exposure to PMN to stimuli leads to rapid changes in specific PL. In addition, they support the concept that neutrophils rapidly “remodel” endogenous PL upon stimulation.     

AGEPC (platelet activating factor) induced stimulation of rabbit platelets: effects on phosphatidylinositol, di- and triphosphoinositides and phosphatidic acid metabolism

Stimulation of washed rabbit platelets with AGEPC (1-O-alkyl-2-acetyl-$\text{sn}$-glyceryl-3-phosphorylcholine) caused a 15–20% decrease in their phosphatidylinositol level within 15 seconds without affecting other major classes of phospholipids. In the same time frame the level of phosphatidic acid (PA) increased dramatically some four fold. LysoGEPC, which is inactive in stimulating rabbit platelets, did not cause any change in PI or PA. When [³²Pi] was present during the stimulation of platelets by AGEPC, the incorporation of radiolabel into PI-4-phosphate (DPI), PI-4,5-bis phosphate (TPI) and PA was enhanced significantly within one minute while the incorporation into PI increased only after one minute. These results clearly established that AGEPC induced stimulation of rabbit platelets was associated with the metabolism of inositol phospholipids and phosphatidic acid. The relevance of these findings to the mode of action of AGEPC and Ca²⁺ mobilization is also discussed.     

Phospholipase C activity in plasma membranes isolated from lapine synovial cells in monolayer culture

Two phosphorylase kinase activities were resolved by DEAE-cellulose chromatography. The main activity peak was enriched 2800-fold, the minor appeared to be an aggregate of the enzyme. Phosphorylase kinase also phosphorylated histone and casein with no changes in phosphorylation ratios throughout the preparation steps but was most active on yeast phosphorylase. The molecular weight was 29000 +/- 2000. ATP, UTP, GTP served as substrates while CTP was inactive. Mg-ions activated the kinase without inhibition at high concentrations (30 mM). In addition to this cAMP-independent kinase, cAMP-dependent protein kinase also phosphorylated phosphorylase. The catalytic subunit and phosphorylase kinase were not identical since the latter was not inhibited by yeast cAMP binding protein.     

Identification of novel phospholipid binding proteins in Saccharomyces cerevisiae

A proteomics approach was used to search for novel phospholipid binding proteins in Saccharomyces cerevisiae. Phospholipids were immobilized on a solid support and the lipids were probed with soluble yeast protein extracts. From this, the phosphatidic acid binding proteins were eluted and identified by mass spectrometry. Thirteen proteins were identified and 11 of these were previously unknown lipid binding proteins. The protein-lipid interactions identified would not have been predicted using bioinformatics approaches as none possessed a known lipid binding motif. A subset of the identified proteins was purified to homogeneity and determined to directly bind phospholipids immobilized on a solid support or organized into liposomes. This simple approach could be systematically applied to perform an exhaustive screen for soluble lipid binding proteins in S. cerevisiae or other organisms.     

Phospholipid alterations elicited by hexachlorobenzene in rat brain are strain-dependent and porphyria-independent

Hexachlorobenzene (HCB) alters phospholipid and heme metabolisms in the liver and Harderian gland. The effects of HCB on phospholipid metabolism, in an organ considered to be non-responsive to its porphyrinogenic effects, remain to be studied. Therefore, as the brain is an organ with this feature, this paper analyzes the effects of HCB on brain phospholipid composition in order to investigate if there is any relationship between HCB-induced porphyrin metabolism disruption and phospholipid alterations. For this purpose, a time-course study of HCB effects on brain phospholipids was performed in two strains of rats differing in their susceptibility to acquire hepatic porphyria: Chbb THOM (low); and Wistar (high). This paper shows for the first time that rat brain phospholipids are affected by HCB exposure. Comparative studies show that HCB-induced disturbances in brain phospholipid patterns are time and strain-dependent. Thus, whereas major phospholipids, phosphatidylcholine and phosphatidylethanolamine were more altered in Wistar rats, minor phospholipids, phosphatidylinositol and phosphatidylserine were more affected in Chbb THOM rats. HCB intoxication led to a sphingomyelin/phosphatidylcholine molar ratio lower than the normal, in both strains. As was expected, brain porphyrin content was not altered by HCB intoxication in either strain. It can be concluded that HCB is able to alter brain phospholipid metabolism in a strain-dependent fashion, and in the absence of alterations in brain heme metabolism. In addition, HCB-induced disturbances in brain phospholipids were not related to the degree of hepatic porphyria achieved by the rats.     

Cardiolipin synthase of Arabidopsis thaliana

Functional expression studies in microorganisms showed that the Arabidopsis thaliana gene At4g04870 represents the cardiolipin synthase (CLS) gene encoding a hydrophobic preprotein of 38 kDa with a cleavable signal peptide for the import into mitochondria. CLS of Arabidopsis over-expressed in Escherichia coli has an alkaline pH optimum, a strict requirement for divalent cations and a distinctly lower K(m) for cytidinediphosphate-diacylglycerol than for phosphatidylglycerol. It displayed a preference for both its substrates esterified with unsaturated acyl groups. Solubilization and purification experiments revealed that the protein requires a defined phospholipid environment, particularly the presence of cardiolipin, to acquire its catalytically active conformation.     

Characterization of the transacylase activity of rat liver 60-kDa lysophospholipase-transacylase. Acyl transfer from the sn-2 to the sn-1 position

Rat liver 60-kDa lysophospholipase-transacylase catalyzes not only the hydrolysis of 1-acyl-sn-glycero-3-phosphocholine, but also the transfer of its acyl chain to a second molecule of 1-acyl-sn-glycero-3-phosphocholine to form phosphatidylcholine (H. Sugimoto, S. Yamashita, J. Biol. Chem. 269 (1994) 6252–6258). Here we report the detailed characterization of the transacylase activity of the enzyme. The enzyme mediated three types of acyl transfer between donor and acceptor lipids, transferring acyl residues from: (1) the sn-1 to -1(3); (2) sn-1 to -2; and (3) sn-2 to -1 positions. In the sn-1 to -1(3) transfer, the sn-1 acyl residue of 1-acyl-sn-glycero-3-phosphocholine was transferred to the sn-1(3) positions of glycerol and 2-acyl-sn-glycerol, producing 1(3)-acyl-sn-glycerol and 1,2-diacyl-sn-glycerol, respectively. In the sn-1 to -2 transfer, the sn-1 acyl residue of 1-acyl-sn-glycero-3-phosphocholine was transferred to not only the sn-2 positions of 1-acyl-sn-glycero-3-phosphocholine, but also 1-acyl-sn-glycero-3-phosphoethanolamine, producing phosphatidylcholine and phosphatidylethanolamine, respectively. 1-Acyl-sn-glycero-3-phospho-myo-inositol and 1-acyl-sn-glycero-3-phosphoserine were much less effectively transacylated by the enzyme. In the sn-2 to -1 transfer, the sn-2 acyl residue of 2-acyl-sn-glycero-3-phosphocholine was transferred to the sn-1 position of 2-acyl-sn-glycero-3-phosphocholine and 2-acyl-sn-glycero-3-phosphoethanolamine, producing phosphatidylcholine and phosphatidylethanolamine, respectively. Consistently, the enzyme hydrolyzed the sn-2 acyl residue from 2-acyl-sn-glycero-3-phosphocholine. By the sn-2 to -1 transfer activity, arachidonic acid was transferred from the sn-2 position of donor lipids to the sn-1 position of acceptor lipids, thus producing 1-arachidonoyl phosphatidylcholine. When 2-arachidonoyl-sn-glycero-3-phosphocholine was used as the sole substrate, diarachidonoyl phosphatidylcholine was synthesized at a rate of 0.23 μmol/min/mg protein. Thus, 60-kDa lysophospholipase-transacylase may play a role in the synthesis of 1-arachidonoyl phosphatidylcholine needed for important cell functions, such as anandamide synthesis.     

Adaptations in the lipid metabolism of the protozoan parasite Trypanosoma brucei

Trypanosomes are unicellular parasites and like all decent parasites, they try to obtain from the host as much material as possible, including lipids. However, the needs of a parasite are not always the same as those of the host, and therefore, mostly, some biosynthetic work still has to be done by the parasite itself. Very often at least modifications of the lipid components that are acquired from the host have to be made. Furthermore, next to the lipids Trypanosoma brucei indeed obtains from the host, some other lipid components have to be synthesized de novo. Especially the processes where the metabolism of T. brucei differs from that of the host, will be discussed, as at least some of them are excellent targets for the development of urgently needed new chemotherapeutics.     

Adaptive changes in lipid composition of rat liver plasma membrane during postnatal development following maternal ethanol ingestion

The fatty acid composition of constituent phospholipids and the cholesterol content of rat liver plasma membranes were determined subsequent to maternal alcohol ingestion during pregnancy and lactation. The alcoholic group was given a liquid Metrecal diet containing 37% ethanol-derived calories. The control group was pair-fed an isocaloric sucrose/Metrecal diet. Litters were killed for lipid analyses at days 5, 15 and 25 after birth. These studies revealed that the total phospholipid phosphorus was similar and increased significantly with age in both groups. Cholesterol also increased significantly with age in both groups but was greater in the alcoholic pups, resulting in a higher cholesterol/phospholipid molar ratio. While the phosphatidylethanolamine (PE) content increased with age in both groups, that of sphingomyelin decreased. Phosphatidylserine + phosphatidylinositol (PS + PI) was significantly higher in the control group at all ages studied. A consistent increase of C22:6 in phosphatidylcholine (PC), sphingomyelin, PS + PI and in the total phospholipid fraction from alcoholic pups was observed. Although other fatty acid changes were found in PC, PS + PI and sphingomyelin, PE was not affected. These results suggest that specific adaptive changes were induced in the liver plasma membrane lipids of the progeny from alcoholic rats.     

A study on the topological distribution of phospholipids in microsomal membranes of chick brain using phospholipase C and trinitrobenzenesulfonic acid

The transbilayer distribution of phospholipids in chicken brain microsomal membranes has been investigated using trinitrobenzenesulfonic acid and phospholipase C from Clostridium weichii. The exposure of intact microsomes to trinitrobenzenesulfonic acid showed that the labelling of aminophospholipids followed biphasic kinetics, indicating that these membranes contain a fast- and a slow-reacting pool of aminophospholipids. Use of microsomes radioiodinated on their surface led to the conclusion that the fast-reacting pool may be located on the outer leaflet of the microsomal vesicles. It contains about 35% of the phosphatidylethanolamine, 29% of the ethanolamine plasmalogens and 18% of the phosphatidylserine. The treatment of intact microsomes with the phospholipase C Cl. welchii produced the hydrolysis of 50% of the phospholipids without any loss of their permeability properties, indicating that they are not permeable to the hydrolase. Phospholipids extracted from the microsomes were hydrolyzed rapidly by the phospholipase C with the exception of phosphatidylserine and phosphatidylinositol. In intact microsomes about 90% of phosphatidylcholine, 32% of ethanolamine phospholipids and 60% of sphingomyelin were accessible to the phospholipase. These results suggest that the phospholipids have an asymmetric distribution in chicken brain microsomes, the external leaflet containing about 75% of the choline phospholipids and 25% of the aminophospholipids, whereas an opposite distribution is observed in the inner leaflet.     

Structural changes of liver microsomes in rat during neonatal life: influence on the glucuronidation rates of various substrates

The Vmax of the membrane bound UDP-glucuronosyltransferase (UDP-GT) towards group 1 substrates (4-nitrophenol, 2-naphthol) was particularly higher in young rats than in adults. On the contrary, activity towards group 2 substrates such as borneol or testosterone was not detectable in fetus liver. The developmental pattern of UDP-GT was related to changes in lipid composition of microsomes, namely in the content in lysophosphatidylcholine which rose at birth. The phospholipid-cholesterol molar ratio also increased 2 fold from the 16th day of fetal life to the 4th day after birth. Measurement of the steady state anisotropy of 1,6-diphenyl-1,3,5-hexatriene (DPH) as well as determination of the order parameter S and the rotation cone angle of the fluorescent probe strongly suggested an increase in membrane fluidity in rat liver microsomes during ontogenesis.     

Identification of the hemoglobin binding sites on the inner surface of the erythrocyte membrane

The hemoglobin binding sites on the inner surface of the erythrocyte membrane were identified by measuring the fraction of hemoglobin released following selective proteolytic or lipolytic enzyme digestion. In addition, binding stoichiometry to and fractional hemoglobin release from inside-out vesicle preparations of human and rabbit membranes were compared since rabbit membranes differ significantly from human membranes only in that they lack glycophorin. Our results show that rabbit inside-out vesicles bind about 65% less human or rabbit hemoglobin under conditions of optimal and stoichiometric binding, despite being otherwise similar in composition. We suggest that this difference is either directly or indirectly due to the absence of glycophorin in rabbit membranes. Further supportive evidence includes demonstrating (a) that neuraminidase treatment of human membranes did not affect hemoglobin binding and (b) that reconstitution of isolated glycophorin into phospholipid vesicles increased the hemoglobin binding capacity in a manner proportional to the fraction of glycophorin molecules oriented with their cytoplasmic sides exposed to the exterior of the vesicle. Proteolysis of human inside-out vesicles either before or after addition of hemoglobin reduced the binding capacity by about 25%. This is consistent with the known proportion of total hemoglobin binding sites involving band 3 protein and the selective lability of the cytoplasmic aspect of band 3 protein to proteolysis. Phospholipid involvement in hemoglobin binding was determined using various phospholipase C preparations which differ in their reactivity profiles. Approximately 38% of the bound hemoglobin was released upon cleavage of phospholipid headgroups. These results suggest that the predominant sites of binding for hemoglobin on the inner surface of the red cell membrane are the two major integral membrane glycoproteins.     

Alteration of phospholipid composition of mouse liver microsomes in vivo and the effect on membrane properties

Administration of the methylation inhibitor periodate-oxidized adenosine to male Swiss-Webster mice on a choline-deficient diet produced a decrease (17%) in phosphatidylcholine to phosphatidylethanolamine ratios compared to saline-injected controls in liver, and also in kidney (11%), but not in muscle microsome preparations. Both intact liver microsomes and reconstituted membranes from lipid extracts showed a higher fluorescence anisotropy of the hydrophobic probe 1,6-diphenyl-1,3,5-hexatriene than control samples in the temperature range of 20–31°C.     

Phosphatidylethanolamine synthesized by three different pathways is supplied to peroxisomes of the yeast Saccharomyces cerevisiae

In the yeast Saccharomyces cerevisiae three pathways lead to the formation of phosphatidylethanolamine (PE), namely decarboxylation of phosphatidylserine (PS) (i) by Psd1p in mitochondria, and (ii) by Psd2p in a Golgi/vacuolar compartment; and (iii) synthesis via CDP–ethanolamine pathway in the endoplasmic reticulum. To determine the contribution of these pathways to the supply of PE to peroxisomes, we subjected mutants bearing defects in the respective metabolic routes to biochemical and cell biological analysis. Despite these defects in PE formation mutants were able to grow on oleic acid indicating induction of peroxisome proliferation. Biochemical analysis revealed that PE formed through all three pathways was supplied to peroxisomes. These analyses also demonstrated that selective as well as equilibrium interorganelle flux of PE appear to be equally important for cellular homeostasis of this phospholipid. Electron microscopic inspection confirmed that defects in PE synthesis still allowed formation of peroxisomes, although these organelles from strains lacking PSD1 were significantly smaller than wild type. The fact that peroxisomes were always found in close vicinity to mitochondria, ER and lipid particles supported the view that membrane contact may play a role in lipid traffic between these organelles.     

Calcium ionophore A 23187 induces arachidonic acid release from phosphatidylcholine in cultured human endothelial cells

Cultured endothelial cells from human umbilical vein were incubated with (³H)arachidonic acid for 24 hours. The label was incorporated into phospholipids (79.3 %), neutral lipids (15.6 %) and non-esterified fatty acids (4.7 %). Upon challenge with the calcium ionophore A 23187, 5.3 % of the total radioactivity were found in supernatant and corresponded to 6-keto-prostaglandin F_1α (1.6 %) and free arachidonic acid (3.7 %). This release was accompanied by a concomitant and selective decrease of phosphatidylcholine. It is concluded that the entry of calcium promoted by A 23187 activates a phospholipase A₂ regulating the availability of arachidonic acid to the prostacyclin synthetase.     

The phospholipid requirement of the (Ca2+ + Mg2+)-ATPase from human platelets

The phospholipid requirement of the ($\text{Ca}{}^{\mathrm{2+}}\text{+}\text{Mg}{}^{\mathrm{2+}}\text{)-}\text{ATPase}$ present in a membrane fraction from human platelets was studied using various purified phospholipases. Only those phospholipases, which hydrolyse the negatively charged phospholipids, inhibited the ($\text{Ca}{}^{\mathrm{2+}}\text{+}\text{Mg}{}^{\mathrm{2+}}\text{)-}\text{ATPase activity}$. The ATPase activity could be restored by adding mixed micelles of Triton X-100 and phosphatidylserine or phosphatidylinositol. Micelles with phosphatidic acid, phosphatidylcholine, phosphatidylethanolamine or sphingomyelin could not be used for reconstitution and inhibited the activity of the native enzyme.     

Opening of holes in liposomal membranes is induced by proteins possessing the FERM domain

The destabilization of vesicles caused by interactions between lipid bilayers and proteins was studied by direct, real-time observation using high-intensity dark-field microscopy. We previously reported that talin, a cytoskeletal submembranous protein, can reversibly open stable large holes in giant liposomes made of neutral and acidic phospholipids. Talin and other proteins belonging to the band 4.1 superfamily have the FERM domain at their N-terminal and interact with lipid membranes via that domain. Here, we observed that band 4.1, ezrin and moesin, members of the band 4.1 superfamily, are also able to open stable holes in liposomes. However, truncation of their C-terminal domains, which can interact with the N-terminal FERM domain, impaired their hole opening activities. Oligomeric states of ezrin affected the capability of the membrane hole formation. Phosphatidylinositol bisphosphate (PIP2), which binds to the FERM domain and disrupts the interaction between the N and C termini of the band 4.1 superfamily, down-regulates their membrane opening activity. These results suggest that the intermolecular interaction plays a key role in the observed membrane hole formation.