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PubMed1985

Phospholipid protection against proteolysis of D-beta-hydroxybutyrate dehydrogenase, a lecithin-requiring enzyme.

D-beta-Hydroxybutyrate dehydrogenase is a lipid-requiring enzyme which is localized on the inner face of the mitochondrial inner membrane. The apodehydrogenase, i.e. the purified enzyme devoid of lipid, has been purified from beef heart mitochondria and as such is inactive. It can be reactivated by insertion into phospholipid vesicles containing lecithin. Proteolytic digestion with different proteases has been carried out to obtain insight into the orientation of the enzyme in the membrane and to assess the extent of immersion of the protein into the phospholipid bilayer. Digestion of the apodehydrogenase with either trypsin, chymotrypsin, Staphylococcus aureus protease, thermolysin, carboxypeptidases A and Y, or Pronase (from Streptomyces griseus) leads to loss of activity, as assayed with phospholipid. Limited digestion with carboxypeptidase results in complete inactivation. Of the proteases tested, only Pronase and chymotrypsin cleave and inactivate the enzyme inserted into phospholipid vesicles (enzyme-phospholipid complex). For the enzyme-phospholipid complex, the loss of activity with Pronase digestion follows a single exponential decay to less than 10% of the initial activity. With chymotrypsin digestion, the staining intensity of the original approximately 31,500-dalton polypeptide decreases more rapidly than the loss of enzymic activity. The enzyme-phospholipid complex, after limited cleavage with chymotrypsin, retains enzymic activity and resonance energy transfer from protein to bound NADH and an approximately 26,000-dalton polypeptide is observed. Phospholipid alters the cleavage pattern with both chymotrypsin and Pronase, and the rate of inactivation of the enzyme-phospholipid complex is slowed in the presence of NAD(H). Moreover, the rate of inactivation of the apodehydrogenase with chymotrypsin is diminished approximately 3-fold in the presence of NAD+. Digestion of submitochondrial vesicles with either trypsin, chymotrypsin, or Pronase rapidly inactivates D-beta-hydroxybutyrate dehydrogenase; the addition of NAD+ or NADH, together with dithiothreitol and increased salt (to 50 mM), decreases the rate of inactivation, and with trypsin, virtually eliminates inactivation.(ABSTRACT TRUNCATED AT 400 WORDS)

Animals↗
PubMed1985

Plasma membrane phospholipid organization in human erythrocytes.

By weight, phospholipids make up approximately 25% of the plasma membrane of mature human erythrocytes. The four major phospholipid species present in the membrane (PC, PE, PS, and SM) are distributed asymmetrically across the bilayer leaflet resulting in an enrichment of choline-phospholipid (PC and SM) in the outer leaflet and of amino-phospholipid (PE and PS) in the cytoplasmic leaflet. This asymmetric organization is preferentially maintained through complex, and at present, poorly understood noncovalent interactions between specific membrane lipids and proteins (in particular, a stabilizing role for the skeletal protein spectrin and band 4.1 have been implicated), although other considerations such as phospholipid net charge, size, and degree of acyl chain unsaturation may also be involved. In certain red cell pathologies, or following experimental manipulation, there is a partial loss of this asymmetry (summarized in Tables XVI, XVII) often resulting in increases in the outer leaflet content of amino-phospholipids and subsequent expression of altered membrane surface properties. Some of these abnormal properties may have pathophysiologic consequence; indeed, red cell membranes displaying increased levels of surface amino-phospholipids have been shown to be potent procoagulants and demonstrate enhanced intermembrane interactions with both model (liposomes) and biologic (mononuclear phagocytes) membranes. Redistribution of membrane phospholipids may not occur homogeneously throughout an entire leaflet but may be restricted to specific membrane regions. These studies strongly suggest that the maintenance of phospholipid asymmetry in human red cell membranes is not a trivial event but probably represents a homeostatic mechanism, the failure of which may lead to alterations in normal erythrocyte functions, and ultimately, survival.

Blood Coagulation↗
PubMed1984

Effects of insulin and protein synthesis inhibitors on phospholipid metabolism, diacylglycerol levels, and pyruvate dehydrogenase activity in BC3H-1 cultured myocytes.

BC3H-1 myocytes were cultured with 32PO4 for 3 days to label phospholipids to constant specific activity. Subsequent treatment with physiological concentrations of insulin provoked 40-70% increases in 32PO4 levels (reflecting increases in mass) in phosphatidic acid, phosphatidylinositol, and polyphosphoinositides, and, lesser, 20-25% increases in phosphatidylserine and the combined chromatographic area containing phosphatidylethanolamine plus phosphatidylcholine plus phosphatidylcholine. Insulin-induced increases in phospholipids were significant within 5 min and near-maximal at 15-30 min. Comparable rapid insulin-induced increases in [3H]phosphatidylinositol were observed in myocytes prelabeled with [3H]inositol. These insulin effects (as per prolonged pulse-chase experiments) were due to increase phospholipid synthesis rather than decreased phospholipid degradation. Cycloheximide (and puromycin) pretreatment prevented insulin-induced increases in phospholipids and rapidly reversed ongoing insulin effects on phospholipids and pyruvate dehydrogenase activity. Insulin also rapidly increased diacylglycerol levels. These findings suggest that: (a) insulin provokes rapid increases in de novo synthesis of phosphatidic acid and its derivatives, e.g. phosphoinositides and diacylglycerol; (b) protein synthesis inhibitors diminish phospholipid levels in insulin-treated (but not control) tissues by increasing phospholipid degradation (?phospholipase(s) activation); and (c) changes in phospholipids and diacylglycerol may be important for changes in pyruvate dehydrogenase and other enzymatic activities during treatment with insulin and/or protein synthesis inhibitors.

Animals↗
PubMed1984

[Absorption by rat fetal tissues of 14C-labeled phospholipids injected into the rat body in the late stages of pregnancy].

The authors studied the possibility of 14C-phospholipid transplacental penetration after 15C-phospholipid injection into rats at the 20th day of pregnancy. The preparation of 14C-phospholipids (total phospholipids) was isolated by thin-layer chromatography from the liver of rats injected with 2-14C-sodium acetate. One hour after its injection into the rat, 14C-phospholipids were detectable in total phospholipids of the pulmonary and cerebral fetal tissues. It was discovered that specific radioactivity of phospholipids contained by these tissues was 2--5 times higher when 14C-phospholipids were injected subcutaneously as compared with intramuscular injection. It is concluded that exogenous phospholipids entrapped in the mother's circulation penetrate the placental barrier of the fetus and the blood-brain barrier of the mature fetus, being consumed by different fetal tissues for forming membrane structures of the fetal tissues.

Animals↗
PubMed1984

Compositional and molecular species analysis of phospholipids by high performance liquid chromatography coupled with chemical ionization mass spectrometry.

High performance liquid chromatography (HPLC) was combined with chemical ionization mass spectrometry (CIMS) by the use of a moving-belt interface. The technique was employed for the analysis of naturally occurring phospholipids. Positive and negative ion mass spectra of various phospholipids such as phosphatidylcholine, phosphatidylethanolamine, phosphatidylserine, phosphatidylinositol, phosphatidylglycerol, and sphingomyelin were obtained in the chemical ionization mode with ammonia or methane as the reagent gas. Specific ions for individual phospholipid "bases" were identified. These ions were used in specific ion monitoring of the phospholipids during HPLC-CIMS. CIMS of each phospholipid also provided extensive information on the molecular species of the individual class of phospholipids. Relative abundance of different molecular species of each phospholipid as determined by CIMS agreed well with the results obtained by gas-liquid chromatography. Rat brain phospholipids were analyzed by HPLC-CIMS in about 15 minutes. Routinely, about 5 micrograms of individual phospholipid was analyzed by HPLC-CIMS, however, with specific ion monitoring the method provides a detection capability at the subnanogram level.

Animals↗
PubMedDate not supplied

[Phospholipid turnover of hepatic subcellular membranes in rats receiving diets with different fat content].

The renewal (turnover) of rat liver mitochondrial an microsomal phospholipids was studied by the pulse-labeled method. As the fat content in the diet was raised (to 9, 26 and 62%), the renewal of mitochondrial phospholipids increased. In the microsomes, the renewal of phospholipids exceeded the control fed both low-fatty and high-fatty diets. The feeding of rats with the low-fatty diet had a substantial effect on the half-life of mitochondrial and microsomal phospholipids, while the half-life of phospholipids labeled in the polar part with 14C-glycerin reduced and that of phospholipids labeled in the fatty acid part by 3H-palmitate considerably rose. It is assumed that under the conditions of inadequate supply of fatty acids with food, the reutilization of fatty acids formed during degradation of membranous phospholipids is activated. As the times of the feeding with experimental diets were raised (up to 60 days), the turnover of membranous phospholipids did not differ significantly from the data obtained in experiments carried out for 30 days. The exception to the rule was the half-life of acyl chains of mitochondrial phospholipids that continued increasing dramatically under 60 days of the low-fatty diet.

Animals↗
PubMedDate not supplied

[Fatty acid composition of the bilayer phospholipids in the photoreceptor membranes and aminophospholipids from the rhodopsin microenvironment of warm-blooded and cold-blooded vertebrates].

Studies have been made on the distribution of phospholipids between rhodopsin and free lipids of photoreceptor membranes of the outer segments of retinal rods from cattle, frog Rana temporaria and fish Teragra chalcogramma. comparative investigation of fatty acid composition of phospholipids from rhodopsin microboundary and lipid bilayer in photoreceptor membranes was made as well. Amino phospholipids from rhodopsin microboundry were revealed using glutaraldehyde. This reagent by means of its aldehyde groups links phospholipid amino groups with amino groups of proteins of photoreceptor membranes. After this treatment, free phospholipids of lipid bilayer were extracted from photoreceptor membranes by methanol-chloroform mixture. It was demonstrated that fatty acid composition of phospholipids of lipid bilayer differs from that of amino phospholipids from rhodopsin microboundary. In the animals investigated, fatty acids of phospholipids from lipid bilayer were found to be more unsaturated than fatty acids of amino phospholipids from rhodopsin microboundary. This difference was more pronounced in photoreceptor membranes from the frog and fish than from cattle.

Animals↗
PubMed1980

The action of human and rabbit serum phospholipase A2 on Escherichia coli phospholipids.

We have compared the properties of phospholipase A (E.C. 3.1.1.4) activity in whole human and rabbit serum toward the phospholipids of Escherichia coli. Using as substrate E. coli labeled during growth with either [1-(14)C]-palmitic acid or [1-(14)C]oleic acid, and then autoclaved to inactivate E. coli phospholipases and to render the labeled phospholipids accessible to exogenous phospholipases, we show that the deacylating activity in both human and rabbit serum is almost exclusively of the A(2) type. Rabbit serum is at least 20-fold more active than human serum. Activity in both sera is maximal at physiological Ca(2+) concentrations (2 mM) and is abolished by ethylenediaminetetraacetic acid. To examine hydrolysis of intact (unautoclaved) E. coli treated with 25% serum, use was made of a phospholipase A-deficient E. coli strain (E. coli S17), thereby eliminating the possible contribution of bacterial phospholipases to degradation. Human and rabbit serum are about equally bactericidal toward E. coli and cause comparable structural damage. However, only rabbit serum produces substantial hydrolysis of the phospholipids of intact E. coli S17. Heated (56 degrees C, 30 min) rabbit serum is non-bactericidal and retains phospholipase A(2) activity toward autoclaved, but not intact E. coli. The ability of heated serum to degrade phospholipids of intact E. coli S17 is restored, however, by adding 25% normal human serum, which is bactericidal. In this combination, doses of heated rabbit serum containing as much phospholipase A(2) activity (toward autoclaved E. coli) as is present in 25% unheated rabbit serum, produce roughly the same extent of hydrolysis of intact E. coli as does normal rabbit serum alone. Low doses with a phospholipase A(2) activity comparable to that of normal human serum elicit little or no hydrolysis. These findings indicate that hydrolysis of the phospholipids of intact E. coli S17 by serum occurs when: 1) the serum is bactericidal, and 2) when sufficient phospholipase A(2) is present. The difference in phospholipid hydrolysis that accompanies killing of E. coli by human or rabbit serum appears to reflect, therefore, the different amounts of phospholipase A(2) activity in the two sera. Phospholipid degradation is not required for the bactericidal action of serum. Bacterial phospholipid breakdown may be important, however, in the overall destruction and digestion of invading bacteria by the host.-Kaplan-Harris, L., J. Weiss, C. Mooney, S. Beckerdite-Quagliata, and P. Elsbach. The action of human and rabbit serum phospholipase A(2) on Escherichia coli phospholipids.

Animals↗
PubMed1981

[The clinical significance of phospholipids in lung pathology].

The phospholipid composition of 35 human lungs with pathological lesions was analysed by means of thin-layer chromatography and densitometric scanning. The pathological conditions studied were: bronchopneumonia, myocardial infarction, chronic heart failure, chronic obstructive airway disease and tuberculosis. The phospholipid composition was compared with that of a control group consisting of sudden death cases (due to unnatural causes), i.e. relatively normal lungs. The phospholipid composition of the lungs in a specific pathological group showed the same pattern. However, significant differences were observed between corresponding phospholipid fractions from lungs in the various pathological groups. Compared with the lipid fractions from relatively normal lungs, these differences were even more marked. From the results it would appear that the abnormal composition of the phospholipid fractions might possibly be a cause of lung pathology. The increase and/or decrease in individual fractions and abnormal ratios between fractions might indicate abnormalities in the biosynthesis and catabolism of the lung phospholipids. Further is necessary to elucidate the association of phospholipids with lung pathology. Phospholipid analysis of lung lavages and lung biopsies could be helpful in the diagnosis of lung diseases. Phospholipids in aerosol form could perhaps be used in treating certain lung disorders.

Bronchopneumonia↗
PubMed1981

Sedimentation of lung-derived phospholipid during low-speed centrifugation of amniotic fluid.

In most methods proposed for the assessment of fetal lung maturity, amniotic fluid is subjected to a preliminary low-speed centrifugation in an attempt to separate whole cells and cell debris from lung-derived surfactant phospholipid (lamellar body phospholipid). However, because lamellar body phospholipid is present in amniotic fluid in a membranous or particulate form, it is also partly sedimented by this procedure. The sedimentation of total phospholipid and lamellar body phospholipid by low-speed centrifugation has been quantitated for 49 samples of amniotic fluid from pregnancies of 30--41 weeks gestation. Isopycnic density-gradient centrifugation in a small air-driven ultracentrifuge was used to isolate lamellar body fractions from whole and centrifuged amniotic fluid. Centrifugation for 5 min at 1000 x g removed 0--70% of total phospholipid or lamellar body phospholipid, the mean values being 34 or 29%, respectively. There was an appreciable increase in lamellar body phospholipid relative to total phospholipid as a result of centrifugation in only 51% of the samples. In general, the effects of centrifugation were not related to gestational age of the fetus or the state of maturity of its lungs.

Amniotic Fluid↗
PubMed1980

The role of phospholipids and factor Va in the prothrombinase complex.

The kinetic parameters of the conversion of bovine prothrombin into thrombin by activated bovine blood clotting factor X (Xa) have been determined in the absence and presence of Ca2+, activated bovine factor V (Va) and phospholipid (dioleoylphosphatidylcholine/dioleoylphosphatidylserine, 1:1; mol/mol). In the absence of accessory components, the Km for prothrombin is 131 microM, which is well above its concentration in bovine plasma of about 1.5 microM. The Vmax of thrombin formation is 0.61 mol min-1 mol of Xa-1 under these conditions. In the presence of 7.5 microM phospholipid, the Km drops to 0.058 microM and the Vmax slightly increases to 2.25 mol min-1 mol of Xa. For the complete prothrombinase complex (Xa, Va, Ca2+, and 7.5 microM phospholipid), a Km for prothrombin of 0.21 microM and a Vmax of 1919 mol min-1 mol of Xa-1 is found. The Vmax of thrombin formation slightly increases when more phospholipid is present in our experiments and there is a considerable increase of the Km for prothrombin at higher phospholipid concentrations. Preliminary calculations show that the prothrombin density at the phospholipid surface at the Km is independent of the phospholipid concentration. This indicates that the Km measured in the presence of phospholipid has to be regarded as an apparent Km and the local prothrombin concentration determines the kinetics of activation. Prothrombin activation by prothrombinase complexes of different compositions was followed by gel electrophoresis in the presence of sodium dodecyl sulfate. Both in the absence and presence of phospholipid but without factor Va, prethrombin 2 is the main product formed during the initial stages of steady state prothrombin activation. In the presence of factor Va, thrombin is the main end product and minute amounts of prethrombin 2 are formed. This shift in the reaction pathway of prothrombin activation caused by factor Va will contribute to the observed increase of the Vmax measured in the presence of factor Va.

Animals↗
PubMed1994

Synthesis of phospholipids by human peritoneal mesothelial cells.

OBJECTIVE: To assess the capacity of cultured human peritoneal mesothelial cells to synthesize choline-containing phospholipids. The study compares the phospholipids secreted from cultured cells with those which we, and others, have identified in the dialysate of patients treated by continuous ambulatory peritoneal dialysis (CAPD). PATIENTS: CAPD effluent was collected from 8 patients who had been receiving CAPD treatment for at least 11 months and who had normal ultrafiltration. CELL CULTURES: Using human omental tissue, homogeneous cultures of mesothelial cells were established. METHODS: Synthesis of phospholipids by mesothelial cells was assessed following incubation with [methyl-14C] choline chloride--a precursor capable of being incorporated into phosphatidylcholine (PtdCho) and sphingomyelin. Lipids from CAPD effluent, cultured cells, and cell medium were extracted in chloroform/methanol. Phospholipids were separated and identified by thin layer chromatography. Synthesis and secretion of PtdCho and other choline-containing lipids by the mesothelial cells were determined by beta scintillation counting of the appropriate bands, while the fatty acid composition of the phospholipids was ascertained by gas liquid chromatography. RESULTS: Synthesis and secretion of PtdCho by mesothelial cells were observed during a 96-hour period. When maintained in medium replete with essential fatty acids, the fatty acid composition of the PtdCho synthesized by cultured mesothelial cells closely resembled that isolated from the peritoneal cavity. CONCLUSION: The demonstration of phospholipid secretion from mesothelial cells, with a fatty acid composition similar to the phospholipids isolated from peritoneal dialysate, lends added support to the hypothesis that the mesothelial cells are the source of the peritoneal phospholipids. As such they offer a useful experimental system in which to study peritoneal phospholipid synthesis.

Cells, Cultured↗
PubMed1996

Cholesterol-loading of membranes of normal erythrocytes inhibits phospholipid repair and arachidonoyl-CoA:1-palmitoyl-sn-glycero-3-phosphocholine acyl transferase. A model of spur cell anemia.

Spur cell anemia may occur in severe liver disease including alcoholic cirrhosis. Spur cell anemia red blood cells (RBCs) have a characteristic morphology, with irregular projections, an increased ratio of membrane cholesterol (Ch) to phospholipid, evidence of oxidative damage, and shortened survival resulting in hemolytic anemia. Normal RBCs may acquire many of the features of spur cells either by transfusion into a spur cell patient or in an in vitro model system that loads the RBC membrane with Ch relative to phospholipid by means of Ch-rich, phospholipid-Ch sonicates. We found evidence of abnormal phospholipid repair metabolism in spur cell anemia RBCs characterized by decreased arachidonate (Ar) uptake into phospholipids and by increased uptake into a fatty acid membrane repair intermediate, acylcarnitine (AcylCn). To study the possible modulation of phospholipid repair metabolism in spur cells by Ch-loading, we compared the Ar metabolism of RBCs loaded with Ch in vitro with that of control cells incubated in autologous serum. Ar, a polyunsaturated fatty acid, is especially sensitive to peroxidation and, thus, is likely to be involved in phospholipid repair. Ch-loading decreased the incorporation of [14C]Ar into total lipids (Ch-loaded, 1,113 +/- 48 pmol/10(10) RBCs; control, 1,525 +/- 48 pmol/10(10) RBCs) including phosphatidylethanolamine, phosphatidylserine, and phosphatidylcholine. Uptake of [14C]Ar into AcylCn increased (control AcylCn, 169 +/- 31 pmol/10(10) RBCs; Ch-loaded AcylCn, 196 +/- 35 pmol/10(10) RBCs; P = .0012). Thimerosal, an inhibitor of arachidonoyl- CoA:l-palmitoyl-sn-glycero-3-phosphocholine acyl transferase or lysophosphocholine acyl transferase (LAT), produced a similar pattern of metabolic abnormality, with decreased incorporation into phospholipid but relative increase into AcylCn. We assayed LAT in RBC membranes from Ch-loaded RBCs, using [14C]arachidonoyl CoA as precursor, and found similar decreased LAT activity at concentrations of 1-palmitoyllysophosphatidylcholine (LPC) from 1 to 30 micromol/L. Similar LAT assay results were obtained using [14C]palmitoyl LPC as the precursor. We conclude that Ch-loading of RBC membranes results in inhibition of LAT in the cell-free system in vitro and may account for the inhibited phospholipid repair in Ch-loaded intact RBCs in vitro and in spur cell anemia RBCs in vivo. Decreased ability to replace peroxidized membrane fatty acid by this metabolic pathway may contribute to the hemolytic process in spur cell anemia.

1-Acylglycerophosphocholine O-Acyltransferase↗
PubMed1996

Differential activity and lack of synergy of lung surfactant proteins SP-B and SP-C in interactions with phospholipids.

This study shows that the hydrophobic lung surfactant proteins (SP)-B and SP-C are not synergistic in enhancing functionally relevant surface behaviors in films and dispersions with phospholipids, and that SP-B is more effective than SP-C in facilitating surface activity. Purified bovine SP-B, SP. C, or SP-B/C (1/1 by wt) were combined with chroma tographically purified calf lung surfactant phospholipids (PPL), or with dipalmitoyl phosphatidylcholine (DPPC) or complex phospholipid mixtures containing 75% or 50% DPPC (75% SPL or 50% SPL). Adsorption was consistently better in corresponding mixtures of phospholipids plus SP-B versus SP-C, but was not improved further by substitution of SP-B/C for SP-B. Interfacial films of DPPC or SPL plus 1.3% SP-B or 1.3% SP-C had improved respreading compared to phospholipids alone (Wilhelmy balance, 23 degrees C and 37 degrees C), but substitution of mixed SP-B/C for either pure apoprotein did not increase respreading further. Surface-excess films of phospholipids plus SP-B had higher maximum surface pressures, or maintained a high maximum pressure through more consecutive compressions, than corresponding films with SP-C. Dispersions of phospholipids plus 1.3% SP-B or mixed (1/1) SP-B/C (2.6%) rapidly lowered surface tension to < 1 mN/m in oscillating bubble studies (20 cpm, 37 degrees C), while corresponding dispersions containing SP-C reduced surface tension more slowly or reached higher minima. Mixtures of 50% SPL with SP-B versus SP-C were also better able to resist inhibition by serum albumin in bubble and adsorption studies, and inhibition resistance was not significantly improved in mixtures containing 2.6% SP-B/C (1/1) versus 1.3% SP-B. The lack of synergy in hydrophobic apoprotein function, coupled with the greater effectiveness of SP-B in improving phospholipid adsorption, dynamic surface activity, and inhibition resistance, suggests that mixtures of phospholipids plus SP-B or related peptides may be particularly relevant its clinical exogenous surfactants.

1,2-Dipalmitoylphosphatidylcholine↗
PubMed1997

Lipid composition and phospholipid asymmetry of membranes from a Schwann cell line.

We report the total lipid composition and phospholipid asymmetry of a plasma membrane preparation isolated from a Schwann cell line (NF1T) derived from a human neurofibroma. The specific activities of three plasma membrane markers (5'-nucleotidase, Na-K-ATPase, and CNPase) were 8-fold, 12-fold, and 16-fold higher, respectively, in the plasma membrane fraction compared to the specific activities found in the total homogenate. The specific activities of the marker enzymes of intracellular membranes in the isolated plasma membrane fraction indicated little contamination with intracellular organelles. The enrichment of cholesterol (3-fold), sphingomyelin (3-fold), and glycolipids (cerebrosides 8-fold, sulfatides 5-fold) also indicated a high degree of purity of the plasma membrane fraction. The high content of phosphatidylinositol and phosphatidylcholine (10% and 44% of total phospholipid) and the low phosphatidylserine and phosphatidylethanolamine content (3% and 14% of the total phospholipid) were also characteristic of the plasma membrane fraction derived from this cell line. The transbilayer phospholipid distribution of the plasma membrane in intact cells and in the isolated plasma membrane fraction was investigated by using phospholipase A2 (bee venom) and sphingomyelinase (S. aureus). The phospholipid asymmetry of NF1T plasma membrane followed the general features of phospholipid asymmetry in eukaryotic cells: sphingomyelin and phosphatidylcholine were preferentially located in the outer leaflet (90% and 89%, respectively) while the aminophospholipids phosphatidylethanolamine, phosphatidylserine, and phosphatidylinositol were in the inner half of the membrane (85%, 96%, and 69%, respectively). A high percentage of the total plasma membrane phosphatidylinositol (31%) was found in the outer side of the membrane indicating a decreased asymmetric distribution for this negatively charged phospholipid. The phospholipid asymmetry found in the plasma membrane vesicle fraction corroborated the phospholipid asymmetry of the intact cells, thus confirming that the plasma membrane vesicles maintained the original orientation and lipid asymmetry after homogenization and/or sonication.

Biomarkers↗
PubMed1976

Phospholipid transfer between plasma and platelets in vitro.

Washed rabbit platelets were resuspended in plasma in which all of the major phospholipids had been isotopically labeled by injection of 32PO4 into rabbits. At certain time intervals during a 6-hr incubation at 37 degrees C, aliquots were removed from the incubation mixture and the platelets were isolated and subjected to lipid extraction and phospholipid analysis. A continuous rise in platelet non-lipid-bound and lipid-bound radioactivity was observed through-out the incubation period. Two platelet phospholipids, lecithin and lysolecithin, were significantly labeled, whereas little or no labeling of the other phospholipids was found. There was no detectable change in total or individual platelet phospholipid content. At 6 hr, 4% of total platelet phospholipid, 43% of platelet lysolecithin, and 7% of platelet lecithin were labeled. Platelets incubated in plasma from rabbits with diet-induced hyperlipidemia took up and incorporated significantly more label into their phospholipids than did platelets in normal plasma. Labeling of both platelet lysolecithin and lecithin could be due to uptake and metabolism of plasma lysolecithin by platelets. However, labeling of platelet lecithin could at least in part be the result of direct exchange of this phospholipid with the plasma. Uptake and incorporation of endogenous plasma lysolecithin by platelets and, possibly, direct exchanged of platelet lecithin may be important mechanisms in the modification by plasma lipids of platelet membrane phospholipid fatty acid composition and platelet function.

Animals↗
PubMed1976

Role of gamma-carboxyglutamic acid. An unusual protein transition required for the calcium-dependent binding of prothrombin to phospholipid.

A first order calcium-dependent transition can be monitored by a decrease in the intrinsic fluorescence of the isolated "pro" (Fragment1) region of prothrombin. The maximum fluorescence change is -40% for Fragment 1, and only about -6% for whole prothrombin. The most remarkable features of this transition are its rate and activation energy. The half-life for the transition at 0 degrees is about 100 min, and the temperature dependence shows an activation energy of 21 kcal/mol. The rate constant for the forward reaction is zero order in calcium and is not affected by the presence of phospholipid membranes. The equilibrium for the transition, however, is affected by phospholipid. At 30 degrees, [Ca]eq (the calcium concentration where half of the protein has undergone the transition) is 0.4 mM and the Hill coefficient is 2.6. Under the same conditions but in the presence of phospholipid [Ca]eq is 0.24 mM and the Hill coefficient is about 4.5. The transition is triggered by binding 3 or 4 calcium ions. The rate of Fragment 1 binding to phospholipid vesicles was tested using gel filtration techniques at 0 degrees. The rate constants, activation energy, and [Ca]eq values for this process were shown to correspond to the properties of the fluorescence change. The rate constants, activation energy, and Hill coefficients for binding of whole prothrombin to phospholipid correspond to the same parameters for Fragment 1 but the [Ca]eq values are lower. At 0 degrees, the [Ca]eq is 0.19 mM for the prothrombin transition and 0.1 mM for the transition in the presence of phospholipid. These results demonstrate that Fragment 1 and prothrombin undergo a transition when exposed to calcium ions which necessarily precedes protein-phospholipid interactions. In addition to its role in determining the correct protein structure, calcium plays a second role in prothrombin-phos-pholipid interaction which is in the actual formation of the protein-phospholipid bond. The [Ca]eq for binding protein (after its transition) to phospholipid is about 0.06 mM.

Binding Sites↗
PubMed1998

The interference effects of hexadecylphosphocholine on proliferation and membrane phospholipid metabolism in human myeloid leukemia cell lines.

Membrane phospholipids are important regulators of cellular function. The phospholipid activities, such as lipid composition and transportation, contribute to cellular homeostasis in the lifespan of cells. Alterations in phospholipids result in the movement of bilayer lipids and the initiation of coagulation, recognition and internalization. Hexadecylphosphocholine (HePC) exerts antitumor potencies and represents a new class of antitumor agents targeted to the cellular membrane. Human myeloid leukemia cell lines HL-60 and K562 employed in this study were inhibited by HePC in vitro. The results indicate that the HL-60 cell line was sensitive, while K562 was resistant to HePC. Synthetic HePC is an alkyllysophospholipid analog which interacted with the cell membrane, thereby altering lipid composition and metabolism of membrane phospholipids and modulating intracellular calcium in human myeloid leukemia HL-60 and K562 cell lines. The contents of membrane phospholipids, including phosphatidylinositol (PI), phosphatidylcholine (PC), phosphatidylserine (PS) and phosphatidylethanolamine (PE), were determined quantitatively with high performance liquid chromatography. The sensitivity of myeloid leukemia HL-60 and K562 cell lines to HePC probably depends on the different distribution of these four phospholipids in the cellular membrane, or on the response of these phospholipids to HePC. The cytosolic free calcium ([Ca++]i) concentration increased by HePC confirmed that [Ca++]i was released from the intracellular calcium pool and is associated with cell differentiation and apoptosis. We investigated the hypothesis that the antiproliferative effect of HePC was mediated through the interference with cellular membrane phospholipids, including choline-containing phospholipids (PC), aminophospholipids (PE and PS) and PI, in eukaryotic cells.

Antineoplastic Agents↗