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PubMed2004

[Effects of soybean phospholipids on learning and memory ability and contents of lipids in mice's brain].

OBJECTIVE: To study the effects of soybean phospholipids on learning and memory abilities and contents of protein and lipids in brain of mice. METHODS: Mice were randomly divided into four groups: low dose (2.5 g/kg), middle dose(5.0 g/kg) and high dose (10.0 g/kg) of soybean Phospholipids in diet and control group (distilled water). The mice were fed with soybean phospholipids everyday while control groups were fed with distilled water for 30 days. After learning and memory abilities were measured, the mice were killed and contents of protein, fatty acids and phospholipids in brain of mice were determined. RESULTS: The learning and memory abilities of mice fed with middle and high dose of soybean Phospholipids were improved obviously(P < 0.05), while memory abilities of low dose's mice were stronger than that of control(P < 0.05). (2) The brain weight v.8 body weight of those in three doses' group was obviously increased, and the protein content in brain was significantly higher compared with control(P < 0.05). (3) The contents of linoleic acid(C18: 2), linolenic acid(C18: 3) and ratio of polyunsaturated fatty acids(PUFA) to total fatty acids (TFA) obviously increased (P < 0.05). In brain of high dose mice, there was obvious change in the content of linolenic acid(C18: 3) in middle's (P < 0.05). (4) The contents of phosphatidylcholine (PS) phosphatidylethanolamine (PE), Phosphatidylcholine (PC) and phospholipids (PL) obviously increase (P < 0.05), while ratio of SM to PC was decreased significantly in brain of high dose mice(P < 0.05). The contents of PS, PC and PL were increased obviously(P < 0.05). CONCLUSION: Soybean phospholipids may increase contents of protein, PUFA and PL in brain of mice and improve learning and memory abilities of mice effectively in a dose-response manner.

Animals↗
PubMed2004

Docosahexaenoic acid-enriched egg phospholipids supplementation induces accretion of arachidonic acid in rat blood lipids.

Animal and humans studies have shown that supplementation with triacylglycerides containing omega3 fatty acids, mainly docosahexaenoic acid (DHA) and eicosapentaenoic acid, can induce a decrease in arachidonic acid (AA) in blood lipids. Interestingly, we observed in a previous work that a supplementation with DHA enriched eggs in a healthy elderly population induced an accretion of AA in their blood lipids. The present study investigates whether purified DHA enriched egg phospholipids could be responsible for this effect. Four groups of rats were supplemented daily, for eight weeks, with DHA phospholipids (10, 30 or 60 mg/kg) or with soybean phospholipids. Red blood cell membranes and plasma fatty acid levels were compared with that of rats without supplementation. Soybean phospholipids supplementation increased the level of AA in blood lipids but decreased that of DHA. The doses of DHA phospholipids, 30 and 60 mg/kg, induced greater amounts of AA without affecting significantly DHA levels. In contrast, DHA phospholipids supplementation, 10 mg/kg, in which there was the greatest amount of AA, induced only a slight increase in AA levels. Moreover, DHA levels were decreased by this supplementation. These results demonstrate that specific increases in AA levels are preferentially associated with DHA phospholipids levels in supplementation.

Animals↗
PubMed2005

The mystery of phospholipid flip-flop in biogenic membranes.

Phospholipid flip-flop is required for bilayer assembly and the maintenance of biogenic (self-synthesizing) membranes such as the eukaryotic endoplasmic reticulum and the bacterial cytoplasmic membrane. Due to the membrane topology of phospholipid biosynthesis, newly synthesized phospholipids are initially located in the cytoplasmic leaflet of biogenic membranes and must be translocated to the exoplasmic leaflet to give uniform bilayer growth. It is clear from many studies that phospholipid flip-flop in biogenic membranes occurs very rapidly, within a period of a few minutes. These studies also reveal that phospholipid translocation in biogenic membranes occurs bi-directionally, independently of the phospholipid head group, via a facilitated diffusion process in the absence of metabolic energy input, and that this type of transport requires specific membrane proteins. These translocators have been termed biogenic membrane flippases, and they differ from metabolic energy-dependent transporters (ABC transporters and MDR proteins). No biogenic membrane flippases have been characterized. This review briefly discusses the importance of biogenic membrane flippases, the various assay methods used for measuring the rate of phospholipid flip-flop, and the progress that has been made towards identifying these proteins.

Animals↗
PubMed1992

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

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

Acylation↗
PubMed1991

Role of adrenergic and cholinergic mediators in gastric mucus phospholipid secretion.

The role of adrenergic and cholinergic mediators in phospholipid secretion by gastric mucosal cells maintained in the presence of [3H]choline was investigated. The secretion of [3H]choline phospholipids over 30 min period averaged 1.98% of the total cellular labeled phospholipids in the absence of any mediator, and was enhanced by beta-adrenergic agonist, isoproterenol, to a greater extent than the cholinergic agonist, pilocarpine. A 2-fold increase in phospholipid secretion was achieved with isoproterenol, while pilocarpine evoked 1.3-fold increase. The stimulatory effect of isoproterenol was inhibited by alprenolol and that of pilocarpine by atropine. The phospholipids secreted in response to isoproterenol exhibited a 30% decrease in lysophosphatidylcholine, while 2.1-fold enrichment in this phospholipid occurred with pilocarpine. The results, for the first time, demonstrate the involvement of neural mediators in the regulation of phospholipid secretion in gastric mucus.

Alprenolol↗
PubMed1976

Gonadotropin receptors in plasma membranes of bovine corpus luteum. II. Role of membrane phospholipids.

The role of phospholipids in the binding of 125I-choriogonadotropin to bovine corpus luteum plasma membranes has been investigated with the use of purified phospholipase A and phospholipase C to alter membrane phospholipids. The phospholipase C-digested plasma membrane preparation showed 85 to 90% inhibition of 125I-choriogonadotropin binding activity when 70% of the membrane phospholipid was hydrolyzed. Similarly treatment of plasma membranes with phospholipase A resulted in 45 to 55% hydrolysis of membrane phospholipid and almost 75% inhibition of receptor activity. Both these enzymes hydrolyzed membrane-associated phosphatidylcholine to a greater extent than phosphatidylethanolamine and phosphatidylserine. Phosphorylaminoalcohols of phospholiphase C end products were completely released into the medium, while phospholipase A by-products remained associated with plasma membranes. Addition of a phospholipids suspension or liposomes to plasma membranes pretreated with phospholipase A and C did not restore gonadotropin binding activity. Soluble phosphorylcholine, phosphorylethanolamine, and phosphorylserine and insoluble diglyceride products of phospholipase C action had no effect on receptor activity. In contrast, end products of the phospholipase A action, such as lysophosphatides and fatty acids, inhibited both on the membrane-associated and solubilized receptor activity. Lysophosphatidylcholine was the most effective end product inhibiting the binding of gonadotropin to the receptor, followed by lysophosphatidylethanolamine and lysophosphatidylserine. The inhibitory effects of phospholipase A or lysophosphatides were completely reversed upon removal of membrane-bound phospholipid end products by washing the membranes with defatted bovine serum albumin. However, phospholipase C inhibition could not be overcome by defatted albumin washings. Solubilization of plasma membranes with detergents which had been pretreated with phospholipase C partially restored the inhibited activity. It is concluded that the phospholipase-mediated inhibition of gonadotropin binding activity was due to hydrolysis and alterations of the phospholipid environment in the case of phospholipase C and by direct inhibition by end products in the case of phospholipase A.

Animals↗
PubMed1990

Regulation of phospholipid biosynthesis during cholesterol influx and high density lipoprotein-mediated cholesterol efflux in macrophages.

We have studied the rate of phospholipid synthesis and turnover in mouse peritoneal macrophages in reaction to cholesterol influx and high density lipoprotein (HDL)-mediated cholesterol efflux, using three different radioactive precursors, 32PO4(3-), [3H]choline, and [14C]oleic acid. The cells were loaded with cholesterol for up to 18 h with acetyl-low density lipoprotein (LDL), and phospholipid synthesis was measured at various time intervals and compared with nonloaded macrophages. In the first 2 h of cholesterol loading, a twofold increase in the rate of synthesis for sphingomyelin, phosphatidylcholine, phosphatidylserine-inositol, and phosphatidylethanolamine was observed. After this initial up-regulation, the rate of phospholipid synthesis continuously declined upon further cholesterol loading, while the turnover rate of cellular phospholipids was not affected under the same conditions. The lysosomal inhibitor chloroquine abolished the down-regulation, revealing a strong correlation between phospholipid synthesis and lysosomal enzyme activity which was presumably dependent on the release of cholesterol from the lysosome. The reduction in phospholipid synthesis induced by cholesterol loading is reversible by the addition of HDL3 to the cells. When HDL3 was added to the culture medium, a two- to threefold increase in phosphatidylcholine synthesis and a twofold increase in sphingomyelin formation was observed after 3 h. Ca2+ antagonists of the dihydropyridine type, which down-regulate HDL-receptor activity and promote the formation and cellular release of lamellar bodies derived from the lysosomal compartment (Schmitz, G., et al. 1988. Arteriosclerosis. 8: 46-56, and Robenek, H., and G. Schmitz. 1988. Arteriosclerosis. 8: 57-67), specifically enhance the synthesis of sphingomyelin in cholesterol-loaded macrophages. Inhibitors of acyl-CoA:cholesterol acyltransferase (Octimibate, progesterone) increase both the synthesis of sphingomyelin and phosphatidylcholine, and enhance HDL-receptor activity. The results indicate that cholesterol and phospholipid metabolism are coordinately regulated in macrophages. Moreover, the formation of phosphatidylcholine and sphingomyelin seems to be an important factor for the promotion of HDL-receptor-mediated cellular cholesterol efflux.

Animals↗
PubMedDate not supplied

The effect of phospholipid vesicles on the NMR relaxation of water: an explanation for the MR appearance of the neurohypophysis?

The normal neurohypophysis is hyperintense relative to brain and adenohypophysis on T1-weighted MR images, but the signal is not chemically shifted with respect to water. The source of the hyperintense MR signal in the normal neurohypophysis has been the subject of recent controversy. To date, an adequate biophysical explanation for the unusual imaging properties of the neurohypophysis has not been found. The purpose of this study was to investigate the effect of two chemical components of the neurohypophysis, phospholipids and vasopressin, on the MR signal. We synthesized phospholipid vesicles of the same size as those found in the neurohypophysis (100-200 nm) and quantitatively measured T1, T2, and chemical shift in a spectrometer at concentrations of 0-250 mg/ml of phospholipid. Imaging of the test materials was performed on a 1.5-T whole-body MR system using T1-weighted images, T2-weighted images, reduced bandwidth, and fat suppression techniques. The experiment was also performed with saline buffer, mineral oil, vasopressin, and vasopressin incorporated into the core of the phospholipid vesicles. We found that a solution containing phospholipid vesicles has T1 and T2 characteristics analogous to the neurohypophysis and that this solution exhibits a single peak that is not chemically shifted with respect to water. Vasopressin had no effect on the signal, neither in solution nor in the vesicles. We conclude that phospholipid acts as a relaxation enhancer of water protons and that the MR characteristics of the phospholipid vesicles can account for the observed MR properties of the neurohypophysis.

Humans↗
PubMed1990

Differential sensitivity of protein kinase C isozymes to phospholipid-induced inactivation.

Interactions of types I, II, and III protein kinase C (PKC) with phospholipids were investigated by following the changes in protein kinase activity and phorbol ester binding. The acidic phospholipids such as phosphatidylserine (PS), phosphatidic acid, phosphatidyl-glycerol, and cardiolipin, which are activators of PKC in the assay of protein phosphorylation, could differentially inactivate PKC I, II, and III during preincubation in the absence of divalent cation. The phospholipid-induced inactivation of PKC was concentration and time dependent and only affected the kinase activity without influencing phorbol ester binding. PKC I was the most susceptible to the phospholipid-induced inactivation, and PKC III was the least. The IC50 values of PS for PKC I, II, and III were 5, 45, and greater than 120 microM, respectively. Addition of divalent cation such as Ca2+ or Mg2+ suppressed the phospholipid-induced inactivation of PKC. In the absence of divalent cation, PKC I, II, and III all formed complexes with PS vesicles, although to a slightly different degree, as analyzed by molecule sieve chromatography. [3H]Phorbol 12,13-dibutyrate binding for PKC I, II, and III was recovered after chromatography; however, the kinase activities of all these enzymes were greatly reduced. In the presence of Ca2+, all three PKCs formed complexes with PS vesicles, and both the kinase and phorbol ester-binding activities of PKC II and III were recovered following chromatography. Under the same conditions, the phorbol ester-binding activity of PKC I was also recovered, but the kinase activity was not. The phospholipid-induced inactivation of PKC apparently results from a direct interaction of phospholipid with the catalytic domain of PKC; this interaction can be suppressed by divalent cations. In the presence of divalent cations, PS interacted preferentially with the regulatory domain of PKC and resulted in the activation of the kinase.

Animals↗
PubMed1990

The interaction of phospholipase A2 with phospholipid analogues and inhibitors.

A series of structurally modified phospholipids have been used to delineate the structural features involved in the interaction between cobra venom (Naja naja naja) phospholipase A2 and its substrate. Special emphasis has been placed on sn-2 amide analogues of the phospholipids. These studies have led to a very potent, reversible phospholipase A2 inhibitor. A six-step synthesis of this compound, 1-palmitylthio-2-palmitoylamino-1,2-dideoxy-sn-glycero-3- phosphorylethanolamine (thioether amide-PE), was developed. Other analogues studied included 1-palmitylthio-2-palmitoylamino-1,2-dideox-sn- glycero-3-phosphorylcholine, 1-palmityl-2-palmitoylamino-2- deoxy-sn-glycero-3-phosphorylcholine, 1-palmitoyl-2-palmitoylamino-2-deoxy-sn-glycero-3- phosphorylcholine, 1-palmitylthio- 2([(tetradecyloxy)carbonyl]amino)-1,2-dideoxy-sn-glycero-3- phosphorylcholine, 1-palmitoyl- 2([(octadecylylamino)carbonyl]amino)-2-deoxy-sn-glycero-3- phosphorylcholine, and sphingomyelin. Inhibition studies used the well defined Triton X-100 mixed micelle system and the spectroscopic thio assay. The phospholipid analogues showed varying degrees of inhibition. The best inhibitor was the thioether amide-PE which had an IC50 of 0.45 microM. In contrast, sphingomyelin, a natural phospholipid that resembles the amide analogues, did not inhibit but rather activated phosphatidylcholine hydrolysis. This systematic study of phospholipase A2 inhibition led to the following conclusions about phospholipid-phospholipase A2 interactions: (i) sn-2 amide analogues bind tighter than natural phospholipids, presumably because the amide forms a hydrogen bond with the water molecule in the enzyme active site, stabilizing its binding. (ii) Inhibitor analogues containing the ethanolamine polar head group appear to be more potent inhibitors than those containing the choline group. This difference in potency may be due solely to the fact that the cobra venom phospholipase A2 is activated by choline-containing phospholipids. Thus, choline-containing non-hydrolyzable analogues both inhibit and activate this enzyme. Both of these effects must be taken into account when studying phosphatidylcholine inhibitors of the cobra venom enzyme. (iii) The potency of inhibition of these analogues is significantly enhanced by increasing the hydrophobicity of the sn-1 functional group.(ABSTRACT TRUNCATED AT 400 WORDS)

Binding Sites↗
PubMed1990

Scavenger receptor-mediated uptake and metabolism of lipid vesicles containing acidic phospholipids by mouse peritoneal macrophages.

We studied the mechanism of uptake and metabolism of exogenous phospholipids in mouse peritoneal macrophages using vesicles composed of various phospholipids and cholesterol. Macrophages in culture were found to actively incorporate and metabolize phosphatidylcholine/cholesterol vesicles containing small amounts of acidic phospholipids such as phosphatidylserine, phosphatidylinositol, or phosphatidic acid and to store the fatty acyl chains and cholesterol in triacylglycerol and cholesteryl ester form in their cytosol. These cells exhibited massive amounts of oil red O-positive lipid droplets, a typical feature of foam cells. The metabolism of exogenous phospholipid vesicles was completely inhibited by chloroquine and cytochalasin B, suggesting that vesicle uptake occurs by endocytosis. A similar type of metabolism was observed in guinea pig peritoneal macrophages, macrophage cell line J774.1, but not in Swiss 3T3 fibroblasts. Competition studies using various ligands for the scavenger receptor showed that acetylated low density lipoprotein (acetyl-LDL), dextran sulfate, or fucoidan was able to compete for up to 60% of the binding of phosphatidylserine-containing vesicles, and that copper-oxidized LDL (oxidized LDL) competed for more than 90% of the vesicle binding. On the other hand, phosphatidylserine-containing vesicles was able to compete for more than 90% of the binding of acetyl-LDL. These results indicate that acidic phospholipids are recognized by the scavenger receptors on the surface of macrophages and that more than one scavenger receptor exists on mouse peritoneal macrophages, i.e. one capable of recognizing acetyl-LDL, oxidized LDL, and an array of acidic phospholipids on membranes, and the other recognizing both acidic phospholipids and oxidized LDL but not acetyl-LDL.

Animals↗
PubMed1986

Inhibition of T lymphocyte activation by cyclosporin A: interference with the early activation of plasma membrane phospholipid metabolism.

Rabbit lymph node and thymus lymphocytes were stimulated with concanavalin A (Con A). Cyclosporin A (CSA) inhibited in a dose-dependent way the induction of RNA and DNA synthesis; nearly complete inhibition was observed at a concentration of 200 ng/ml. Results of kinetic studies suggested that the immunosuppressive drug interfered with an early event occurring in activated lymphocytes. Among the earliest changes detectable in activated lymphocytes, the turnover of plasma membrane phospholipids is increased, predominantly of their fatty acid moieties, catalyzed by the membrane-bound lysophosphatide acyltransferase. CSA, at concentrations identical with those inhibiting macromolecular synthesis, also inhibited the Con A-stimulated specific increase in the incorporation of labeled fatty acids into plasma membrane phospholipids. When lymphocytes were stimulated with Con A for 1 hr, incorporation of labeled oleic acid and arachidonic acid approximately doubled in plasma membrane phospholipids. CSA at a concentration of 200 ng/ml prevented the elevated incorporation of labeled fatty acids into plasma membrane phospholipids of Con A-stimulated thymocytes. Concomitantly, the activation of lysolecithin acyltransferase, the key enzyme for the incorporation of long-chain fatty acids into phospholipids, was strongly inhibited. Up to high concentrations, CSA had no effect on the phospholipid metabolism of unstimulated lymphocytes. The results suggest that CSA inhibits the activation of T lymphocytes by interfering with the early activation of plasma membrane phospholipid metabolism.

1-Acylglycerophosphocholine O-Acyltransferase↗
PubMed1987

Synthesis of chromatin phospholipids.

The presence of a phospholipid fraction associated with chromatin has been demonstrated by biochemical technique in rat hepatocytes. The composition of this fraction determined by chromatography with respect to that of the nuclei is characterized by low content of phosphatidylserine and high content in phosphatidylethanolamine. Also the synthesis and turnover studied after injection of [32P]O4(2-) show a different behaviour: the peak of activity is after 6 hrs in nuclei and microsomes, whereas in chromatin it occurs after 9 hrs. A second peak is evident after 24 hrs in chromatin and microsome phospholipids. Differences have been also shown by analyzing the single phospholipid radioactivity in time. The behaviour of chromatin phospholipids has also been studied during DNA premitotic synthesis in regenerating liver. It has been shown that there is no difference in synthesis in relation to that of DNA in nuclear phospholipids, whereas the specific activity of chromatin phospholipids begins to increase twelve hours after hepatectomy and continues throughout the period of the first mitotic wave, thus bringing to a summation with the beginning of the second wave. The role of this phospholipid fraction in relation to DNA synthesis and gene expression is discussed.

Animals↗
PubMed1989

Phospholipid composition of Mycobacterium smegmatis susceptible and resistant to antitubercular drugs.

The phospholipid composition, distribution and metabolism in mono drug resistant mutants towards antitubercular drugs, viz, streptomycin, ethambutol and isoniazid, were investigated. Though their total phospholipid content was not altered significantly, changes were observed in their individual phospholipid content. Reduced biosynthesis and degradation of phospholipids (monitored by pulse and chase technique using [32P]orthophosphoric acid as a precursor) was observed in all the mutants studied. The subcellular distribution of phospholipids revealed accumulation of phospholipids in the cell walls and reduction in cell membranes of the drug-resistant mutants. Similar alterations were seen in individual phospholipids of these subcellular fractions.

Antitubercular Agents↗
PubMed1989

Changes in phospholipid and cholesterol concentrations of the rat microvillus membrane during maturation.

We studied the cholesterol and phospholipid content and the cholesterol-to-phospholipid molar ratio of the small intestinal microvillus membrane in fetal, weanling, and adult rats. We also investigated the effect of glucocorticoid administration on these lipids and the ontogeny of 1,2-diglycerol-CDP choline phosphocholine transferase (PCT). Cholesterol and phospholipid concentrations decreased with maturation, phospholipids declining more than cholesterol. Thus, the cholesterol-to-phospholipid molar ratio rose with maturation. A similar decline was seen with PCT activity. Glucocorticoid treatment had no effect on cholesterol, phospholipids, or PCT. However, the maturational increase in the cholesterol-to-phospholipid molar ratio was blunted by glucocorticoid administration in the weanling and adult rats.

Age Factors↗
PubMed1989

Rapid separation and quantitation of major phospholipids in biological samples by combined high-performance liquid chromatography and automated phosphorus analyzer.

Phospholipids extracted from tissue samples were separated by an isocratic high-performance-liquid-chromatographic (HPLC) method and simultaneously quantitated by an automated phosphorus analyzer. Results from various tissues were compared with previously published data obtained by thin-layer chromatography (TLC). Liver, heart, skeletal muscle, kidney and brain cortex synaptosomes from rats were examined. Optimal separation of major phospholipids of these tissues was achieved in a single HPLC run using a mobile phase of acetonitrile, methanol and sulfuric acid 100:2.1:0.05 (v:v:v). Recoveries of pure phospholipids injected onto the column averaged 75-80%. Similar recoveries were obtained with heart and skeletal muscle phospholipids, whereas liver, kidney and synaptosomes yielded lower recoveries (50-66%), suggesting the presence of other phospholipids in these tissues which did not elute from the column. The composition of total and individual phospholipids varied among the tissues and was generally similar to previously reported findings with TLC. The intraassay coefficients of variation ranged from 5 to 11%. We conclude that this technique is a reliable, rapid, and reproducible method for separation and quantitation of the major phospholipid species of tissues and subcellular fractions.

Animals↗
PubMed1989

The interaction of bone Gla protein (osteocalcin) with phospholipid vesicles.

The binding interaction of bone Gla protein (BGP), or osteocalcin, to phospholipid vesicles in the presence of calcium has been investigated. Two separate indirect methodologies involving displacement of pyrene-modified Factor Va bound to phospholipid vesicles, and competition with several coagulation proteins in a prothrombin activation assay were performed. Titration of BGP into a cuvette containing phospholipid vesicles (75:25, L-alpha-phosphatidylcholine/L-alpha-phosphatidylserine (PCPS] saturated with pyrene-modified Factor Va resulted in a systematic decrease in steady-state anisotropy, suggesting competition for membrane binding sites with pyrene-modified Factor Va. BGP was also found to inhibit thrombin generation in the prothrombin activation assay. Approximately 50% inhibition was observed at 3 microM BGP under phospholipid-limiting (0.5 microM PCPS) concentrations. No inhibition was observed under phospholipid excess (30 microM PCPS) concentrations. Direct measurement of phospholipid binding was measured using equilibrium gel filtration. Elution profiles using fixed lipid (3.4 mumol of PCPS) and varying BGP concentrations (1-17 microM) in the presence of 3 mM CaCl2 showed a BGP-phospholipid association. Quantitation of determined isotherm yielded a dissociation constant of 6 +/- 1 microM with a stoichiometry of 102 +/- 9 BGP molecules/vesicle at saturation (35 PCPS lipids/BGP) in the presence of 3 mM CaCl2. These results support the hypothesis that protein gamma-carboxylation events are coincident with membrane binding potential.

Binding, Competitive↗
PubMed1988

Influence of vitamin D status on the content of complexed acidic phospholipids in chick diaphyseal bone.

Calcium acidic phospholipid phosphate complexes cause hydroxyapatite formation in vitro and in vivo. Previous studies have shown that the amount of these complexed acidic phospholipids is altered in the bones of animals depleted of vitamin D and phosphate. To help differentiate the effects of vitamin D from those due to altered mineral metabolism, the amount of complexed acidic phospholipids present in diaphyseal bone was determined in five groups of chicks varying in calcium, phosphate and vitamin D status. Highest levels of complexed acidic phospholipids were found in chicks receiving vitamin D and having normal serum levels of Ca and P. Lowest levels were found in vitamin D-deficient chicks; levels were not altered in vitamin D-deficient chicks when their serum Ca was normalized. Intermediate levels were found in vitamin D-treated chicks fed a low Ca diet. Levels of the complexed acidic phospholipids were low in bones of hypophosphatemic, vitamin D-treated chicks but, when related to total lipid phosphorus content, values were similar to those of hypocalcemic chicks. These data suggest that while disturbances in calcium and phosphate metabolism which are present in rickets influence the bone content of these complexed acidic phospholipids, vitamin D may also play a direct role in their formation. Further, the ability of vitamin D to increase bone complexed acidic phospholipid content does not seem to be related to its hypercalcemic effect.

Animals↗