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PubMed1996

Regulation of phosphatidate phosphatase activity from the yeast Saccharomyces cerevisiae by phospholipids.

Regulation of Saccharomyces cerevisiae membrane-associated phosphatidate phosphatase (3-sn-phosphatidate phosphohydrolase, EC 3.1.3.4) activity by phospholipids was examined using purified enzyme and Triton X-100/phospholipid-mixed micelles. Anionic phospholipids activated phosphatidate phosphatase activity whereas zwitterionic phospholipids had a slight inhibitory effect on activity. Cardiolipin (A0.5 = 1.9 mol %), CDP-diacylglycerol (A0.5 = 2.6 mol %), and phosphatidylinositol (A0.5 = 5.5 mol %) were the most potent anionic phospholipid activators. Enzyme activation by cardiolipin (n=2.8), CDP-diacylglycerol (n=2.1), and phosphatidylinositol (n=3.3) followed positive cooperative kinetics. A kinetic analysis was performed to determine the mechanism of phosphatidate phosphatase activation by anionic phospholipids. The dependence of phosphatidate phosphatase on phosphatidate was cooperative (n approximately 2.2) in the absence and presence of phospholipid activators. Cardiolipin, CDP-diacylglycerol, and phosphatidylinositol were mixed competitive activators of phosphatidate phosphatase activity. The major effect of the activators was to cause a decrease in the Km for phosphatidate. Sphinganine, a positively charged sphingoid base, inhibited phosphatidate phosphatase activity and antagonized the activation of the enzyme by cardiolipin and phosphatidylinositol. Sphinganine caused an increase in the cooperativity of cardiolipin activation, but had little effect on the A0.5 value for cardiolipin. On the other hand, sphinganine had little effect on the cooperativity of phosphatidylinositol activation, but caused an increase in the A0.5 value for phosphatidylinositol. The activation constants for cardiolipin, CDP-diacylglycerol, and phosphatidylinositol were within the range of their cellular concentrations. These results suggested that the activation of phosphatidate phosphatase activity by anionic phospholipids may be physiologically relevant.

Cardiolipins↗
PubMed1997

Importance of phospholipid in the folding and conformation of phosphatidylinositol transfer protein: comparison of apo and holo species.

The significance of noncovalently bound phospholipid as a structural component of phosphatidylinositol transfer protein (PITP) and its role in acquisition and maintenance of the native conformation of the protein have been addressed by studying the refolding of PITP after exposure to 6 M guanidinium chloride (GdnCl). Protein conformations were characterized by (1) the intrinsic tryptophan fluorescence, circular dichroism, and absorbance spectroscopy, (2) the degree of binding of the fluorescent probe 1,8-ANS, and (3) limited proteolytic digestion. When the GdnCl concentration was reduced 100-fold by rapid dilution at 25 degrees C, practically all of the native transfer activity was regained within 20 min. Endogenous phospholipid demonstrated a strong interaction with the native PITP. Separation of the phospholipid from the protein by chromatography on a lipophilic matrix was achieved only under denaturing conditions and resulted in spontaneous oxidation of the apo-protein, accompanied by almost complete loss of recoverable transfer activity. Under reducing conditions, however, apo-PITP recovered more than 80% of the native transfer activity and was similar to holo-PITP in the kinetics of phospholipid transfer. Renatured apo-PITP demonstrated a significant relaxation of the tertiary structure, compared to native and renatured holo-PITP. Incubation of apo-PITP with phospholipid vesicles resulted in a more compact protein conformation. We conclude that the polypeptide can spontaneously fold to a native-like conformation, sufficient for interaction with a lipid membrane and acquisition of a phospholipid ligand. Binding of a phospholipid ligand brings about the final adjustments of protein conformation to the more compact native structure.

Carrier Proteins↗
PubMed1998

Binding of quinacrine to acidic phospholipids and pancreatic phospholipase A2. Effects on the catalytic activity of the enzyme.

Binding of quinacrine to phospholipids and porcine pancreatic phospholipase A2 (PLA2) was investigated using fluorescence resonance energy transfer, Langmuir films, assay for the enzymatic activity, and molecular modeling. No significant binding of this drug to the zwitterionic phosphatidylcholine was observed whereas a high affinity for acidic phospholipids was revealed by quenching of pyrene-labeled phospholipid analogues. Partial reversal of this binding was observed due to the addition of 4 mM CaCl2. Quinacrine efficiently and independently of the lipid surface pressure penetrated into monolayers of phosphatidylglycerol while only a weak penetration into phosphatidylcholine films was evident. Quinacrine also bound to eosin-labeled PLA2, and the addition of 4 mM CaCl2 reversed this interaction almost completely. In the presence of acidic phospholipids both the drug and the enzyme were attached to the lipid surface. Studies on the influence of quinacrine on the activity of PLA2 toward pyrene-labeled phospholipid analogues revealed that the hydrolysis of phosphatidylcholine was progressively reduced as a function of increasing [quinacrine]. At low [CaCl2] and low quinacrine:lipid molar ratios (<1:5) quinacrine enhanced slightly the rate of hydrolysis of acidic phospholipids whereas at higher drug:lipid molar ratios (>1:2) an inhibition was observed. In the presence of 1 mM CaCl2 quinacrine inhibited PLA2-catalyzed hydrolysis of phosphatidylglycerol only when the drug:lipid molar ratio exceeded 1:1. The presence of 4 mM CaCl2 abolished nearly completely the inhibition with all the substrate analogues used. Our data suggest that the inhibition of PLA2 by quinacrine is due to its binding to the enzyme. This is supported also by molecular modeling which suggested a binding site for quinacrine close to the active site and Ca2+ binding site of the enzyme. Importantly, our data indicate that quinacrine binds avidly to acidic phospholipids and their presence may influence the drug-enzyme interaction and the inhibition of the enzyme action. Accordingly, presence of quinacrine may interfere also with other processes that require the presence of acidic lipids and/or Ca2+, such as the function of the nicotinic acetylcholine receptor.

Animals↗
PubMed1998

Nonessential activation and competitive inhibition of bacterial phosphatidylinositol-specific phospholipase C by short-chain phospholipids and analogues.

Phosphatidylinositol-specific phospholipase C (PI-PLC) from Bacillus thuringiensis is an allosteric enzyme with both a phospholipid activator site and an active site. The activation of PI-PLC enzyme is optimal with phosphatidylcholine (PC) binding to the activator site and anchoring the enzyme to the interface [Zhou, C., et al. (1997) Biochemistry 36, 347-355; Zhou, C., et al. (1997) Biochemistry 36, 10089-10091]. In contrast to PC, anionic short-chain phospholipids with smaller headgroups [phosphatidylmethanol (PMe) and phosphatidic acid (PA)] as well as phosphatidylglycerol (PG) can bind to both sites playing dual roles: nonessential activation and competitive inhibition of cyclic-(1, 2)-inositol phosphate hydrolysis. PG is also a substrate, albeit a poor one, for PI-PLC, and is cleaved slowly to form alpha-glycerol phosphate. Analysis of enzyme kinetics using cIP as the substrate coupled with effects of different short-chain phospholipids on enzyme intrinsic fluorescence indicates that anionic phospholipids with small headgroups bind to the two sites with different affinities. If no interface is present, all dihexanoylphospholipids bind to the activator site more strongly than to the active site. When the activator site is occupied, it is likely that the enzyme undergoes a conformational change that allows phospholipids to bind easily to the active site. Such behavior is consistent with the observation that enzyme activation is detected at low short-chain anionic phospholipid concentrations with inhibition observed at higher concentrations, and that only inhibition is seen with these phospholipids added as monomers in the presence of a PC interface that optimally activates the PI-PLC. A kinetic model is used to extract the affinity of short-chain lipids for the active site from experimental data.

Bacillus thuringiensis↗
PubMed2005

Changes produced in the antioxidative activity of phospholipids as a consequence of their oxidation.

The antioxidative activities of native and oxidized soybean phosphatidylcholine (PC), phosphatidylthanolamine (PE), and phosphatidylinositol (PI) in the protection of soybean oil heated in the dark under air at 60 degrees C were studied in an attempt to clarify the consequences that phospholipid oxidation has on antioxidative activities. The three native phospholipids protected the oil when assayed at 200 ppm, and phospholipid oxidation decreased the antioxidative activity of both PC and PI. However, slightly oxidized PE was more antioxidative than native PE, most likely as a consequence of the formation by amino-carbonyl reactions of pyrrolized phospholipids, which were determined and for which antioxidative properties are known. Nevertheless, further increases in PE oxidation produced a decrease in its antioxidative activity. These results suggest that two opposite reactions are competing in the antioxidative activity of amino phospholipids upon oxidation: fatty acid chain oxidation, which decreases phospholipid antioxidative activity, and amino-carbonyl reactions, which produce derivatives with antioxidant properties. This last property may be useful to increase the antioxidative activity of commercial lecithins containing amino phospholipids.

Antioxidants↗
PubMed2004

In situ photopolymerization of a polymerizable poly(ethylene glycol)-covered phospholipid monolayer on a methacryloyl-terminated substrate.

We have prepared a chemically anchored monolayer of PEG (poly(ethylene glycol)) and phospholipid mixture (PEG/phospholipid) on a methacryloyl-terminated substrate by in situ photopolymerization. Both monoacryloyl phospholipid (acryloyl-PC, 1-palmitoyl-2-[12-(acryloyloxy)dodecanoyl]-sn-glycero-3-phosphocholine) and monoacryloyl PEG (acryloyl-PEG, 12-(acryloyloxy)dodecanoyl-PEG) were synthesized by modifyingphospholipid and PEGwith 12-(acryloyloxy)-1-dodecanoic acid and 12-(acryloyloxy)-1-dodecanol, respectively. The surface pressure-area (pi-A) isotherm showed that acryloyl-PEG molecules were stable in the phospholipid monolayer and that they could be evenly inserted into a phospholipid monolayer at the air/water interface. By adding 10 mol % acryloyl-PEG into phosholipid vesicles, we could produce a PEG/phosholipid monolayer on methacryloyl-terminated substrates using vesicle fusion for 3 h. Then, this polymerizable PEG/phospholipid monolayer was in situ photopolymerized onto a methacryloyl-terminated substrate with eosin Y/triethanolamine as co-initiators. Optimal vesicle fusion and irradiation condition were determined with respect to the vesicle fusion time and duration of irradiation. As confirmed by atomic force microscopy and X-ray reflectivity studies, the polymerized PEG/phosholipid surface formed a PEG-covered phospholipid monolayer with thicknesses of 3 and 6 nm for the base phospholipid monolayer and the covering PEG layer, respectively. The chemical anchoring efficiency ofpolymerized PEG and phospholipid molecules, which was calculated by the relative carbon ratio of each surface before and after methanol washing using X-ray photoelectron spectroscopy, was 98%. This polymerized PEG/phosholipid monolayer showed good stability in organic solution due to firm chemical anchoring to a solid surface.

Carbon↗
PubMed1999

Reduced palmitate turnover in brain phospholipids of pentobarbital-anesthetized rats.

Our laboratory has reported that pentobarbital-induced anesthesia reduced the incorporation of intravenously injected radiolabeled palmitic acid into brain phospholipids. To determine if this decrease reflected a pentobarbital-induced decrease in palmitate turnover in phospholipids, we applied our method and model to study net flux and turnover of palmitate in brain phospholipids (1). Awake, light and deep pentobarbital (25-70 mg/kg, iv) anesthetized rats were infused with [9,10-3H]palmitate over a 5 min period. Brain electrical activity was monitored by electroencephalography. An isoelectric electroencephalogram characterized deep pentobarbital anesthesia. Net incorporation rates (J(FA,i)) and turnover rates (Fi) of palmitate were calculated. J(FA,i) for palmitate incorporated into phospholipids was dramatically reduced by pentobarbital treatment in a dose-dependent manner, by 70% and 90% respectively for lightly and deeply anesthetized animals, compared with awake controls. Turnover rates for palmitate in total phospholipid and individual phospholipid classes were decreased by nearly 70% and 90% for lightly and deeply anesthetized animals, respectively. Thus, pentobarbital decreases, in a dose-dependent manner, the turnover of palmitate in brain phospholipids. This suggests that palmitate turnover is closely coupled to brain functional activity.

Adjuvants, Anesthesia↗
PubMed2002

Secretory phospholipase A2 and phospholipids in neural membranes in an experimental epilepsy model.

OBJECTIVES: Previous studies have revealed an increase in several forms of phospholipase A2 activity associated with cell injury, but the secretory form of phospholipase A2 has not previously been studied in neurological disorders. We investigated the influence of seizures on secretory phospholipase A2 and phospholipid breakdown in synaptosome fractions prepared from rat hippocampus, cortex and cerebellum in pentylenetetrazol-induced epilepsy. MATERIAL AND METHODS: Secretory phospholipase A2 concentration was measured by a photometric enzyme immunoassay. The synaptosomes underwent extraction, and the phospholipids fractions for phosphatidylcholine, phosphatidylethanolamine and lysophosphatidylcholine were recovered from the thin layer chromatography plates. The amount of each phospholipid was quantified using the amount of recovered phosphate in each phospholipid spot. RESULTS: Secretory phospholipase A2 concentration was found to be significantly higher in the epileptic group when compared with the control group. The amounts of phospholipids were found to be highly variable in different brain regions. CONCLUSION: Our results suggest that epileptic seizures enhanced phospholipid breakdown and induced alterations in the distribution of phospholipids in different brain regions.

Animals↗
PubMed1997

Serum phospholipid fatty acid composition and habitual intake of marine foods registered by a semi-quantitative food frequency questionnaire.

OBJECTIVE: To examine the relation between consumption of fish and fish products registered by a comprehensive food frequency questionnaire and the composition of fatty acids in serum phospholipids. DESIGN: Cross-section study. SETTING: Cardiovascular screening centre in Trondheim, Mid-Norway. SUBJECTS: Of 256 eligible women 242 agreed to participate in the present study. Altogether 234 middle-aged women (91.4%) completed the questionnaire and gave a valid blood sample. RESULTS: Total frequency consumption of fish for dinner showed only weak association with serum phospholipid fatty acid composition. In separate analyses of lean and fatty fish, consumption of fatty fish was negatively associated with n-6 and positively associated with n-3 fatty acids in serum phospholipids, while no significant associations were found for lean fish consumption. Cod liver oil consumption was strongly related to the phospholipid fatty acid composition. The associations improved moderately when adding portion size information. Spearman's correlation coefficient between dietary intake of eicosapentaenoic acid (EPA) and serum phospholipid EPA was 0.58, and Spearman's correlation coefficient between intake of docosahexaenoic acid (DHA) and serum phospholipid DHA was 0.53. CONCLUSIONS: This study suggests that in populations with a high consumption of fish and cod liver oil, habitual intake can be reflected in serum phospholipids. However, as the fat content of fish is highly variable, separate registration of lean and fatty fish consumption is needed.

Adult↗
PubMed1970

Co-ordination between membrane phospholipid synthesis and accelerated biosynthesis of cytoplasmic ribonucleic acid and protein.

1. The rate of synthesis of membrane phospholipid was studied in rat liver and seminal vesicles by following the incorporation of [(32)P]orthophosphate, [(14)C]choline and [(14)C]glycerol. Particular emphasis was laid on the endoplasmic reticulum, which was fractionated into smooth microsomal membranes, heavy rough membranes, light rough membranes and free polyribosomes. 2. Phospholipid labelling patterns suggested a heterogeneity in the synthesis and turnover of the different lipid moieties of smooth and rough endoplasmic membranes. The major phospholipids, phosphatidylcholine and phosphatidylethanolamine, were labelled relatively rapidly with (32)P over a short period of time whereas incorporation of radioisotope into the minor phospholipids, sphingomyelin, lysolecithin and phosphatidylinositol proceeded slowly but over a longer period of time. 3. The incorporation of orotic acid into RNA and labelled amino acids into protein of the four submicrosomal fractions was also studied. 4. Rapid growth of the liver was induced by the administration of growth hormone and tri-iodothyronine to hypophysectomized and thyroidectomized rats and by partial hepatectomy. Growth of seminal vesicles of castrated rats was stimulated with testosterone propionate. 5. The rate of labelling of membrane phospholipids was enhanced in all major subcellular particulate fractions (nuclear, mitochondrial and microsomal) during induced growth. However, it was in the rough endoplasmic reticulum that the accumulation of phospholipids, RNA and protein was most marked. The effect of hormone administration was also to accelerate preferentially the labelling with (32)P of sphingomyelin relative to that of phosphatidylcholine or phosphatidylethanolamine. 6. Time-course analyses showed that, in all four growth systems studied, the enhancement of the rate of membrane phospholipid synthesis coincided with the rather abrupt increase in the synthesis of RNA and protein of the rough endoplasmic reticulum. Growth hormone and tri-iodothyronine administered to hypophysectomized rats had additive effects in all the biosynthetic processes. The latent period of action of each hormone was maintained so that two waves of proliferation of endoplasmic reticulum occurred if the hormones were administered simultaneously. 7. It is concluded that there is some mechanism in the cell that tightly co-ordinates the formation of membranes, especially those of the endoplasmic reticulum, when an increased demand is made for protein synthesis.

Amino Acids↗
PubMed1970

Phospholipid synthesis and exchange in isolated liver cells.

1. The [(32)P]phosphate incorporated into the phospholipids of isolated rat hepatic cells is present in phosphatidic acid and to a smaller extent in phosphatidylinositol. 2. The ability to synthesize nitrogen-containing phospholipids is restored by adding a liver supernatant fraction, and it is suggested that the metabolic deficiency is caused by the leakage of cytoplasmic enzymes of the synthetase system from the cells. 3. Fortified cell preparations were pulse-labelled with [(32)P]phosphate, [Me-(14)C]choline, [2-(14)C]ethanolamine and [U-(14)C]inositol and the subsequent fate of the labelled microsomal and mitochondrial phospholipids followed. 4. A fall in the specific radioactivity of microsomal phospholipids and a rise in that of mitochondrial phospholipids is interpreted as providing evidence of a transfer of labelled phospholipid molecules from the synthetic site (endoplasmic reticulum) to the mitochondrial membranes in the intact cells. 5. The formation of the phospholipids of mitochondrial membranes is discussed.

Amino Alcohols↗
PubMed1971

The interaction of insulin with phospholipids.

1. A simple two-phase chloroform-aqueous buffer system was used to investigate the interaction of insulin with phospholipids and other amphipathic substances. 2. The distribution of (125)I-labelled insulin in this system was determined after incubation at 37 degrees C. Phosphatidic acid, dicetylphosphoric acid and, to a lesser extent, phosphatidylcholine and cetyltrimethylammonium bromide solubilized (125)I-labelled insulin in the chloroform phase, indicating the formation of chloroform-soluble insulin-phospholipid or insulin-amphipath complexes. Phosphatidylethanolamine, sphingomyelin, cholesterol, stearylamine and Triton X-100 were without effect. 3. Formation of insulin-phospholipid complex was confirmed by paper chromatography. 4. The two-phase system was adapted to act as a simple functional system with which to investigate possible effects of insulin on the structural and functional properties of phospholipid micelles in chloroform, by using the distribution of [(14)C]glucose between the two phases as a monitor of phospholipid-insulin interactions. The ability of phospholipids to solubilize [(14)C]glucose in chloroform increased in the order phosphatidylcholine<sphingomyelin<phosphatidylethanolamine<phosphatidic acid. Insulin decreased the [(14)C]glucose solubilized by phosphatidylcholine, phosphatidylethanolamine and phosphatidic acid, but not by sphingomyelin. 5. The significance of these results and the molecular requirements for the formation of insulin-phospholipid complexes in chloroform are discussed.

Anti-Infective Agents, Local↗
PubMed1974

Phospholipid synthesis in rat liver endoplasmic reticulum after the administration of phenobarbitone and 20-methylcholanthrene.

1. Phenobarbitone injection did not affect the concentration of phospholipids in the liver endoplasmic reticulum, but it increased the rate of incorporation of [(32)P]orthophosphate into the phospholipids. 20-Methylcholanthrene caused a transient increase in total phospholipid but a decrease in the turnover rate of the phospholipids. 2. Incorporation of [(32)P]orthophosphate into phosphatidylcholine, compared with that into phosphatidylethanolamine, was increased by phenobarbitone injection but decreased by 20-methylcholanthrene injection. 3. The activity of S-adenosylmethionine-phosphatidylethanolamine methyltransferase increased 12h after phenobarbitone injection, when incorporation of [(32)P]orthophosphate into phosphatidylcholine was a maximum, but at other times, and after 20-methylcholanthrene injection, the activity of the enzyme did not correlate with the rate of phosphatidylcholine synthesis. 4. [(14)C]Glycerol was incorporated more rapidly into phosphatidylcholine than into phosphatidylethanolamine, whereas [(32)P]orthophosphate and [(14)C]ethanolamine were incorporated more rapidly into phosphatidylethanolamine than into phosphatidylcholine. 5. Incorporation of [(32)P]orthophosphate into phosphatidylethanolamine of liver slices incubated in vitro was much more rapid than into phosphatidylcholine, and incorporation into phosphatidylcholine was markedly stimulated by addition of methionine to the medium. Changes in the incorporation of [(32)P]orthophosphate into phospholipids observed in vivo after injection of phenobarbitone or methylcholanthrene could not be reproduced in slices incubated in vitro. 6. It is concluded that phenobarbitone injection causes an increased rate of turnover of total phospholipids in the endoplasmic reticulum and an increased conversion of phosphatidylethanolamine into phosphatidylcholine, whereas 20-methylcholanthrene injection depresses both the turnover rate of total phospholipids and the formation of phosphatidylcholine.

Animals↗
PubMed1981

Hepatic uptake of phospholipid-depleted chylomicrons in vivo. Comparison with the uptake of chylomicron remnants.

1. Rats pretreated with Triton WR-1339 to prevent the formation of remnants were injected with [3H]cholesterol-labelled remnants, intact chylomicrons or chylomicrons depleted of most of their surface phospholipids by treatment with phospholipase A2. Within 5 min about 80% of the injected label of remnants and phospholipid-depleted chylomicrons was incorporated into the livers compared with less than 10% of the injected radioactivity of intact chylomicrons. A similar rapid hepatic uptake of radioactivity occurred when rats not pretreated with Triton were injected with [3H]cholesterol-labelled phospholipid-depleted chylomicrons. This rapid hepatic uptake of phospholipid-depleted chylomicrons occurred apparently without any alteration in the apoprotein composition of the particles. 2. The participation of hepatocytes in the uptake of remnants and phospholipid-depleted chylomicrons was examined. Both types of particles were taken up by the hepatocytes. However, small chylomicrons (Sf less than 400) were taken up more efficiently than were large chylomicrons (Sf greater than 400), but neither was taken up as efficiently as the remnants. 3. The results of this study lend support to the hypothesis that phospholipid-depleted chylomicrons and chylomicron remnants are taken up by the liver by a similar mechanism, which depends on the loss of surface phospholipids.

Animals↗
PubMed1984

Investigation of phospholipids of the pulmonary extracellular lining by electron paramagnetic resonance. The effects of phosphatidylglycerol and unsaturated phosphatidylcholines on the fluidity of dipalmitoyl phosphatidylcholine.

The membranous structures of the pulmonary extracellular lining were removed from the lungs of rabbits by pulmonary lavage and isolated by differential centrifugation. This membranous fraction contained 93% of the total extracellular phospholipids present in lavage effluents and consisted of membranous vesicles, membrane fragments, tubular myelin and secreted lamellar bodies. The fraction was rich in phosphatidylcholine (79.4%) containing 85.2% palmitic acid in the 1-position and 57.4% palmitic acid in the 2-position. Phosphatidylglycerol was the next most abundant phospholipid, accounting for 9.4% of the total. E.p.r. spectra, obtained by using 5-doxylmethylstearate as a probe, showed that the extracellular phospholipids of the pulmonary lining were organized into structures which were much more fluid than erythrocyte-ghost membranes. The fluidity of phosphatidylcholine isolated from the membranous fraction was similar to that of the fraction itself, indicating that the minor phospholipids had very little influence on the fluidity of the major phospholipid. At physiological temperature, the fluidity of dipalmitoyl phosphatidylcholine was relatively low, but could be markedly increased by the presence of 1-palmitoyl-2-oleoyl phosphatidylcholine or phosphatidylglycerol (10%). Protein present in the extracellular phospholipid fraction did not affect the fluidity of the fraction. These studies indicate that the unsaturated phosphatidylcholines could play a major role in determining the fluidity of the important surface-tension-lowering phospholipids such as dipalmitoyl phosphatidylcholine.

Animals↗
PubMed1986

Incorporation into phospholipid vesicles of pore-like properties from Golgi membranes of lactating-rat mammary gland.

The ability of rat mammary-gland Golgi membranes to produce monosaccharide-specific pores in phospholipid vesicles was investigated. The apparent ability of Triton X-100 extracts of Golgi membranes to form such pores was re-evaluated, since we have now found that an apparent pore is produced by the detergent alone. We therefore incorporated intact Golgi membranes (1 mg of protein) into egg-yolk phospholipid vesicles by direct sonication in the absence of any detergent. These vesicles retained about 0.6% of the total sucrose, but demonstrated selective permeability towards glucose compared with sucrose, with 19.8% of the glucose being lost during gel filtration on Sepharose 4B. This phenomenon seemed to be enhanced by the presence of acidic phospholipids and lysophosphatidylcholine, but was inhibited by inclusion of cholesterol in the vesicles. The best mixture of phospholipids comprised 6.5 mg of egg-yolk phospholipid, 1 mg of phosphatidylserine and 0.05 mg of lysophosphatidylcholine, where 32.9% of the glucose was lost. By using this optimum phospholipid mixture the pores were shown to be permeable to both glucose and mannitol, whereas sucrose and lactose were retained by the vesicles. Chaps (3- [(3-cholamidopropyl)dimethylammonio] propane-1-sulphonate)-solubilized membranes produced similar permeability in vesicles produced by dialysis of a solution of the phospholipids mixed with the membrane extract. This technique resulted in a greater loss of glucose, 33% loss requiring about 0.25 mg of protein. The pore-forming ability of both intact Golgi membranes and Chaps extracts was sensitive to boiling and proteolysis, indicating that a membrane protein was likely to be involved in pore formation.

Animals↗
PubMed1988

Phospholipid turnover during cell-cycle traverse in synchronous Chinese-hamster ovary cells. Mitogenesis without phosphoinositide breakdown.

The turnover of phospholipids was investigated in quiescent serum-starved Chinese-hamster ovary (CHO-K1) cells stimulated to progress through the cell cycle by the addition of dialysed bovine serum. A variety of radiolabelling techniques were employed to study the rapid effects of serum on phospholipids and later events during G1 and S phases of the cell cycle. Pulse-labelling studies using [32P]Pi revealed that there was a stimulation of the synthesis rate of all phospholipids investigated during the initial few hours after serum addition. The greatest stimulation (20-fold) was observed in phosphatidylcholine, and the smallest in the polyphosphoinositides (PPIs). Mock stimulation with serum-free medium caused a similar increase in PPI turnover, but little or no effect on turnover of other phospholipids. This effect could be accounted for by a stimulation of the turnover of cellular ATP pools increasing [32P]ATP specific radioactivity. Late G1 and S phases were associated with a decrease in the rate of synthesis of all phospholipids. Phosphatidic acid was the only phospholipid whose labelling fell below that in mock-stimulated cells during the period of the cell cycle. Stimulation of serum-starved cells that had been prelabelled with myo-[2-3H]inositol caused no change in the amounts of inositol trisphosphate, but both serum-stimulated and mock-stimulated cells exhibited similar small decreases in both inositol bisphosphate and inositol monophosphate, of approx. 30% after 30 s. When cells were serum-stimulated in the presence of 10 mM-Li+, there was no increase in the size of the total inositol phosphate pool. We conclude that mitogenic stimulation and cell-cycle traverse cause profound and complex effects on phospholipid turnover in CHO-K1 cells, but there is no evidence for a role of inositol lipid turnover in the proliferative response to serum in this cell line.

Adenosine Triphosphate↗
PubMed1988

Phosphorylation of phospholipids in isolated guinea pig hearts stimulated with isoprenaline.

Phosphorylation of phospholipids was studied in Langendorff perfused guinea pig hearts subjected to beta-adrenergic stimulation. Hearts were perfused with Krebs-Henseleit buffer containing [32P]Pi and freeze-clamped in a control condition or at the peak of the inotropic response to isoprenaline. 32P incorporation into total phospholipids, individual phospholipids and polyphosphoinositides was analysed in whole tissue homogenates and membranes, enriched in sarcoplasmic reticulum, prepared from the same hearts. Isoprenaline stimulation of the hearts did not result in any significant changes in the levels of phosphate incorporation in the total phospholipid present in cardiac homogenates (11.6 +/- 0.4 nmol of 32P/g for control hearts and 12.4 +/- 0.5 nmol of 32P/g for isoprenaline-treated hearts; n = 6), although there was a significant increase in the degree of phospholipid phosphorylation in sarcoplasmic reticulum (3.5 +/- 0.3 nmol of 32P/mg for control hearts and 6.7 +/- 0.2 nmol of 32P/mg for isoprenaline-treated hearts; n = 6). Analysis of 32P incorporation into individual phospholipids and polyphosphoinositides revealed that isoprenaline stimulation of the hearts was associated with a 2-3-fold increase in the degree of phosphorylation of phosphatidylinositol monophosphate and bisphosphate as well as phosphatidic acid in both cardiac homogenates and sarcoplasmic reticulum membranes. In addition, there was increased phosphate incorporation into phosphatidylinositol in sarcoplasmic reticulum membranes. Thus, perfusion of guinea pig hearts with isoprenaline is associated with increased formation of polyphosphoinositides and these phospholipids may be involved, at least in part, in mediating the effects of beta-adrenergic agents in the mammalian heart.

Animals↗