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PubMed1986

Site-site interaction in the phospholipid activation of D-beta-hydroxybutyrate dehydrogenase.

D-beta-Hydroxybutyrate dehydrogenase is a lipid-requiring enzyme with absolute specificity for phosphatidylcholine (PC). The enzyme devoid of lipid, the apodehydrogenase, inserts spontaneously into phospholipid vesicles where it exists as a tetramer. We now find the lipid activation to be limited by the mole fraction of PC in the total phospholipid. These studies suggest that the concentration of the enzyme-PC complex, which is essential for enzymic activity, becomes diffusion limited at lower PC concentration. The lipid activation and the tryptophan fluorescence of purified D-beta-hydroxybutyrate dehydrogenase were studied in the presence of a constant "bilayer background" of approximately 100 nonactivating phospholipid molecules/enzyme monomer. Activation by PC was half-maximal at 20 PC molecules/enzyme monomer. This value was doubled when the amount of "background" phospholipid was doubled. Activation proceeded with positive cooperativity having a Hill coefficient of approximately 2.4. These data indicate interactions between at least three PC-binding sites. The quenching of tryptophan fluorescence by the phospholipid activator, 1-palmitoyl-2-(1-pyrenyl)-decanoyl-PC (2-pyrenyl-PC), gives a saturation curve with half-maximal quenching of 6 quencher molecules/enzyme monomer. This value is equivalent to an apparent phospholipid-protein dissociation constant in the two-dimensional membrane and corresponds to approximately 6 mol % of total phospholipid. In distinct contrast to the phospholipid activation curve, the fluorescence quenching saturation curve was hyperbolic and there was no specificity for PC. The fluorescence quenching by 2-pyrenyl-PC could be diminished by using a several-fold excess of PC or other phospholipids so as to reduce the mole fraction of quencher in the bilayer. It would appear that formation of enzyme-PC complex is a dynamic process consisting of at least two discernible steps: 1) a primary interaction, as measured by tryptophan quenching, which is hyperbolic and not specific for lecithin. This interaction is independent from and precedes 2) phospholipid activation of D-beta-hydroxybutyrate dehydrogenase, which is cooperative in nature and specific for lecithin.

Binding Sites↗
PubMed1986

Phospholipid transfer proteins: mechanism of action.

Phospholipid transfer proteins are generally localized in the cytosolic fraction of cells and are capable of catalyzing the flux of phospholipid molecules among membranes. Artificial membranes also participate in protein-catalyzed phospholipid movements. In this review the major phospholipid transfer proteins are discussed with respect to their phospholipid substrate specificity and the contributions of membrane physical properties to this process. The phenomenon of net transfer of phospholipids is described. The use of various kinetic approaches to the study of these catalysts is reviewed. A detailed consideration of the distinct phospholipid binding and membrane interaction domains of one phospholipid transfer protein is presented. Finally, some recent applications of phospholipid transfer proteins to the examination of membrane structure and function and further directions for the continued research activity with this class of proteins are summarized.

Animals↗
PubMed1997

Transbilayer movement of fluorescent phospholipids in Bacillus megaterium membrane vesicles.

We investigated the transbilayer movement or flip-flop of phospholipids in vesicles derived from the cytoplasmic membrane of Bacillus megaterium. Since common assay techniques were found to be inapplicable to the Bacillus system, we exploited and elaborated a newly described method in which fluorescent phospholipids (1-myristoyl-2-C6-NBD phospholipids) are used as tracers to monitor flip-flop. These lipids were introduced into Bacillus vesicles from synthetic donor vesicles containing a fluorescence quencher. Transport was measured by monitoring the increase in fluorescence as the tracers departed the quenched environment of the donor vesicle and entered first the outer membrane leaflet and subsequently the inner leaflet of Bacillus vesicles. Independent experiments involving cobalt quenching of NBD fluorescence provided results consistent with the existence of pools of fluorescent phospholipid in the outer and inner leaflets of Bacillus vesicles at the completion of transport. Using the assay we show that phospholipid flip-flop in Bacillus vesicles occurs rapidly (half-time approximately 30 s at 37 degrees C) with no preference for a particular phospholipid headgroup and that it is sensitive to proteolysis. We also establish that flip-flop does not occur in synthetic phospholipid vesicles or vesicles made from Bacillus phospholipids. We conclude that Bacillus vesicles possess the ability to promote rapid transbilayer movement of phospholipids, and that the transport is probably protein (flippase)-mediated.

4-Chloro-7-nitrobenzofurazan↗
PubMedDate not supplied

Developmental changes in phosphatidylinositol transfer protein concentration and phospholipid transfer activities in rat type II cells.

The phospholipid transfer proteins (PLTPs) are cytosolic proteins that have been characterized by their ability to facilitate the transfer of phospholipids between membranes in vitro. The goals of this study were to determine whether PITP alpha concentration and phospholipid transfer activities are enriched in type II cells compared with whole lung and to determine the developmental changes in PITP alpha concentration and phospholipid transfer activities during late gestation and newborn period. The concentration of PITP alpha in type II cell cytosol measured by enzyme-linked immunosorbent assay (ELISA) increased during late fetal gestation to 2.2-fold adult levels and declined 41% during the first postnatal day. However, compared to whole adult lung cytosol, type II cell cytosol was not significantly enriched with PITP alpha. Phospholipid transfer activities were determined by a vesicle-rat lung membrane transfer assay. In adult lung, transfer activities for all the phospholipids were enriched in adult type II cell cytosol compared to whole lung cytosol (phosphatidylglycerol [PG], 12.5-fold; phosphatidylinositol [PI], 9.2-fold; phosphatidylcholine [PC], 6.5-fold; and phosphatidylethanolamine [PE], 6.6-fold; P < .05 in each case). The rate of phospholipid transfer in type II cell cytosol increased during late fetal gestation to levels 4.9 (PG), 3.7 (PI), and 2.8 (PC) times greater than adult levels. In cytosol from cells from different stages, the order of transfer rate was PG > PI > PC > PE. PITP alpha immunodepletion of adult type II cytosol did not significantly affect phospholipid transfer activities, suggesting that other PLTPs are responsible for the majority of the observed transfer activities in these cells. Developmental increases in PITP alpha concentration and other PLTPs parallel developmental changes in type II cell surfactant phospholipid metabolism, suggesting a possible role of these transfer proteins in the unique function of the type II cell.

Animals↗
PubMed1999

Flow cytometric detection of transbilayer movement of fluorescent phospholipid analogues across the boar sperm plasma membrane: elimination of labeling artifacts.

Reliable protocols were established for investigating asymmetric distributions of 6-(7-nitrobenz-2-oxa-1,3-diazol-4-yl)amino-caproyl (C6NBD) phospholipids in the plasma membrane of boar sperm cells under physiological conditions. A method based on fluorescence resonance energy transfer was used to ensure that incorporation of the fluorescent phospholipids into the sperm proceeded via monomeric transfer. The total amount of incorporated phospholipid fluorescence and the proportion of translocated phospholipid fluorescence were determined by flow cytometric analysis before, and after, dithionite destruction of outer leaflet fluorescence. Catabolism of incorporated fluorescent phospholipids was blocked with phenylmethylsulfonyl fluoride. Membrane-damaged cells were detected with impermeant DNA stains, thereby enabling their exclusion from subsequent analyses of the flow cytometric data, whence it could be demonstrated that the labeled phospholipids were incorporated only via the outer plasma membrane leaflet in living sperm cells. Phospholipid uptake and internalization was followed at 38 degrees C. After 1 hr of labeling, about 96% of the incorporated C6NBD-phosphatidylserine, 80% of C6NBD-phosphatidylethanolamine, 18% of C6NBD-phosphatidylcholine, and 4% of C6NBD-sphingomyelin were found to have moved across the plasma membrane bilayer to the interior of the spermatozoa. These inward movements of fluorescent phospholipids were ATP-dependent and could be blocked with sulfhydryl reagents. Movements from the inner to the outer leaflet of the sperm plasma membrane were minimal for intact fluorescent phospholipids, but were rapid and ATP-independent for fluorescent lipid metabolites. The described method enables, for the first time, assessment of changes in lipid asymmetry under fertilizing conditions.

4-Chloro-7-nitrobenzofurazan↗
PubMed1996

Tissue phospholipids during human pregnancy by 31P NMR: myometrium, decidua, placenta and fetal membranes.

The biophysical environment formed by phospholipids, rather than the amount of functional proteins, can be rate limiting for factors controlling myometrial contractility and pregnancy maintenance. We therefore studied myometrial, decidual, placental and fetal membrane phospholipids using the 31P NMR spectrum. This enabled us to identify bulk phospholipids over 0.05 mmol/kg. The method was checked for reliability for the reproductive tissues studied. The chemical shift of phospholipid standards was slightly different according to whether a single compound or a mixture was analyzed. The bulk phospholipids found were phosphatidylcholine (PC), phosphatidylethanolamine, sphingomyelin (SM) and phosphatidylinositol. The ratio PC/SM decreased during pregnancy in the decidua, placenta and fetal membranes, but not in the myometrium. Pregnancy did not induce significant changes in the total myometrial phospholipids. Their composition was stable even during clinical labor. The fetal tissues, placenta and fetal membranes contained about twice as much phospholipid as the maternal tissues, myometrium and decidua. There was no sign of lysocompounds, cardiolipin or phosphatidic acid. This supports the view that the extraction and analyzing techniques used earlier probably created artefacts. The increased fluidity of the myometrial and placental phospholipids during pregnancy may depend on factors other than the composition of phospholipids.

Decidua↗
PubMed1992

Transfer of phospholipids from fat body to lipophorin in Rhodnius prolixus.

32P-Labeled fat bodies (32P-fat bodies) of Rhodnius prolixus females were incubated in the presence of non radioactive purified lipophorin and the release of radioactivity to the medium was analysed to answer the question of whether lipophorin is a reusable shuttle for phospholipids. The radioactivity found in the medium was associated with lipophorin phospholipids. When the 32P-fat bodies were incubated in the absence of lipophorin, only a small amount of radioactivity was released and it was not associated with lipophorin, indicating that there was no release of pre-labeled 32P-lipophorin by the tissue. Analysis of 32P-phospholipids transferred from fat bodies to the lipophorin particles by thin-layer chromatography revealed a predominance of phosphatidylethanolamine and phosphatidylcholine, with minor amounts of phosphatidylserine, phosphatidylinositol, and sphingomyelin. The transfer of phospholipids to lipophorin was linear with time up to 45 min and the process was inhibited at low temperature and by the metabolic inhibitors azide and fluoride. The transfer of phospholipids from the fat bodies to lipophorin was saturable with respect to the concentration of lipophorin, which was half-maximal at about 8 mg/ml. A directional movement of phospholipids from the fat body to lipophorin was observed. The net gain of phospholipids in 2 h of incubation with fat body was 8.54 nmol per insect, which corresponds to 6.69% of increase in the lipophorin phospholipid content. The rate of 32P-phospholipid transfer from fat body to lipophorin particles varied during the days after a blood meal increasing up to day 10 and then decreasing in parallel with the process of oogenesis.

Animals↗
PubMed1995

Loading of lipophorin particles with phospholipids at the midgut of Rhodnius prolixus.

32P-Labelled midguts (32P-midguts) of Rhodnius prolixus females were incubated in the presence of nonradioactive purified lipophorin and the release of radioactivity to the medium was analysed. The radioactivity found in the medium was associated with lipophorin phospholipids. When the 32P-midguts were incubated in the absence of lipophorin, no 32P-phospholipids were found in the medium. Comparative analysis by thin-layer chromatography of 32P-phospholipids derived from metabolically labelled 32P-midgut or lipophorin particles after incubation with 32P-midgut showed some differences, revealing a possible selectivity in the process of phospholipids transfer. The transfer of phospholipids to lipophorin was linear with time up to 45 min, was saturable with respect to the concentration of lipophorin, and was half-maximal at about 5 mg/ml. The binding of 32P-lipophorin to the midgut at 0 degrees C reached the equilibrium at about 1 h of incubation. The binding of 32P-lipophorin was inhibited by an excess of nonradioactive lipophorin, which suggests a specific receptor for lipophorin. The capacity of midguts and fat bodies to transfer phospholipids to lipophorin varied during the days following the meal. When lipophorin enzymatically depleted of phospholipids by treatment with phospholipase A2 was incubated with 32P-midguts, the same amount of phospholipids was transferred, indicating a net gain of phospholipids by the particle.

Animals↗
PubMed1988

Phospholipid profile of the human brain: 31P NMR spectroscopic study.

Phosphatidylcholine, phosphatidylethanolamine, phosphatidylserine, phosphatidylinositol, sphingomyelin, and ethanolamine plasmalogen represent the six most abundant phospholipids of brain cell membrane. The ratio of the phospholipid contents (phospholipid profile) of the brain is remarkably consistent under various metabolic conditions and alteration of the phospholipid profile is believed to reflect changes in the membrane system. We describe here a simple but sensitive method to analyze the phospholipid profile of the human brain utilizing the acidified chloroform-methanol lipid extraction method of Folch et al. and 31P nuclear magnetic resonance (NMR) spectroscopy. Unique regional phospholipid profiles were consistently obtained. Although the large chemical-shift anisotropy of the 31P confined to rigid structures such as the cell membrane precludes direct observation of phospholipid profiles in living tissue, a reflection of the membrane phospholipid profiles can nevertheless be obtained by studying "NMR visible" water soluble intermediate metabolites of membrane phospholipids in vivo.

Adult↗
PubMed1986

Maintenance of epithelial surface membrane lipid polarity: a role for differing phospholipid translocation rates.

Large differences in lipid composition of apical and basolateral membranes from epithelial cells exist. To determine the responsible mechanism(s), rat renal cortical brush border and basolateral membrane phospholipids were labeled using 32P and either [3H]-glycerol or [2-3H] acetate for incorporation and degradation studies, respectively. Brush border and basolateral membrane fractions were isolated simultaneously from the same cortical homogenate. Different phospholipid classes were degraded at variable rates with phosphatidylcholine having the fastest decay rate. Decay rates for individual phospholipid classes were, however, similar in both brush border and basolateral membrane fractions. In phospholipid incorporation studies, again, large variations existed between individual phospholipid classes with phosphatidylcholine and phosphatidylinositol showing the most rapid rates of incorporation. Sphingomyelin and phosphatidylserine showed extremely slow incorporation rates and did not enter into the isotopic decay phase for 48 hr. In contrast to degradation studies, however, the same phospholipid class labeled the two surface membrane domains at highly variable rates. The difference in these rates, with the exception of phosphatidylinositol, were identical to the differences in phospholipid compositions between the two membranes. For example, phosphatidylcholine was incorporated into the basolateral membrane 2.5 X faster than into the brush border membrane and its relative composition was 2.5 X greater in the basolateral membrane. The opposite was true for sphingomyelin. These results indicate incorporation and not degradation rates of individual phospholipids play a major role in regulating the differing phospholipid composition of brush border and basolateral membranes.

Animals↗
PubMed1981

Phospholipid synthesis in human embryo fibroblasts infected with herpes simplex virus type 2.

The effect of herpes simplex virus type 2 infection on the synthesis of phospholipids in human embryo fibroblasts was determined at temperatures permissive (35 C) or nonpermissive (42 C) for virus replication. Incorporation of [32P]i was decreased by herpes simplex virus type 2 infection after 6 hr, which corresponds to the time of initiation of progeny virus production. No differences were observed in the relative incorporation of [32P]i phospholipid classes. In another series of experiments cells were labeled with [3H]ethanolamine before infection and with [14C]ethanolamine after infection. The incorporation of [14C]ethanolamine was also decreased after 6 hr of infection. When choline was substituted for ethanolamine, a similar, although less pronounced, decrease in incorporation was seen in infected cells compared to mock-infected cells. During abortive infections at 42 C, incorporation of [3H]thymidine into cellular DNA was stimulated, but the incorporation of phospholipid precursors was decreased. Total phospholipid composition and phospholipid acyl group composition were not changed appreciably during abortive or productive infection, regardless of whether the cells were labeled before or after infection. In conclusion, these data indicated that, during herpes simplex virus type 2 infection, the incorporation of lipid precursors into phospholipid was decreased. The stimulation of cellular DNA synthesis previously observed during abortive infection at 42 C was not paralleled by a detectable stimulation of total phospholipid synthesis. Neither productive nor abortive infection resulted in significant phospholipid compositional changes in the host cell; however, both resulted in a marked inhibition of phospholipid synthesis.

Cells, Cultured↗
PubMed1989

Effect of aluminum inhalation on alveolar phospholipid profiles in experimental silicosis.

In the sheep tracheal lobe model of silicosis, we have recently reported that total phospholipid, lecithin, and phosphatidylglycerol levels were elevated in lung lavage. To investigate further this observation, we obtained complete phospholipid profiles of lung lavage in 10 sheep exposed to saline only (Sa group), 10 sheep exposed to aluminum lactate inhalation only (Al group), 10 sheep exposed to 100 mg Minusil-5 in saline followed by monthly saline inhalation (Si group), and 10 sheep exposed to 100 mg Minusil-5 in saline followed by monthly aluminum lactate inhalation (Si-Al group). The following phospholipid components were measured: total phospholipids, phosphatidylglycerol (PG), phosphatidylethanolamine (PE), phosphatidylinositol (PI), phosphatidylcholine, disaturated phosphatidylcholine, sphingomyelin, and lyso-phosphatidylcholine. All values were comparable in the Sa group, Al group, and Si-Al group. In the Si group, there was a significant increase in total phospholipid to approximately 200% of the control values. The phospholipid profile of this group demonstrated an increase in all of the phospholipid components with some enrichment of the fraction of PG, PE, and PI. We concluded that lung exposure to silica dust significantly increases the concentration of phospholipids in the alveoli. This increase is of a large spectrum of alveolar phospholipids and is completely suppressed by aluminum lactate inhalation.

Administration, Inhalation↗
PubMed1999

Dietary phospholipid alters biliary lipid composition in formula-fed piglets.

Plasma cholesterol, arachidonic acid (AA, 20:4n-6), and docosahexaenoic acid (DHA, 22:6n-3) are higher in breast-fed infants than in infants fed formula without cholesterol, AA, or DHA. This study investigated differences in plasma, hepatic, and bile lipids and phospholipid fatty acids, and expression of hepatic proteins involved in sterol metabolism that result from feeding formula with cholesterol with egg phospholipid to provide AA and DHA. For this study, three groups of piglets were evaluated: piglets fed formula with 0.65 mmol/L cholesterol, the same formula with 0.8% AA and 0.2% DHA from egg phospholipid, and piglets fed sow milk. Piglets fed the formula with phospholipid AA and DHA had higher plasma high density lipoprotein, but not apoprotein (apo) B cholesterol or triglyceride; higher bile acid and phospholipid concentrations in bile; and higher liver and bile phospholipid AA and DHA than piglets fed formula without AA and DHA (P < 0.05). Hydroxy methylglutaryl (HMG)-CoA reductase and 7-alpha-hydroxylase, the rate-limiting enzymes of cholesterol and bile acid synthesis, respectively, and low density lipoprotein receptor mRNA levels were not different between piglets fed formula without and with phospholipid AA and DHA, but HMG-CoA reductase and 7alpha-hydroxylase mRNA were higher, and plasma apo B containing lipoprotein cholesterol was lower in all piglets fed formula than in piglets fed milk. These studies show that supplementing formula with AA and DHA from egg phospholipid alters bile metabolism by increasing the bile AA and DHA, and bile acid and phospholipid.

Animals↗
PubMed1975

Effects of local anesthetics on membrane properties. I. Changes in the fluidity of phospholipid bilayers.

The effect of the local anesthetic dibucaine on the solid to liquid-crystalline phase transition in phospholipid vesicles was studied by calorimetry and fluorescence polarization. The partition coefficient (greater than 3000) of dibucaine in the membranes of vesicles prepared from acidic phospholipids was more than 20 times higher than in neutral phospholipid membranes under the same conditions. Calorimetric measurements on vesicles prepared form acidic phospholipids (bovine brain phosphatidylserine; dipalmitoylphosphatidylglycerol) showed that dibucaine (1 with 10(-4) M) produced a significant reduction in the gel-liquid crystalline transition temperature (Tc). This fluidizing effect of dibucaine on acidic phospholipid membranes was even more marked in the presence of Ca2+. In contrast, dibucaine at the same concentration did not alter the Tc of neutral phospholipids (dipalmitoylphosphatidylcholine). Significant increase in the fluidity of neutral phospholipid membranes occurred only at higher dibucaine concentrations (2 with 10(-3) M). Measurements of the fluorescence polarization and lifetime of the probe, 1,6-diphenylhexatriene, in acidic phospholipid vesicles revealed that dibucaine (1 with 10(-4) M) caused an increase in the probe rotation rate indicating an increase in the fluidity of the phospholipid membranes. A good correlation was obtained between fluorescence polarization data on dibucaine-induced changes in membrane fluidity and calorimetric measurements on vesicles of the same type.

Anesthetics, Local↗
PubMed1981

The role of phospholipids in the modulation of enzyme activities in the chromaffin granule membrane.

(1) 93% of protein of chromaffin granule membranes can be solubilized by 1.3% (w/v) sodium cholate. The solubilized material can be substantially delipidated by ammonium sulphate precipitation. After three such cycles less than 2% of the endogenous phospholipids remain. (2) The chromaffin granule membrane Mg2+-ATPase depends on the presence of phospholipids for retention of its full activity. Soybean and extracted chromaffin granule phospholipids fully reactivate the delipidated enzyme provided only one delipidation step is used. (3) Successive ammonium sulphate precipitation steps result in a delipidated, and deactivated ATPase preparation which can be only partially reactivated on re-addition of phospholipids. (4) The phospholipid specificity for reactivation of the Mg2+-ATPase is broad. Although acidic phospholipids allow higher activities than neutral phospholipids, the main requirement appears to be the hydrophobic environment provided by the phospholipid hydrocarbon chains. (5) Correlations between changes in slope in the Arrhenius plot of the Mg2+-ATPase, and phase transitions in the phospholipid used for reactivation suggest that the 'fluidity' of the hydrocarbon chains modulates the activity of the enzyme.

Adenosine Triphosphatases↗
PubMed1995

Antigenicity of hepatitis B surface antigen proteins reconstituted with phospholipids.

Hepatitis B surface antigen (HBsAg) has been reconstituted with different phospholipid classes. All epitopes defined by a panel of monoclonal antibodies which recognize both group- and subtype-specific antigenic determinants showed specificity for acidic phospholipids. Electrostatic interactions between HBsAg proteins and acidic phospholipids are partly responsible for the complete recovery of the antigenic properties. In addition to the nature of the polar head group, the fatty acid composition of the phospholipid also influenced the recovery of the antigenic activity. Negatively charged phospholipids must bear at least one unsaturated fatty acid in order to be effective in recovering full antigenic activity of HBsAg. The results reported herein support the conclusion that the antigenic activity is dependent on the physical state of the phospholipid moiety. The appropriate membrane fluidity is required for optimum conformation but, once this conformation is established, additional interactions imparted by the various phospholipids give a difference in the patterns of antigenicity. The analysis of binding of the monoclonal antibodies allowed the classification of the epitopes into two groups according to their dependence on the lipid moiety. Of all the antigenic determinants only those close to the lipid-protein interface would change upon direct interaction with the phospholipids. The rest would depend on the correct protein conformation determined by the appropriate phospholipid composition.

Antibodies, Monoclonal↗
PubMed1976

Interaction of glutathione-insulin transhydrogenase (disulfide interchange enzyme) with phospholipids.

The effect of the addition of several phospholipids (lysophosphatidylcholine, alpha-lecithin, phosphatidylserine, phosphatidylethanolamine, lysophosphatidyl-ethanolamine, sphingomyelin, and disphosphatidylglycerol and phosphatidic acid) and related compounds (glycerophosphocholine, alpha- and beta-glycerophosphate, choline, serine, glycerol, dipalmitoylglycerol, and stearic acid) on the ability of purified (from beef pancreas) and microsomal (rat liver) glutathione-insulin transhydrogenase (Glutathione:protein-disulphide oxidoreductase, EC 1.8.4.2) to degrade insulin has been examined. With purified enzyme, except for phosphatidic acid and phosphatidylserine, all other phospholipids tested caused a slight activation at low concentration with phosphatidylethanolamine causing the highest activation. Lysophosphatidylcholine and phosphatidic acid are the only agents which cause inhibition of activity. The reaction rate as a function of concentration of inhibitor is hyperbolic for phosphatidic acid ([I]0.5 = 25 muM) and biphasic for lysophosphatidylcholine ([I]0.5 = 270 muM). Kinetic studies show that the two phospholipids are noncompetitive versus both substrates (insulin and GSH). Further, the structures of the phospholipids are quite different from the substrates and products of the reaction catalyzed by the enzyme. These dats, together with the data obtained with microsomes (see below), support the possibility that phospholipids, in particular lysolecithin and phosphatidic acid, might function by an interaction at an allosteric site or sites to bring about a conformational change in the enzyme. With a microsomal fraction, four phospholipids (lysophosphatidylcholine, lysophosphatidylethanolamine, phosphatidylethanolamine, and phosphatidic acid) caused an increase in GSH-insulin transhydrogenase activity. At low concentration the addition of each of these phospholipids led to a 2.5-fold increase in GSH-insulin transhydrogenase activity. At higher concentration, lysophosphatidylcholine almost totally inhibited the microsomal GSH-insulin transhydrogenase activity, as it did with purified enzyme, while phosphatidic acid showed only a slight inhibition, in contrast to its effect on purified enzyme. With the microsomal fraction in which GSH-insulin transhydrogenase activity had been previously unmasked by Triton X-100 treatment, the addition of small amounts of lysophosphatidylcholine and phosphatidic acid produced, as expected, only slight increase in the transhydrogenase activity for both phospholipids; again, only lysophosphatidylcholine but not phosphatidic acid caused inhibition when higher levels were used. It is concluded that the four phospholipids and Triton X-100 increase the GSH-insulin transhydrogenase activity in the microsomes by unmasking the catalytic site without fully unmasking the allosteric site, the point of reaction with the phosphatidic acid.

Animals↗
PubMed1984

Studies on ether phospholipids. I. A new method of determination using phospholipase A1 from guinea pig pancreas: application to Krebs II ascites cells.

A new method for ether phospholipid analysis has been devised, based on the selective destruction of diacyl phospholipids by guinea pig phospholipase A1 and of plasmalogens by acidolysis. The paper describes optimal conditions allowing a specific degradation of diacyl phospholipids by the enzyme(s). This requires the incubation of a total lipid extract in the presence of 2.4 mM sodium deoxycholate, at pH 8.0, at a temperature of 42 degrees C. As shown with various radioactive markers, all the diacyl phospholipids become degraded, whereas sphingomyelin and ether phospholipids remain refractory to phospholipase A1 attack. Phospholipids are then separated by a bidimensional thin-layer chromatography involving the exposure of the plates to HCl fumes between the two runs, in order to hydrolyse plasmalogens. Selectivity of both hydrolytic procedures is further demonstrated upon analysis of acetyl diacylglycerol derived from phospholipids. Various phospholipids can thus be determined by phosphorus measurement using sphingomyelin as an internal standard. By this way, it is shown that Krebs II cells present a very high content of ether phospholipid species (around 25% of total). Among these, about 50% are alkyl forms in ethanolamine phosphoglycerides, whereas this value reaches 70% in choline phosphoglycerides.

Animals↗