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PubMed1999

Phospholipid metabolism of hypothermically stored rat hepatocytes.

Isolated rat hepatocytes were suspended and stored in either Liebovitz-15 medium (37 degrees C or 4 degrees C) or University of Wisconsin (UW) solution (4 degrees C) containing [(3)H] arachidonic acid (AA). At varying times, membrane phospholipids were separated by thin layer chromatography. AA labeled phospholipids similarly at both 4 degrees C and 37 degrees C. Analysis of the ratios of [(3)H] AA and [(14)C] glycerol incorporated into phosphatidic acid or other phospholipids in dual-labeled cells indicated that the deacylation/reacylation cycle was the major route of AA incorporation at hypothermia. This was supported by showing that blocking phospholipase A(2) (PLA(2)) activity by trifluoperazine suppressed AA incorporation into phospholipids. PLA(2) activity, measured by determining the release of AA, was slow during 48-hour cold storage, but increased significantly when ATP was depleted by inhibition of mitochondria and glycolysis. In the whole rat liver, there was no significant loss of phospholipids during 48-hour storage (total phospholipids [micromol phosphorus/L/mg] : 0.197 +/-. 001 at 0 hours) unless energy blockers were used (0.155 +/-.005 at 48 hours) or glycogen depleted by fasting the rat (0.167 +/-.001 at 48 hours). This study shows that a net PLA(2) stimulated hydrolysis of phospholipids is seen only when ATP is depleted and its generation from anaerobic glycolysis inhibited. Thus, PLA(2) hydrolysis of phospholipids is not a significant cause of liver cell injury during cold storage when livers are obtained in optimal condition. However, conditions affecting the generation of ATP during cold storage could alter PLA(2) leading to membrane damage.

Adenosine↗
PubMed1994

High-performance liquid chromatographic analysis of phospholipids from different sources with combined fluorescence and ultraviolet detection.

An isocratic high-performance liquid chromatographic (HPLC) system was developed for the separation of major phospholipid classes, i.e., phosphatidylcholine, sphingomyelin, lysophosphatidylcholine, phosphatidylglycerol, phosphatidylethanolamine, phosphatidylinositol, and phosphatidylserine. Phospholipids were detected with ultraviolet absorption at 205 nm and subsequent fluorescence detection. Fluorescence of the phospholipids (excitation, 340 nm; emission, 460 nm) was achieved by postcolumn formation of mixed micelles with 1,6-diphenyl-1,3,5-hexatriene. For ultraviolet absorption there were great differences depending on the saturation of phospholipid fatty acids but for fluorescence the sensitivity was almost identical for all phospholipids except phosphatidylinositol and lysophosphatidylcholine. Dipalmitoylphosphatidylcholine showed nearly no ultraviolet but good fluorescence response. Ultraviolet to fluorescence ratio was characteristic for different phospholipids and for identical phospholipids from different sources. Quantification of phosphatidylcholine and phosphatidylethanolamine with HPLC using N-monomethylphosphatidylethanolamine (dioleoyl) as an internal standard gave the same results as phospholipid phosphorus quantification after thin-layer chromatography.

Chromatography, High Pressure Liquid↗
PubMed1994

Colorimetric phospholipid determination with erythrosin B.

Binding of phospholipid vesicles to erythrosin B results in a shift of the absorption maximum of the dye from 528 to 549 nm. This effect was employed to develop a simple, rapid, and sensitive quantification method for unilaminar phospholipid vesicles. At room temperature the color development of the phospholipid-dye complex at 549 nm is essentially complete in 5 min and only a slight decrease is observed in the following hours. The pH optimum of 4.5 for the assay is related to the tight binding at this pH (Kd = 3.6 micrograms/ml). A maximal binding of one erythrosin to seven phospholipid molecules is found. The sensitivity of the assay is high for zwitterionic phospholipids (e.g., 0.023 delta A549 x ml/micrograms for dioleoyl phosphatidyl choline) and lower for anionic phospholipids. The assay depends on the size of the phospholipid liposomes, indicating the importance of the phospholipid surface area for binding.

Colorimetry↗
PubMed2002

Phospholipid composition of cell-derived microparticles determined by one-dimensional high-performance thin-layer chromatography.

Microparticles in the circulation activate the coagulation system and may activate the complement system via C-reactive protein upon conversion of membrane phospholipids by phospholipases. We developed a sensitive and reproducible method to determine the phospholipid composition of microparticles. Samples were applied to horizontal, one-dimensional high-performance thin-layer chromatography (HPTLC). Phospholipids were separated on HPTLC by chloroform:ethyl acetate:acetone:isopropanol:ethanol:methanol:water:acetic acid (30:6:6:6:16:28:6:2); visualized by charring with 7.5% Cu-acetate (w/v), 2.5% CuSO(4) (w/v), and 8% H(3)PO(4) (v/v) in water; and quantified by photodensitometric scanning. Erythrocyte membranes were used to validate the HPTLC system. Microparticles were isolated from plasma of healthy individuals (n = 10). On HPTLC, mixtures of (purified) phospholipids, i.e., lysophosphatidylcholine, phosphatidylcholine (PC), sphingomyelin (SM), lysophosphatidylserine, phosphatidylserine, lysophosphatidylethanolamine, phosphatidylethanolamine (PE), and phosphatidylinositol, could be separated and quantified. All phospholipids were detectable in erythrocyte ghosts, and their quantities fell within ranges reported earlier. Quantitation of phospholipids, including extraction, was highly reproducible (CV < 10%). Microparticles contained PC (59%), SM (20.6%), and PE (9.4%), with relatively minor (<5%) quantities of other phospholipids. HPTLC can be used to study the phospholipid composition of cell-derived microparticles and may also be a useful technique for the analysis of other samples that are available only in minor quantities.

Chromatography, Thin Layer↗
PubMed1994

Phospholipid peroxidation after 60 min of global ischaemia and 10 min of reperfusion. A study in the isolated rat heart.

Peroxidation of polyunsaturated fatty acids in cell membranes is thought to be a crucial factor in the cascade leading to reperfusion damage in the myocardium. However, some studies also describe increased lipid peroxidation in ischaemic tissue. The present study therefore examines phospholipid peroxidation after 60 min of global ischaemia and during the initial phase of reperfusion in isolated Langendorff-perfused rat hearts. Lipids were extracted from these hearts and separated into phospholipid, triglyceride and non-esterified fatty acid fractions. The phospholipid fraction was hydrolysed with phospholipase A2, and reverse-phase high performance liquid chromatography of the fatty acids derived from the phospholipids was performed. Peroxidized polyunsaturated fatty acids were separated from unchanged fatty acids and amounts of monohydroxy or monohydroperoxy isomers were quantified by measuring conjugated dienes by UV absorption (235 nm). Phospholipids from ischaemic as well as free-radical-exposed tissue contained increased levels of peroxidized polyunsaturated fatty acids (20.7 +/- 2.4 and 20.5 +/- 2.3 respectively, v 11.8 +/- 1.4 units/mg dry weight in controls). After 2-10 min of reperfusion, a significant increase in phospholipid peroxidation was no longer detected (12.5 +/- 1.2 units/mg). The amount and the composition of non-esterified fatty acids were examined by gas chromatography. Ischaemia significantly increased both the amount of non-esterified fatty acids (1.5 +/- 0.8 v 4.9 +/- 1.8 nmol/mg dry wt) as well as the percentage composed of arachidonic acid (3.4 +/- 3.2% v 7.4 +/- 1.4%). Fatty acid levels remained elevated during reperfusion (5.5 +/- 1.9 nmol/mg and 7.0 +/- 1.4%). In conclusion, our results have demonstrated that prolonged ischaemia alone caused phospholipid peroxidation as well as accumulation of non-esterified arachidonic acid. There was no sign of further phospholipid peroxidation during reperfusion.

Animals↗
PubMed2001

Comparison of in vitro surface properties of clove oil-phospholipid suspensions with those of ALEC, Exosurf and Survanta.

Dipalmitoyl phosphatidylcholine, the main component of lung surfactant is ineffective as a replacement surfactant due to its poor adsorption. We studied clove oil as a possible additive for improving the surface activity of protein-free phospholipid suspensions. We added low doses of clove oil, to phospholipid suspensions and studied the surface properties by in vitro analysis using a pulsating bubble surfactometer and a Wilhelmy balance. Survanta, ALEC and Exosurf were used as controls for comparison. The test surfactants, which were phospholipid-oil suspensions at 1% concentration, in buffer containing either 2 or 5 mM calcium, were pulsated at 40 cpm in a pulsating bubble surfactometer. The phospholipids studied were dipalmitoyl phosphatidylcholine (PC), phosphatidylethanolamine (PE), phosphatidylglycerol (PG), binary mixtures of PC:PE (2:3) and PC:PG (2:3). The addition of clove oil (CO) to each of the above phospholipids was in the ratio of nine parts of phospholipid to one part of oil. The presence of CO caused a significant improvement in the adsorption and minimum surface tension of all the phospholipid suspensions studied. The mixtures PC with CO, both in the presence of 2 and 5 mM calcium, and PCPE with CO at 2 mM calcium concentration had surface properties significantly better than those of ALEC and Exosurf and equivalent to those of Survanta. The addition of clove oil helps improve the surface properties of phospholipids.

Adsorption↗
PubMed1975

Subcellular distribution of phospholipids during liver damage induced by rare earths.

After intravenous injection of praseodymium nitrate, female Wistar rats develop fatty livers. In contrast to the marked increase of triglycerides, the phospholipid content was only increased by 50%. The subcellular distribution of phospholipids showed that major changes occur in the microsomal fraction within the first 24 hrs. Among the individual phospholipids only phosphatidylcholine and phosphatidylethanolamine concentrations were elevated. Further subfractioning revealed that phospholipid concentration increased in the smooth endoplasmic reticulum, whereas it decreased in the rough endoplasmic reticulum. The individual phospholipids in the smooth endoplasmic reticulum increased to the same degree as did the total phospholipids. On the other hand, in the rough endoplasmic reticulum only the lecithin fraction decreased, while all other phospholipids remained unchanged. Cytochrome P450, cytochrome b5, and glucose 6-phosphatase activity were drastically reduced in the rough endoplasmic reticulum, while no changes could be observed in the smooth endoplasmic reticulum. In the serum, phospholipid concentration fell to half the normal value within the first 24 hrs after praseodymium intoxication.

Animals↗
PubMed1985

Renal cortical brush-border and basolateral membranes: cholesterol and phospholipid composition and relative turnover.

A new procedure for the rapid isolation of renal cortical brush-border and basolateral membranes from the same homogenate is described. Brush-border membranes isolated using Mg2+-EGTA precipitation were enriched 18-fold for leucine aminopeptidase and had a recovery of 32.5%. Basolateral membrane fractions were isolated using a discontinuous sucrose gradient and showed an enrichment of 10.7-fold and recovery of 12.8% using (Na+,K+)-ATPase as a marker enzyme. Lipid analysis using two-dimensional TLC separation of phospholipids and gas liquid chromatography for cholesterol showed marked differences in the lipid composition of the brush-border and basolateral membranes. The brush-border membrane had increased sphingomyelin, phosphatidylserine, ethanolamine plasmalogens, and an increased cholesterol-to-phospholipid and sphingomyelin-to-phosphatidylcholine ratio compared to the basolateral membrane. The relative turnover of total membrane and individual phospholipid species using a double isotope ratio method was carried out. Phospholipids were labeled with either phosphorus 32 and 33 or acetate (3H, 1-14C). The relative turnover of phospholipid species and cholesterol differed strikingly. Phosphatidylcholine showed a high turnover, phosphatidylethanolamine and phosphatidylinositol had intermediate values and sphingomyelin, phosphatidylserine and cholesterol had low relative turnover rates. The order of phospholipid class relative turnover was independent of the labeled precursor used. The brush-border membrane had a significantly reduced relative turnover rate for total membrane phospholipids, sphingomyelin and cholesterol compared to the basolateral membrane. These data show marked differences in the lipid composition and relative turnover rates of the phospholipid species of the brush-border and basolateral membranes.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1978

In vitro and in vivo effects of exogenous lipids on the enzymatic hydrolysis of rat bile phospholipids.

The addition of total phospholipids, phosphatidylcholines, triglycerides, cholesterol or glycerol to incubation media containing rat pancreatic juice and bile labeled with [9, 10 3H2] oleic acid (90% of the radioactivity present as phospholipids) had no effect on the hydrolysis of bile endogenous phospholipids. The introduction of 2 or 10 mg of phosphatidylcholines and 0.5 ml of bile (approximately 1.5 mg of phospholipids) into the rat upper duodenum decreased the rate absorption of native bile phospholipids. It was not followed by an increase of free fatty acids released from biliary phospholipids in the intestinal lumen. The introduction of bile (0.5 ml) and small amounts of triolein (1.4--3.5 mg) into the duodenum had little effect on the rate of hydrolysis and absorption of native bile phospholipids, but caused a reduced absorption of the free fatty acids released or those coming from initial nonphosphorus biliary lipids. The introduction of bile (0.5 ml) and large amounts of triolein (30 mg) into the duodenum increased the rates of hydrolysis and absorption of endogenous bile phospholipids. These observations suggest that luminal lipid components can modify the organization of luminal micelles and, consequently, the action of the pancreatic phospholipase A2 and the absorption of bile lipids.

Animals↗
PubMed1987

Abnormal metabolism of polyunsaturated fatty acids and phospholipids in diabetic glomeruli.

Studies were done on changes in phospholipid content and fatty acid composition of phospholipids and on the role of the acylation pathway in synthesis of phospholipids in the development of abnormal fatty acid composition in the glomeruli of rats 2 and 10 mo after induction of diabetes with streptozotocin. The proportions of individual phospholipids in the glomeruli of rats were not changed 2 mo after induction of diabetes, but the proportion of phosphatidylethanolamine (PE) decreased and that of sphingomyelin increased 10 mo after induction of diabetes. In contrast, in liver the proportion of PE was increased and that of phosphatidylcholine was decreased. These results showed that changes of individual phospholipids in glomeruli were time-dependent and tissue-specific. Two mo after induction of diabetes, the main change in the phospholipid fatty acid composition of diabetic glomeruli was a decrease in arachidonic acid (AA); the main change in serum free fatty acids (FFA) was an increase in linoleic acid (LA) and a decrease in AA. Ten mo after induction of diabetes, the main changes in the phospholipid fatty acid composition of glomeruli were an increase in LA and a decrease in AA; the main change of the serum FFA composition was a decrease in AA. Thus, the fatty acid composition of glomerular phospholipids was not directly correlated to that of the serum in diabetic rats. Acyl-CoA synthetase and acyltransferase activities increased in diabetic glomeruli with either AA or LA as substrate, but activity toward LA increased more at 2 mo after induction of diabetes.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1995

Molecular architecture and biophysical properties of phospholipids during thermal adaptation in fish: an experimental and model study.

Phospholipids from livers of carps (Cyprinus carpio L.) adapted to winter (5 degrees C) and summer (25 degrees C) temperatures were isolated, and the fatty acid composition of total phospholipids, as well as molecular species composition of diacyl phosphatidylcholines and ethanolamines, were determined. Order parameter of 5-doxyl stearic acid and steady-state fluorescence anisotropy of different anthroyloxy fatty acids--[2-, 12(N-9-anthroyloxy)stearic acid and 16(N-9-anthroyloxy)palmitic acid--embedded in native and synthetic (16:0/16:0, 16:0/22:6, 18:0/22:6, 18:1/22:6, 20:4/20:4, 22:6/22:6 phosphatidylcholines and 16:0/18:1, 18:1/22:6 phosphatidylethanolamines) phospholipid vesicles was also determined between -30 and 30 degrees C and 5 and 30 degrees C, respectively. There is an accumulation of 1-monoenoic, 2-polyenoic diacyl phosphatidylcholine and ethanolamine with a concomitant reduction of 1-stearoyl,2-docosahexaenoyl species in the cold-adapted state. Despite a 30% accumulation of long-chain polyunsaturated fatty acids in phospholipids in cold, there is only a 5 degrees C downshift in the solid-gel to liquid-crystalline phase transition temperature (-8 vs. -13 degrees C). Vesicles from total phospholipids of cold-adapted fish proved to be more disordered in all segments than from the warm-adapted ones when assayed using 2,12-(N-9-anthroyloxy)stearic and 16-(N-9-anthroyloxy)palmitic acid. Vesicles made from purified phosphatidylcholines showed the same pattern, but they were more disordered than the corresponding total phospholipids. This could be modelled using mixed phospholipid vesicles made of synthetic 16:0/22:6 phosphatidylcholine (75%) and either 18:1/22:6 phosphatidylethanolamine (25%) vs. 16:0/18:1 phosphatidylethanolamine (25%) and comparison of the anisotropy parameters of 100% 16:0/22:6 and 100% 18:1/22:6 phosphatidylcholine vesicles. Mixing either 16:0/18:1 (25%) or 18:1/22:6 (25%) phosphatidylethanolamines to 18:0/22:6 (75%) phosphatidylcholine shifted down or up, respectively, the transition temperature of vesicles compared to 100% 18:0/22:6 vesicles assayed by electron spin resonance spectroscopy using 5-doxylstearic acid. It is concluded that it is not the gross amount of long-chain polyunsaturated fatty acids in phospholipids, but rather their specific combination with cis delta 9 monounsaturated fatty acids in the position sn-1, especially in phosphatidylethanolamines, that is important in determining the physical properties of biomembranes in relation to adaptational temperature.

Adaptation, Physiological↗
PubMed1996

Effect of dietary alpha-linolenic acid intake on incorporation of docosahexaenoic and arachidonic acids into plasma phospholipids of term infants.

The fractional conversion rates of plasma phospholipid alpha-linolenic acid (18:3n-3) and linoleic acid (18:2n-6) to docosahexaenoic acid (22:6n-3) and arachidonic acid (20:4n-6), respectively, and the fractional rates of incorporation of 22:6n-3 and 20:4n-6 into plasma phospholipids were determined in 27 healthy 3-wk-old term infants who had received formulas with approximately 16% of fat as 18:2n-6 and 0.4% (n = 6), 1.0% (n = 11), or 3.2% (n = 10) as 18:3n-3 from birth. The infants were given a single dose of both [U-13C] 18:2n-6 and [U-13C] 18:3n-3 with a feeding, and blood samples were collected 8, 12, and 24 h afterward for determination of the isotopic enrichments of the [M + 18] isotopomers of plasma phospholipid fatty acids by negative chemical ionization gas chromatography/mass spectrometry. A simple precursor/product compartmental model was used to estimate fractional rates of conversion and incorporation. All infants converted 18:3n-3 to 22:6n-3 and 18:2n-6 to 20:4n-6. Although the fractional rate of conversion of 18:3n-3 to 22:6n-3 did not differ among groups, the fractional rate of incorporation of 22:6n-3 into the plasma phospholipid fraction was greater in infants who received 3.2% vs. 0.4% or 1.0% 18:3n-3 (4.1 +/- 2.2 vs. 1.6 +/- 1.5 or 2.0 +/- 1.0% of the plasma phospholipid 22:6n-3 pool daily). The fractional rate of conversion of 18:2n-6 to 20:4n-6 was less in infants who received the 3.2% 18:3n-3 intake (0.4 +/- 0.3% of the plasma phospholipid 18:2n-6 pool daily vs. 1.1 +/- 0.7% and 0.8 +/- 0.5% in those who received 0.4 and 1.0% 18:3n-3, respectively). The fractional rate of incorporation of 20:4n-6 into plasma phospholipid also was less in the 3.2% vs. the 0.4 and 1.0% 18:3n-3 groups (2.7 +/- 1.4% vs. 5.9 +/- 2.6 and 4.4 +/- 1.7%, respectively, of the plasma phospholipid 20:4n-6 pool daily).

Arachidonic Acid↗
PubMed2005

Modulation of ecto-5'-nucleotidase by phospholipids in human umbilical vein endothelial cells (HUVEC).

Ecto-5'-nucleotidase, the major enzyme controlling extracellular adenosine production, can be activated by phospholipids, e.g. lysophosphatidylcholine (LPC). This study examined the structural requirements of phospholipids to evoke this enzyme activation and figured out two new activators of ecto-5'-nucleotidase: platelet activating factor (PAF) and sphingosylphosphorylcholine (SPC). Potential signal transduction pathways including an involvement of protein kinase C and PAF-receptor were evaluated on the model of human umbilical vein endothelial cells (HUVEC). Cells were pre-incubated with 10 microM of various phospholipids including lysophosphatidylcholine, beta-arachidonyl-gamma-palmityl-alpha-phosphatidylcholine, beta,gamma-dipalmityl-alpha-phosphatidyl-choline, beta,gamma-dipalmityl-alpha-phosphatidylethanolamine, beta,gamma-dipalmityl-alpha-phosphatidylserine, gamma-acyl-beta-lyso-alpha-phosphatidylethanolamine, beta-acetyl-gamma-O-hexadecyl-alpha-phosphatidylcholine (platelet activating factor), lysophosphatidylic acid, sphingosine-1-phosphate and sphingosylphosphorylcholine. In the cell supernatant the extracellular dephosphorylation rate of the fluorescent AMP-analogue 1,N6-etheno-5'AMP to 1,N6-etheno-adenosine was measured by HPLC. Out of these ten structurally related phospholipids only lysophosphatidylcholine, sphingosylphosphatidylcholine and platelet activating factor dose-dependently increased the activity of ecto-5'-nucleotidase. Pharmacological blocking experiments revealed that neither the activation of PAF-receptor nor of protein kinase C were important for mediating the activation of ecto-5'-nucleotidase. Thus, using information on the known molecular structures of tested phospholipids, a phosphatidylcholine residue in alpha-position and a short chain length fatty acid esterified in beta-position seem essential for activation of ecto-5'-nucleotidase by glycerophospholipids. Since all tested phospholipids have similar fatty acid chain lengths and residues in alpha-position, they should act similarly on membrane fluidity. It is concluded that the observed effects are not based on changes in membrane fluidity by the added phospholipids, but rather involve a yet to be determined phospholipid-receptor.

5'-Nucleotidase↗
PubMed1998

Epidermal and dermal phospholipids of the human eyelid: a 31P nuclear magnetic resonance spectroscopy study.

The phospholipids of the skin are difficult to quantify because they represent only a small fraction of the skin tissue. In this study, 31P nuclear magnetic resonance, which permits precise profiling of these phospholipids, was used to compare the phospholipids of upper eyelid epidermal and dermal lipid extracts (n = 13 profiles). Phospholipid profiles included alkylacylphosphatidylcholine (AAPC), dihydrosphingomyelin (DHSM), diphosphatidylglycerol (cardiolipin), ethanolamine plasmalogen (EPLAS), lysophosphatidylcholine, phosphatidic acid, phosphatidylcholine (PC), phosphatidylethanolamine, phosphatidylinositol, phosphatidylserine, sphingomyelin, and uncharacterized phospholipids (U1 and U2, particularly enriched in the epidermis). The computed phospholipid metabolic index (n = 86 indexes) findings can be summarized as follows: a lower content of the en-ol and ether phospholipids in the epidermis relative to the dermis, internal compensation among the component phospholipids so as to maintain the choline functional group ratio, and a greater concentration of hydroxyl-containing functional groups in the epidermis. A membrane index (fmem) value of -0.37 for the epidermis deviated considerably from the value of -0.06 characteristic of living membranes and the dermis. The production of the reduced phosphatides, EPLAS and AAPC, indicates the use of alternative pathways between the two tissues. Relative to the dermis, increased PC in the epidermis coupled with decreased DHSM, EPLAS, and AAPC are factors enabling the epidermis of eyelid tissue to be an effective water barrier.

Dermis↗
PubMed2002

Platelet phospholipids are differentially protected against oxidative degradation by plasmalogens.

The oxidative degradation of phospholipids in the presence and absence of plasmalogens (plasmenyl phosphatidylethanolamine: PPE) was followed by chemical analysis. Human platelet phospholipids, either intact or after removal of PPE by acid treatment, were oxidized with 28 mM 2,2'-azobis(2-amidinopropane di-HCl in Triton X-100 micelles (detergent/phospholipid 5:1, mol/mol). PPE (12% of all phospholipids, mol/mol) disappeared about three times more rapidly than glycerophospholipids, whereas sphingomyelin remained unaltered and the lysophosphatidylethanolamine (lysoPE) generated became progressively more unsaturated. After 60 min oxidation, the FA compositions of PS, PC, and PI were similar in extracts with or without plasmalogens. In contrast, diacyl phosphatidylethanolamine (DPE) became more saturated in the absence of PPE. The rate of phospholipid destruction was always unique to each class, but for all phospholipids slowed down in the presence of PPE. This protective effect increased in the order DPE < PS < PC < PI and did not seem to be simply related to the class unsaturation. Alpha-tocopherol had no influence on the time courses of the quantities and compositions of the phospholipids, even at a molar ratio of alpha-tocopherol to phospholipids four times higher than in platelet membranes. Thus, PPE protected phospholipids efficiently but differentially against peroxidative attack, whereas the contribution of alpha-tocopherol appeared to be negligible even at a concentration four times greater than in platelet membranes.

Blood Platelets↗
PubMed1985

Acidic phospholipids may inhibit rat brain hexokinase by interaction at the nucleotide binding site.

Rat brain hexokinase (ATP:D-hexose 6-phosphotransferase; EC 2.7.1.1) is inhibited by acidic phospholipids such as phosphatidylinositol, phosphatidylserine, and cardiolipin. Several aspects of this inhibition are atypical when compared to inhibition by established reversible inhibitors of this enzyme such as the product, Glc-6-P. Maximal inhibition is attained rather slowly (approximately 30 min at 22 degrees C), and is not reversed by simple dilution of the enzyme-lipid mixture. Ligands such as ATP or Glc-6-P can protect the enzyme against inhibition by acidic phospholipids; addition of protective ligands after mixing of enzyme and lipids does not, however, reverse inhibition that occurred prior to ligand addition. Inhibition can be prevented but not reversed by elevated (0.1-0.2 M) [NaCl], indicating a probable role for electrostatic forces in the interaction of lipid with enzyme. Greater inhibition is seen at 22 degrees C than at 3-4 degrees C, suggesting that hydrophobic interactions may also be involved. It is suggested that acidic phospholipids inhibit brain hexokinase by binding at the nucleotide-binding site of the enzyme. The effectiveness of ATP (or the ATP analog, Cibacron Blue) in protecting against inhibition by acidic phospholipids is attributed to direct competition between ATP and the phospholipid for a common binding site. The effectiveness of Glc-6-P (or analogs) in preventing the inhibition is attributed to a conformational change, induced by the binding of this ligand, which prevents binding of ATP or acidic phospholipids to the enzyme. The pH dependency of the inhibition has suggested involvement of the protonated form of a dissociable group (pK approximately 7) on the enzyme in the interaction with acidic phospholipids; this may be the histidyl residue implicated by Solheim and Fromm [Biochemistry 19, 6074-6080 (1984)] in the binding of ATP to brain hexokinase. Structural similarities in the nucleotide-binding sites of several nucleotide-binding enzymes suggest that similar inhibition by acidic phospholipids may be seen with other enzymes of this type; there are already some reports to this effect.

Adenosine Triphosphate↗
PubMed1980

31P-NMR studies on membrane phospholipids in microsomes, rat liver slices and intact perfused rat liver.

1. The 36.4 and 81 MHz 31P-NMR spectra of isolated rat liver microsomes, rat liver slices and perfused rat liver have been recorded in the 4-40 degree C temperature range. 2. In isolated microsomes at 37 degrees C the majority of the phospholipids undergo isotropic motion, whereas at 4 degrees C most of the phospholipids give rise to typical 'bilayer' spectra. 3. Isolated hydrated rat liver microsomal phosphatidylethanolamine is organised in the hexagonal HII phase above 7 degrees C. 4. The Mn2+ permeability of the microsomal membrane is strongly temperature dependent. At 37 degrees C Mn2+ addition eliminates the entire 31P-NMR spectrum, demonstrating that all phospholipids interact with Mn2+. At 4 degrees C a 43% reduction in signal intensity is observed, indicating that at this temperature 43% of the phospholipids are located in the outer monolayer of a lipid bilayer. 5. In liver slices incubated in oxygenated Krebs-Ringer buffer at 4 degrees C there is a rapid decrease in ATP level such that within 20 min almost all ATP is degraded. In these ATP-depleted liver slices at 4 degrees C, virtually all phospholipids in all membranes have 31P-NMR spectra indicating bilayer structure. At 37 degrees C approx. 14% of the phospholipids undergo isotropic motion. 6. Preliminary experiments on perfused rat liver show stable ATP levels for 4 h at 37 degrees C. The spectra, furthermore, suggest similar behavior for the membrane phospholipids to that observed in the liver slices. 7. The possible sources of teh observed isotropic motion of the membrane phospholipids are discussed.

Animals↗
PubMed1981

Transbilayer distribution of phospholipid fatty acyl chains in photoreceptor membrane.

The transverse distribution of the fatty acyl chains of the major phospholipids over the two faces of the photoreceptor membranes has been determined in bovine rod outer segment (stacked disk) preparations. For this purpose, the fatty acid composition of the phospholipids has been analyzed before and after treatment with trinitrobenzenesulfonic acid and phospholipase D. The latter agents are used under conditions in which they have been demonstrated to attack only the outer (cytoplasmic) face of the membrane. After treatment with trinitrobenzenesulfonic acid or phospholipase D, the fatty acid composition of the unreacted phospholipids is the same as that before treatment, regardless of the extent of modification or hydrolysis attained. The fatty acid composition of phosphatidic acid, resulting from phospholipase D action, also remains unchanged during progressive hydrolysis. These results indicate that the fatty acyl chains of the major phospholipids have the same composition on either side of the disk membrane. Together with our previously published evidence for the distribution of the major phospholipids in rod outer segment disk membranes, this means that both the phospholipids and their fatty acyl chains have a remarkably symmetrical distribution over the two membrane faces. On the basis of literature data it is concluded that this approximate symmetry reflects the high mobility of the entire phospholipid pool of disk membranes, thus including appreciable transbilayer movements of the phospholipids.

Animals↗