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PubMed1979

Cyclopropane fatty acid synthase of Escherichia coli. Stabilization, purification, and interaction with phospholipid vesicles.

The cyclopropane fatty acid (CFA) synthase of Escherichia coli catalyzes the methylenation of the unsaturated moieties of phospholipids in a phospholipid bilayer. The methylene donor is S-adenosyl-L-methionine. The enzyme is loosely associated with the inner membrane of the bacterium and binds to and is stabilized by phospholipid vesicles. The enzyme has been purified over 500-fold by flotation with phospholipid vesicles and appears to be a monomeric protein having a molecular weight of about 90 000. The enzyme binds only to vesicles of phospholipids which contain either unsaturated or cyclopropane fatty acid moieties. CFA synthase is active on phosphatidylglycerol, phosphatidylethanolamine, and cardiolipin, the major phospholipids of E. coli, and also has some activity on phosphatidylcholine. The enzyme is equally active on phospholipid vesicles in the ordered or the disordered states of the lipid phase transition. Studies with a reagent that reacts only with the phosphatidylethanolamine molecules of the outer leaflet of a phospholipid bilayer indicate that CFA synthase reacts with phosphatidylethanolamine molecules of both the outer and the inner leaflets of phospholipid vesicles.

Cyclopropanes↗
PubMed1976

Complete retention of phospholipid acyl groups by mammalian cells in culture.

Radiolabeled phosphate, acetate, and glycerol are incorporated into strain L-fibroblast phospholipids. The acetate and glycerol specifically label the fatty acid and glycerol moieties, respectively, of the phospholipids. To study the metabolic fate of the various moieties of phospholipids, cells incubated with the above radiolabeled compounds were transferred to unlabeled medium, and the rate at which phospholipid radioactivity per 10(6) cells decreased was determined. The rate of decrease expected on the basis of cell division alone was estimated either by monitoring increases in cell number, or by measuring the rate at which radiolabeled DNA per 10(6) cells decreased. Both phospholipid phosphorus and glycerol are lost at a rate greater than can be accounted for by cell division alone. By contrast, nearly all phospholipid acyl chains were retained by the cell to the same extent as radiolabeled DNA. While presence of nonradioactive glycerol in the medium increased the rate at which glycerol was lost from phospholipid, the addition of exogenous fatty acid was without effect on the retention of phospholipid acyl groups. The acyl-glycerol bond of phosphatidylcholine is metabolically more labile than that of phosphatidylethanolamine. Together the data suggest that although L-fibroblast phospholipids undergo deacylation-reacylation reactions, the acyl chains do not equilibrate with either extracellular or intracellular pools of unesterified fatty acid.

Acetates↗
PubMed2003

Evidence that hepatic lipase and endothelial lipase have different substrate specificities for high-density lipoprotein phospholipids.

Hepatic lipase (HL) and endothelial lipase (EL) are both members of the triglyceride lipase gene family. HL hydrolyzes phospholipids and triglycerides in triglyceride-rich lipoproteins and high-density lipoproteins (HDL). EL hydrolyzes HDL phospholipids and has low triglyceride lipase activity. The aim of this study was to determine if HL and EL hydrolyze different HDL phospholipids and whether HDL phospholipid composition regulates the interaction of EL and HL with the particle surface. Spherical, reconstituted HDL (rHDL) containing either 1-palmitoyl-2-oleoylphosphatidylcholine (POPC), 1-palmitoyl-2-linoleoylphosphatidylcholine (PLPC), 1-palmitoyl-2-arachidonylphosphatidylcholine (PAPC), or 1-palmitoyl-2-docosahexanoylphosphatidylcholine (PDPC) as the only phospholipid, apolipoprotein A-I as the only apolipoprotein, and either cholesteryl esters (CE) only or mixtures of CE and triolein (TO) in their core were prepared. The rHDL were similar in size and had comparable core lipid/apoA-I molar ratios. The CE-containing rHDL were used to determine the kinetics of HL- and EL-mediated phospholipid hydrolysis. For HL the V(max) of phospholipid hydrolysis for (POPC)rHDL > (PLPC)rHDL approximately (PDPC)rHDL > (PAPC)rHDL, while the K(m)(app) for (POPC)rHDL > (PDPC)rHDL > (PLPC)rHDL > (PAPC)rHDL. For EL the V(max) for (PDPC)rHDL > (PAPC)rHDL > (PLPC)rHDL approximately (POPC)rHDL, while the K(m)(app) for (PAPC)rHDL approximately (PLPC)rHDL > (POPC)rHDL > (PDPC)rHDL. The kinetics of EL- and HL-mediated TO hydrolysis was determined using rHDL that contained TO in their core. For HL the V(max) of TO hydrolysis for (PLPC)rHDL > (POPC)rHDL > (PAPC)rHDL > (PDPC)rHDL, while the K(m)(app) for (PLPC)rHDL > (POPC)rHDL approximately (PAPC)rHDL > (PDPC)rHDL. For EL the V(max) and K(m)(app) for (PAPC)rHDL > (PDPC)rHDL > (PLPC)rHDL > (POPC)rHDL. These results establish that EL and HL have different substrate specificities for rHDL phospholipids and that their interactions with the rHDL surface are regulated by phospholipids.

Animals↗
PubMed1977

Effects of analogles of ethanolamine and choline on phospholipid metabolism in rat hepatocytes.

1. Analogues of ethanolamine and choline were incubated with different labelled precursors of phospholipids and isolated hepatocytes and the effects on phospholipid synthesis were studied. 2. 2-Aminopropan-1-ol and 2-aminobutan-1-ol were the most efficient inhibitors of [(14)C]ethanolamine incorporation into phospholipids, whereas the incorporation of [(3)H]choline was inhibited most extensively by NN-diethylethanolamine and NN-dimethylethanolamine. 3. When the analogues were incubated with [(3)H]glycerol and hepatocytes, the appearance of (3)H in unnatural phospholipids indicated that they were incorporated, at least in part, via CDP-derivatives. The distribution of [(3)H]glycerol among molecular species of phospholipids containing 2-aminopropan-1-ol and 1-aminopropan-2-ol was the same as in phosphatidylethanolamine. In other phospholipid analogues the distribution of (3)H was more similar to that in phosphatidylcholine. 4. NN-Diethylethanolamine stimulated both the conversion of phosphatidylethanolamine into phosphatidylcholine and the incorporation of [Me-(14)C]methionine into phospholipids. Other N-alkyl- or NN-dialkyl-ethanolamines also stimulated [(14)C]methionine incorporation, but inhibited the conversion of phosphatidylethanolamine into phosphatidylcholine. This indicates that phosphatidyl-NN-diethylethanolamine is a poor methyl acceptor, in contrast with other N-alkylated phosphatidylethanolamines. 5. These results on the regulation of phospholipid metabolism in intact cells are discussed with respect to the possible control points. They also provide guidelines for future experiments on the manipulation of phospholipid polar-headgroup composition in primary cultures of hepatocytes.

Animals↗
PubMed1986

The effect of phospholipids on the activation of protein C by the human thrombin-thrombomodulin complex.

Human thrombomodulin, an endothelial-cell-membrane glycoprotein, has been purified from placenta by Triton X-100 extraction and by affinity chromatography on concanavalin A-Sepharose and thrombin-Sepharose. It has been characterized by its ability to promote the activation of human protein C by human alpha-thrombin in the presence of Ca2+ and fulfilled the requirements of a cofactor. Reconstitution of thrombomodulin into phospholipid vesicles containing anionic phospholipids resulted in an increased rate of activation of protein C. Cardiolipin and vesicles containing phosphatidylcholine/phosphatidylserine (1:1, w/w) were the most effective. The apparent Km of the thrombin-thrombomodulin complex for protein C was 2 microM. It was not changed in the presence of phospholipid, whereas the Vmax. could be apparently increased up to 3.2-fold depending on the phospholipid and on its concentration, the catalytic-centre activity reaching 15.7 mol of activated protein C formed/min per mol of thrombin. Above their optimal concentrations, phospholipids inhibited the amidolytic activity of activated protein C. Phospholipids had no effect on the activation of 4-carboxyglutamic acid-domainless protein C, a proteolytic derivative of protein C lacking the 4-carboxyglutamic acid residues. These results show that the positive effect of anionic phospholipids in the activation of protein C by the thrombin-thrombomodulin complex involves a Ca2+-dependent interaction between protein C and phospholipids. They suggest that the enhancement of thrombomodulin activity by such phospholipids may be of functional significance.

Calcium↗
PubMed1997

Measurement of total phospholipids in urine of patients treated with gentamicin.

AIMS: The excretion of phospholipids in urine may be a marker of the early renal toxicity of the aminoglycoside antibiotics. Urinary phospholipids are formed in myeloid bodies which develop in the lysosomes of proximal tubules during treatment with the aminoglycosides, and overflow into the urine. METHODS: Published assays were modified in order to measure the total phospholipid concentrations in human urine. Phospholipids were extracted from freeze-dried urine samples, digested in concentrated sulphuric acid, and the inorganic phosphorus content determined by complexing with ammonium molybdate and measuring the absorbance at 820 nm. Ten septicaemic patients treated with gentamicin for 5-7 days had significantly higher urine phospholipid concentrations than 10 healthy untreated control subjects (P < 0.0001). There was a negative linear relationship between phospholipid excretion and creatinine clearance (r2 = 0.71). RESULTS: In 34 patients with acute pyelonephritis, increased phospholipid concentrations were observed prior to treatment compared with healthy controls (P < 0.001) and did not alter during treatment with gentamicin. However, the phospholipid concentrations decreased significantly after treatment was completed (P < 0.03). CONCLUSIONS: These studies suggest that urinary phospholipids may indicate early aminoglycoside toxicity but with poor specificity, as many of the infections being treated may themselves be associated with phospholipiduria.

Adolescent↗
PubMed2002

The fatty acid composition of the serum phospholipids of children with sickle cell disease in Nigeria.

The purpose of this study was to determine the fatty acid composition of the serum phospholipids of children with sickle cell disease (SCD) in Nigeria and to compare the relative fluidity of the acyl chains of the serum phospholipids of controls versus the subjects with SCD. It is widely accepted that the fatty acid composition of an individual's serum phospholipids reflects that of their tissue phospholipids. An alteration in the fatty acid composition of membrane phospholipids could affect critical membrane-dependent enzymes and processes (e.g., ion and solute transport, hormone-receptor interactions, signal transduction pathways). We found a significant reduction in the content of polyunsaturated n-3 fatty acids in the phospholipids of subjects with SCD which could result in a reduction of the fluidity of their tissue membranes. Specifically, there was a 40-50% reduction in the proportion of total n-3 fatty acids in subjects with SCD. On the basis of calculated melting points and double bond indices of the acyl chains of the serum phospholipids, the phospholipids of the children with SCD are less fluid relative to those of their healthy counterparts. In addition, we determined that linoleic acid, arachidonic acid, and stearic acid were the major determinants of the fluidity of the acyl chains of the serum phospholipids of the healthy controls and children with SCD.

Adolescent↗
PubMed1979

Phospholipid methylation in macrophages is inhibited by chemotactic factors.

Chemotaxis by human monocytes has been shown to require methylation mediated by S-adenosyl-L-methionine(AdoMet), but the specific transmethylation reaction necessary for this function was not elucidated. In an attempt to define the methylation requirement for chemotaxis, we examined the effect of chemotactic agonists and antagonists on protein carboxy-O-methylation of protein and methylation of phospholipid in guinea pig macrophages. Chemotactic agents tested over a wide dose and time range produced no alteration in carboxy-O-methylation. However, these agents did produce an effect on the methylation of phosphatidylethanolamine by macrophages. AdoMet-mediated phospholipid methylation was inhibited by as much as 73% by chemotactic factors, and there was excellent correlation (r = 0.99) between their concentrations for producing half-maximal chemotactic responses and for inhibiting phospholipid methylation. The inhibition of methylation by chemotactic factors was observed at all incubation times and could not be explained by an increased turnover of membrane phospholipid. Neither the chemotaxis antagonist fPhe-Met nor the nonchemotactic tripeptide Met-Met-Met significantly depressed phospholipid methylation. Immune phagocytosis by macrophages similarly did not alter phospholipid methylation. The chemotactic factors produced no alteration in total macrophage phospholipid synthesis or in the phospholipid methylation in a nonchemotactic cell type. The formation of newly methylated derivatives of phosphatidylethanolamine in macrophages was decreased by a biologically active dose of chemotactic factor. These findings indicate that chemotactic factors are capable of altering the methylation of phosphatidylethanolamine in chemotactically responsive cells. The inhibition of phospholipid methylation by chemotactic factors may be necessary for the translation of a chemotactic signal on the surface of the cell into directional cell movement.

Animals↗
PubMed1994

Electrospray ionization mass spectroscopic analysis of human erythrocyte plasma membrane phospholipids.

Electrospray ionization mass spectrometry (ESI-MS) was utilized for the structural determination and quantitative analysis of individual phospholipid molecular species from subpicomole amounts of human erythrocyte plasma membrane phospholipids. The sensitivity of ESI-MS was 2-3 orders of magnitude greater than that achievable with fast-atom bombardment mass spectrometry (FAB-MS). Phospholipid structure determination and quantitative analysis with ESI-MS can be performed directly from chloroform extracts of biologic samples, obviating the need for prior chromatographic separation of phospholipid classes which has been necessary in FAB-MS phospholipid analyses. Furthermore, ESI-MS is uncomplicated by differential fragmentation of molecular ions and idiosyncratic surface desorption, allowing the quantitation of phospholipids with coefficients of determination (r2) > 0.99 and accuracies > 95%. More than 50 human erythrocyte plasma membrane phospholipid constituents were identified by direct ESI-MS analysis of chloroform extracts of plasma membranes derived from the equivalent of < 1 microliter of whole blood. The major ethanolamine glycerophospholipid subclass in erythrocyte plasma membranes was plasmenylethanolamine that was highly enriched in polyunsaturated fatty acids at the sn-2 position. Collectively, these results demonstrate that ESI-MS of phospholipids is an enabling strategy for the direct structural determination and quantitative analysis of subpicomole amounts of phospholipids from biologic samples.

Erythrocyte Membrane↗
PubMed1996

Efflux of cellular cholesterol and phospholipid to apolipoprotein A-I mutants.

Human plasma apolipoprotein A-I (apoA-I) and recombinant full-length proapoA-I (apoA-I-(-6-243)) as well as four truncated forms of proapoA-I were used as acceptors to study cholesterol and phospholipid efflux from HepG2 cells. Efflux of both cholesterol and phospholipid to the lipid-free plasma apoA-I was twice that of apoA-I-(-6-243). When apoA-I was incorporated into reconstituted high density lipoprotein, cholesterol efflux increased, phospholipid efflux decreased and the difference between plasma apoA-I and apoA-I-(-6-243) disappeared. Truncation of recombinant apoA-I to residues 222 (apoA-I-(-6-222)) and 210 (apoA-I-(-6-210)) resulted in a 70-95% decrease in their ability to promote the efflux of both intracellular and plasma membrane cholesterol. Further truncation to residues 150 (apoA-I-(-6-150)) and 135 (apoA-I-(-6-135)) fully restored the ability of apoA-I to promote cholesterol efflux. Phospholipid efflux closely paralleled the efflux of cholesterol. Interaction of 125I-labeled apoA-I with the cells was similar for apoA-I-(-6-243), apoA-I-(-6-222), and apoA-I-(-6-210), but slightly higher for apoA-I-(-6-150) and apoA-I-(-6-135). When complexed with phospholipid, all forms except apoA-I-(-6-210) formed discoidal reconstituted high density lipoprotein particles. When the same amounts of free or lipid-associated apoA-I were compared, association of apoA-I with phospholipid increased cholesterol efflux and decreased phospholipid efflux, and the difference in the ability of different mutants to promote cholesterol and phospholipid efflux disappeared. We conclude that the capacity of lipid-free apoA-I to promote cholesterol efflux is related to its ability to mobilize cellular phospholipid, which apparently involves a region around residues 222-243. A second lipid-binding region is exposed when the carboxyl-terminal half of apoA-I is absent.

Apolipoprotein A-I↗
PubMed1998

The influence of apolipoproteins on the hepatic lipase-mediated hydrolysis of high density lipoprotein phospholipid and triacylglycerol.

This study describes the influence of apolipoproteins on the hepatic lipase (HL)-mediated hydrolysis of phospholipids and triacylglycerol in high density lipoproteins (HDL). HL-mediated hydrolysis was assessed in well characterized, homogeneous preparations of spherical reconstituted high density lipoproteins (rHDL). The rHDL were comparable in size and lipid composition and contained either apoA-I ((A-I)rHDL) or apoA-II ((A-II)rHDL) as their sole apolipoprotein constituent. Preparations of rHDL containing only cholesteryl esters (CE) in their core, (A-I/CE)rHDL and (A-II/CE)rHDL, were used to assess phospholipid hydrolysis. Preparations of rHDL that contained triacylglycerol as their predominant core lipid, (A-I/TG)rHDL and (A-II/TG)rHDL, were used to assess both triacylglycerol and phospholipid hydrolysis. The rHDL contained trace amounts of either radiolabeled phospholipid or radiolabeled triacylglycerol. Hydrolysis was measured as the release of radiolabeled nonesterified fatty acids (NEFA) from the rHDL. Kinetic analysis showed that HL had a greater affinity for the phospholipids in (A-II/CE)rHDL (Km(app) = 0.2 mM) than in (A-I/CE)rHDL (Km(app) = 3.1 mM). This was also evident when hydrolysis was measured directly by quantitating NEFA mass. HL also had a greater affinity for the phospholipids and triacylglycerol in (A-II/TG)rHDL than in (A-I/TG)rHDL. The Vmax for phospholipid hydrolysis was, by contrast, greater for (A-I/CE)rHDL than for (A-II/CE)rHDL: 309.3 versus 49.1 nmol of NEFA formed/ml of HL/h. Comparable Vmax values were obtained for the hydrolysis of the phospholipids in (A-II/TG)rHDL and (A-I/TG)rHDL. In the case of triacylglycerol hydrolysis, the respective Vmax values for (A-I/TG)rHDL and (A-II/TG)rHDL were 1154.8 and 240.2 nmol of NEFA formed/ml of HL/h. These results show that apolipoproteins have a major influence on the kinetics of HL-mediated phospholipid and triacylglycerol hydrolysis in rHDL.

Apolipoprotein A-I↗
PubMed2004

Phospholipid barrier to fibrinolysis: role for the anionic polar head charge and the gel phase crystalline structure.

The massive presence of phospholipids is demonstrated in frozen sections of human arterial thrombi. Purified platelet phospholipids and synthetic phospholipids retard in vitro tissue-type plasminogen activator (tPA)-induced fibrinolysis through effects on plasminogen activation and plasmin function. The inhibition of plasminogen activation on the surface of fibrin correlates with the fraction of anionic phospholipid. The phospholipids decrease the amount of tPA penetrating into the clot by 75% and the depth of the reactive surface layer occupied by the activator by up to 30%, whereas for plasmin both of these parameters decrease by approximately 50%. The phospholipids are not only a diffusion barrier, they also bind the components of the fibrinolytic system. Isothermal titration calorimetry shows binding characterized with dissociation constants in the range 0.35-7.64 microm for plasmin and tPA (lower values with more negative phospholipids). The interactions are endothermic and thermodynamically driven by an increase in entropy, probably caused by the rearrangements in the ordered gel structure of the phospholipids (in line with the stronger inhibition at gel phase temperatures compared with liquid crystalline phase temperatures). These findings show a phospholipid barrier, which should be overcome during lysis of arterial thrombi.

Anions↗
PubMed1999

Effect of intravenous bilirubin infusion on biliary phospholipid secretion, hepatic P-glycoprotein expression, and biliary cytotoxicity in pigs.

BACKGROUND: Infusion of large intravenous bilirubin loads in bile acid-depleted pigs reduces P-glycoprotein-dependent biliary phospholipid secretion and increases the cytotoxicity of bile. The reasons for the diminution of biliary phospholipid secretion and the increase in biliary cytotoxicity are not known. This study was undertaken to determine whether the bilirubin-induced lowering of biliary phospholipid secretion is associated with alterations in hepatic P-glycoprotein (P-gp) expression and to determine why bilirubin infusions increase biliary cytotoxicity. METHODS: Hepatic bile was collected from bile acid-depleted pigs before and during intravenous bilirubin infusion. Hepatic P-gp expression was measured with protein blot analysis, using the P-gp-specific antibody C219. Biliary cytotoxicity was assayed against erythrocytes. The biliary phospholipid fatty acid profile was determined by means of gas chromatography. RESULTS: Bilirubin infusions lowered biliary phospholipid secretion by 69% without changing hepatic P-gp expression, suggesting that bilirubin infusions have an inhibitory effect on hepatic P-gp activity. Bilirubin infusions did not cause P-gp losses into bile. An unequivocal, proportional relationship (r2 = 0.80) pertained between cytotoxicity and the bile acid to phospholipid ratio in bile secreted before and during bilirubin infusion and in phosphatidylcholine-supplemented bile. Unconjugated bilirubin in bile did not contribute to biliary cytotoxicity. Biliary phospholipids were always phosphatidylcholine >> phosphatidylethanolamine, mainly of C16:0, 18:2 and C16:0, 18:1 fatty acid configuration. CONCLUSIONS: Intravenous bilirubin loads reduce biliary phospholipid secretion without changing hepatic P-gp expression. Bilirubin infusions increase biliary cytotoxicity by augmenting the biliary bile acid to phospholipid ratio.

ATP Binding Cassette Transporter, Subfamily B↗
PubMed2001

Phospholipids released from activated platelets improve platelet aggregation and endothelial cell migration.

This study was undertaken to isolate phospholipids released from activated platelets and to investigate their biological activities. Freshly washed platelets were activated with freezing/thawing, thrombin, ionophore 23187, and arachidonic acid. Thrombin was incubated with platelet-rich plasma to promote synthesis and release of phospholipids from platelets. Phospholipids in supernatants of activated platelets were extracted with butanol and separated by thin-layer chromatography. Release of phosphatidylserine (PS) and phosphatidic acid (PA) increased when platelets were treated with freezing/thawing, ionophore, and thrombin. The lysophosphatidyl ethanolamine (LPE) appeared not to be induced with freezing/thawing, but increased significantly by thrombin, ionophore, and arachidonic acid. The effects of platelet phospholipids on hemostasis and angiogenesis were studied with platelet aggregation and endothelium chemotaxis. Phospholipids isolated from thrombin-stimulated platelet-rich/platelet-poor plasmas were used as synergistic agonists in platelet aggregation and as chemotactic agents in endothelial cell migration. Several phospholipids increased chemotaxis and platelet aggregation; these were PS, PA, LPE, and sphingosine-1-phosphate. Also, chemotaxis of those phospholipids increased when combined with charcoal-stripped fetal bovine serum, suggesting that cofactors in serum enhanced phospholipid-induced cell migration. These observations suggest that activated platelets release biologically active phospholipids into the blood stream, where they may play an important role in thrombosis and angiogenesis.

Animals↗
PubMed1971

Assembly of lipids into membranes in Acanthamoeba palestinensis. I. Observations on the specificity and stability of choline- 14 C and glycerol- 3 H as labels for membrane phospholipids.

In order to determine the feasibility of using radioactive precursors as markers for membrane phospholipids in Acanthamoeba palestinensis, the characteristics of phospholipids labeled with choline-(14)C and glycerol-(3)H were examined. Choline-(14)C was found to be a specific label for phosphatidyl choline. There was a turnover of the radioactive moiety of phosphatidyl choline at a rate that varied with the concentration of nonradioactive choline added to the growth medium. Radioactivity was lost from labeled phosphatidyl choline into the acid-soluble intracellular pool and from the pool into the extracellular medium. This loss of radioactivity from cells leveled off and an equilibrium was reached between the label in the cells and in the medium. Radioactive choline was incorporated into phosphatidyl choline by cell-free microsomal suspensions. This incorporation leveled off with the attainment of an equilibrium between the choline-(14)C in the reaction mixture and the choline-(14)C moiety of phosphatidyl choline in the microsomal membranes. Therefore, a choline exchange reaction may occur in cell-free membranes, as well as living A. palestinensis. In contrast to choline-(14)C, the apparent turnover of glycerol-(3)H-labeled phospholipids was not affected by large concentrations of nonradioactive choline or glycerol in the medium. The radioactivity in lipids labeled with glycerol-(3)H consisted of 33% neutral lipids and 67% phospholipids. Phospholipids labeled with glycerol-(3)H turned over slowly, with a concomitant increase in the percentage of label in neutral lipids, indicating a conversion of phospholipids to neutral lipids. Because most ( approximately 96%) of the glycerol-(3)H recovered from microsomal membranes was in phospholipids, whereas only a minor component ( approximately 2%) of the glycerol-(3)H was in the phospholipids isolated from nonmembrane lipids, glycerol-(3)H was judged to be a specific marker for membrane phospholipids.

Amoeba↗
PubMed1990

Platelet activating factor activity in the phospholipids of bovine spermatozoa.

Platelet activating factor (PAF) has been detected in sperm from several mammalian species and can affect sperm motility and fertilization. Because bovine sperm contain a high percentage of ether-linked phospholipid precursors required for PAF synthesis, a study was undertaken to determine the PAF activity of bovine sperm phospholipids. Total lipids of washed, ejaculated bull sperm were extracted, and phospholipids were fractionated by thin-layer chromatography. Individual phospholipid fractions were assayed for PAF activity on the basis of [3H]serotonin release from equine platelets. PAF activity was detected in the PAF fraction (1.84 pmol/mumol total phospholipid) and in serine/inositol (PS/PI), choline (CP), and ethanolamine phosphoglyceride (EP) and cardiolipin (CA) fractions. Activity was highest in the CP fraction (8.05 pmol/mumol total phospholipid). Incomplete resolution of PAF and neutral lipids may have contributed to the activity in the PS/PI and CA fractions, respectively. Phospholipids from nonsperm sources did not stimulate serotonin release. Platelet activation by purified PAF and by sperm phospholipid fractions was inhibited by the receptor antagonist SRI 63-675. These results indicate that bovine sperm contain PAF and that other sperm phospholipids, especially CP and EP, which are high in glycerylether components, are capable of receptor-mediated platelet activation.

Animals↗
PubMed1997

Reduction of sperm cholesterol:phospholipid ratio is a possible mechanism for enhancement of human sperm binding to the zona pellucida following incubation with phosphatidylcholine liposomes.

TEST (TES (N-tris[hydroxymethyl]methyl-2-aminoethanesulfonic acid) and Tris) yolk buffer (TYB) has recently been shown to improve the binding capacity of spermatozoa to zona pellucidae. The present study had two objectives: 1) to elucidate which component(s) of TYB dominates this effect and 2) to define the responsible mechanism. Sperm samples obtained from subfertile men were incubated for 2 h in either TYB or media containing egg yolk lipoproteins or phospholipids. After incubation, sperm binding was tested by the hemizona assay. Yolk lipoprotein-treated spermatozoa bound hemizonae with efficiency equal to that of the spermatozoa incubated in control medium. Conversely, incubation of spermatozoa in media containing either TYB, yolk-phospholipids, or pure phosphatidylcholine (PC) resulted in a 2- to 3-fold increased binding capacity (p < 0.01). A close correlation was found between the effect of yolk-phospholipids and TYB on the binding capacity of the same sperm samples, compared to spermatozoa incubated in control medium. Incubation of spermatozoa in yolk phospholipid medium caused a dose-dependent increase of sperm binding capacity (p < 0.05). Treatment of sperm samples with 1 mg/ml or more of purified PC preparation also resulted in a reduction of the sperm cholesterol:phospholipid molar ratio. Significant correlations between the effects of the treatments on sperm cholesterol: phospholipid molar ratio and sperm binding were obtained with yolk-phospholipids (r = -0.55) or 1 mg/ml purified PC (r = -0.61). We conclude that 1) the enhanced binding capacity of human spermatozoa following TYB treatment is probably due to yolk-phospholipids, mainly egg yolk PC; and 2) it appears that the enhanced binding capacity of human spermatozoa following treatment with egg yolk-containing media may be a result of the reduction of the cholesterol:phospholipid molar ratio in the sperm cells.

Acrosome↗
PubMed1990

Heterogeneity of binding reactivity to different phospholipids of antibodies from patients with systemic lupus erythematosus (SLE) and with syphilis.

The binding specificities were investigated of anti-phospholipid antibodies derived from sera from 55 patients with SLE and related diseases, and from 33 patients with syphilis. Antibodies from both these groups of patients bind strongly to cardiolipin in solid-phase immunoassays, but only antiphospholipid antibodies from patients with autoimmune diseases are associated with thrombotic complications and recurrent spontaneous abortions. IgG anti-phospholipid antibodies from both groups of patients cross-reacted with a range of negatively charged phospholipids, but binding to neutral phospholipids was largely restricted to sera from patients with syphilis. A monoclonal IgM lambda anti-cardiolipin antibody, derived from a patient with autoimmunity, was used to inhibit binding of anti-phospholipid antibodies to cardiolipin and to phosphatidic acid. This antibody inhibited the binding of autoimmune sera to cardiolipin more strongly than sera from syphilis patients, but the converse pattern of inhibition of binding to phosphatidic acid was observed. The VDRL titre correlated with anti-phospholipid antibody activity in sera from syphilis patients, but not from those with autoimmunity. Lupus anti-coagulant activity correlated weakly with IgG antibody levels to each of the negatively charged phospholipids among the patients with autoimmunity. Lupus anticoagulant activity did not correlate uniquely with any anti-phospholipid antibody specificity. These results provide further documentation of the great heterogeneity of anti-phospholipid antibodies associated with autoimmune disease and syphilis.

Antibody Specificity↗