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PubMed1985

The side-chain cleavage of cholesterol sulfate--II. The effect of phospholipids on the oxidation of the sterol sulfate by inner mitochondrial membranes and by a reconstituted cholesterol desmolase system.

This study compares the side-chain cleavage of aqueous suspensions of cholesterol sulfate with the side-chain cleavage of cholesterol sulfate which is incorporated into phospholipid vesicles. Three different cholesterol desmolase systems are examined: the membrane-bound cholesterol side-chain cleavage system present in inner mitochondrial membranes isolated from bovine adrenal mitochondria; a soluble, lipid-depleted, reconstituted side-chain cleavage system prepared from cytochrome P-450scc, adrenodoxin and adrenodoxin reductase; a membrane associated side-chain cleavage system prepared by adding phospholipid vesicles, prepared from adrenal mitochondrial, to the reconstituted system. Soluble cholesterol sulfate, in low concentration, is a good substrate for the lipid-depleted reconstituted side chain cleavage system. However, at concentrations above 2 microM, in the absence of phospholipids, the sterol sulfate appears to bind at a non-productive site on cytochrome P-450scc which leads to substrate inhibition. Phospholipids, while inhibiting the binding of cholesterol sulfate to the cytochrome, also appear to prevent non-productive binding of the sterol sulfate to the cytochrome. Thus the addition of phospholipids to the lipid-depleted enzyme system leads to an activation of side-chain cleavage of high concentrations of the sterol sulfate. Soluble cholesterol sulfate is a good substrate for both the native and reconstituted membrane-bound systems and no substrate inhibition is observed when the membrane bound enzyme systems are employed in the assay of side-chain activity. However, the cleavage of cholesterol sulfate, which is incorporated into phospholipid vesicles, by both membrane bound enzyme systems appears to be competitively inhibited by the phospholipids of the vesicles. The results of this study suggest that the regulation of the side-chain cleavage of cholesterol sulfate may be entirely different than the regulation of the side-chain cleavage of cholesterol, if cholesterol sulfate exists intracellularly as a soluble non-complexed substrate. If, on the other hand, cholesterol sulfate is present in the cell in lipid droplets as a complex with phospholipids, its metabolism may be under the same constraints as the side-chain cleavage of cholesterol.

Adrenal Glands↗
PubMed1987

The influence of gonadal steroids and the pituitary on the levels and composition of plasma phospholipids in the rat.

Gonadal steroids have been shown to influence plasma phospholipids. In the present study, the possible interaction between gonadal steroids and the pituitary in the regulation of plasma phospholipids was studied in rats. The total phospholipid concentration (higher in females) and the fatty acid composition of plasma lecithin was different in male compared to female rats. Gonadectomy resulted in a "feminization" of plasma phospholipids (total concentration and fatty acids in lecithin) in male rats but had no effect in females. Testosterone treatment of gonadectomized males or intact females resulted in a "masculinization" of plasma phospholipids, whereas estrogen treatment of intact males resulted in a "feminization." Hypophysectomy resulted in a marked decrease in plasma phospholipid concentration and the fatty acid composition of lecithin showed a "masculine" pattern in both males and females. Neither testosterone nor estrogen treatment had any effects on plasma phospholipids in hypophysectomized male and female rats, respectively. It is concluded that gonadal steroids and the hypothalamic-pituitary axis interact in the regulation of the synthetic and perhaps also degradative pathways controlling plasma phospholipids.

Animals↗
PubMedDate not supplied

A quantitative method for measuring regional in vivo fatty-acid incorporation into and turnover within brain phospholipids: review and critical analysis.

An experimental method and its associated mathematical model are described to quantitate in vivo incorporation rates into and turnovers of fatty acids (FAs) within stable brain metabolic compartments, particularly phospholipids. A radiolabeled FA is injected i.v. in a rat, and arterial plasma unacylated FA radioactivities and unlabeled concentrations are sampled until the animal is killed after 15 min, when the brain is analyzed biochemically or with quantitative autoradiography. Unbound unacylated label in blood easily crosses the blood-brain barrier; rapidly equilibrates in the unacylated FA, acyl-CoA and phosphatidate-diacylglycerol brain pools; then is incorporated into phospholipids and other stable metabolic compartments. Uptake and incorporation of labeled FAs are independent of cerebral blood flow at constant brain blood volume. Different labeled FAs enter specific sn positions of different brain phospholipids, suggesting that a combination of probes can be used to investigate metabolism of these phospholipids. Thus, [9,10-3-H]palmitate preferentially labels the sn1 position of phosphatidylcholine; [1-14C]arachidonate the sn2 positions of phosphatidylinositol and phosphatidylcholine; and [1-14C]docosahexaenoate the sn2 positions of phosphatidylethanolamine and phosphatidylcholine. The FA model provides an operational equation for rates of incorporation of FAs into brain phospholipids, taking into account intracerebral recycling and de novo synthesis of the FA, as well as entry into brain of FA from acylated blood sources. The equation is essentially independent of specific details of the proposed model, and can be used to calculate turnovers and half-lives of FAs within different phospholipid classes. For the model to be most applicable, experiments should satisfy conditions for pulse-labeling of the phospholipids, with brain sampling times short enough to minimize exchange of label between stable metabolic compartments. A 15-20 min sampling time satisfies these criteria. The FA method has been used to elucidate the dynamics of brain phospholipids metabolism in relation to brain development, brain tumor, chronically reduced auditory input, transient ischemic insult, axotomy with and without nerve regeneration, and cholinergic stimulation in animals with or without a chronic unilateral lesion of the nucleus basalis magnocellularis.

Animals↗
PubMed1989

Insulin-like growth factor I stimulates phospholipid synthesis in renal cortical slices without production of inositol phosphate.

The effects of insulin-like growth factor I (IGF-I) on the metabolism of phospholipids in renal cortical slices were examined using either sodium [32P]orthophosphate or myo-[3H]inositol. IGF-I was found to increase the incorporation of phosphate into phospholipids about 2-3-times above control values, leading to an increase in the concentration of total phospholipid phosphorus of 20% above control value after 1 h of incubation. The increased incorporation of phosphate into phospholipids could be prevented by 10 microM cycloheximide, while with 1 microM TPA (12-O-tetradecanoylphorbol 13-acetate) it could not. Insulin was also found to increase the incorporation of phosphate into phospholipids, but only if its concentration was at least 100-times higher than that of IGF-I. When phospholipids were prelabelled, IGF-I neither decreased the level of 32P in phospholipids nor stimulated the formation of inositol phosphates. The results show that IGF-I stimulates phospholipid synthesis without production of inositol phosphates in renal cortical slices.

Animals↗
PubMed1984

Decreased cytochrome oxidase activity in hepatic mitochondria after chronic ethanol consumption and the possible role of decreased cytochrome aa3 content and changes in phospholipids.

In ethanol-fed baboons, hepatic mitochondrial cytochrome oxidase activity and cytochrome aa3 content were significantly decreased by 58.3 and 50.5%, respectively, compared to their pair-fed controls. However, there was no significant correlation between the two, suggesting that other factors in addition to cytochrome aa3 may be responsible for the depression in cytochrome oxidase activity. The total phospholipid content of the mitochondrial membranes was significantly decreased (0.24 +/- 0.03 mumol of phospholipid phosphorus/mg of protein vs. 0.32 +/- 0.04 in controls). This change was accounted for, in part, by the significant decrease in the levels of phosphatidylcholine and cardiolipin. In addition, the fatty acid pattern of the phospholipids was changed. There was a marked increase in the relative amounts of oleic and linoleic acids and a decrease in arachidonic acid. These changes were associated with an increase in the activity of phospholipase A2. The reactivation rate of phospholipid-depleted cytochrome oxidase by endogenous phospholipids from ethanol-fed baboons was significantly lower than that by phospholipid from pair-fed controls, when measured at an optimal phospholipid to protein ratio. Thus, it appears that alterations in the phospholipid composition of the mitochondrial membranes are responsible, at least in part, for the depression of cytochrome oxidase activity produced by chronic ethanol consumption.

Animals↗
PubMed1985

Gas chromatography-mass spectrometry method for determination of phospholipid peroxides; I. Transesterification to form methyl esters.

The purpose of this study is to develop methods for determining the chemical species of lipid peroxides that occur in various types of tissue pathology. Experiments are aimed at determining the phospholipid peroxides associated with retinal degeneration as the initial test case. Phospholipid hydroperoxides are synthesized by photosensitized oxidation, chemically characterized and used to develop an effective and simplified method to identify and measure phospholipid hydroperoxides by gas chromatography-mass spectrometry (GC-MS). A sensitive reverse phase high performance liquid chromatography (HPLC) method is also presented to separate peroxidized phospholipids from phospholipids. For GC-MS, phospholipid peroxides are reduced with sodium borohydride and transesterified to form fatty acid methyl esters using a mild quaternary ammonium hydroxide catalyst. The hydroxyl groups produced by reducing the hydroperoxides are formed into trimethylsilyl ethers and GC-MS is employed (with electron ionization and negative ion chemical ionization) to identify oxidized fatty acids at the 10 ng level. Photooxidation of (palmitoyl)(linoleoyl) phosphatidylcholine yielded equal amounts of the conjugated (9 and 13 isomers) and the nonconjugated (10 and 12 isomers) linoleoyl hydroperoxides. Photooxidation of rat retina total lipids yielded oxidation products of oleolyl (18:1) esters as well as the conjugated and nonconjugated oxidation products of arachidonoyl (20:4) and docosahexaenoyl (22:6) esters virtually all of which arise from phospholipids. The nonconjugated products are of interest as indicators of photosensitized light damage in retina and other tissues. It is notable that all the possible singly oxidized products are found with the exception of the 4, 5 and 7 hydroperoxides of 22:6 and the 5 hydroperoxide of 20:4. It appears that the approach of singlet oxygen is strongly inhibited in the sterically hindered region near the phospholipid head groups.

Animals↗
PubMed1994

Distribution of arachidonic acid among phospholipid subclasses of lone star tick salivary glands.

The subclass composition of choline- and ethanolamine-containing phospholipids was determined by analysis of acyl-linked fatty acids released by base hydrolysis of diradylglycerobenzoates formed from lone star tick salivary gland diacyl, alkylacyl and alkenylacyl phospholipids. The diacyl subclass comprises 87% of all choline-containing phospholipids, while th alkylacyl subclass comprises c. 9% and the alkenylacyl subclass c. 4%. The diacyl subclass comprises 72-77% of ethanolamine-containing phospholipids and about 14 and 13% of this subclass of phospholipid are alkylacyl and alkenylacyl lipids, respectively. Arachidonic acid (20:4) is the most abundant fatty acid (28% of all fatty acids) esterified in the alkylacyl form of phosphatidylcholine (PC) and it comprises 17% of the fatty acids in alkenylacyl-PC. The alkylacyl form of phosphatidylethanolamine (PE) is also rich in 20:4 (24%) while the alkenylacyl-PE subclass contains only 9% 20:4. Despite the relatively high amounts of 20:4 within the ether-linked phospholipids, the majority of the salivary gland 20:4 (> 83%) is found in the diacyl phospholipid subclass because of the preponderence of this subclass in tick salivary glands. Isolated salivary glands incorporated [3H]-20:4 primarily (> 98%) into the sn-2 position of diacyl PC > PE, with some incorporation into triglycerides. Continued incubation in the absence of labeled 20:4 demonstrated remodeling of [3H]-20:4 from PC into PE, and from the diacyl subclass to the alkylacyl subclass in the choline containing phospholipids.

Animals↗
PubMed2005

Characterization of phospholipid-protein interactions by capillary isoelectric focusing with whole-column imaging detection.

The integration of functional proteins in the phospholipid bilayer is one of the most crucial features of biological membrane architecture. Phospholipid-protein interactions play an important role in the functions of bounded proteins in the phospholipid membrane. When the phospholipid-protein interactions occur, the protein structure tends to alter, which can result in a change in the isoelectric points (pI) of protein. Capillary isoelectric focusing (cIEF) with whole-column imaging detection (WCID) is an attractive technique that has the features of simple operation, high resolution, and fast separation without focused band mobility for detection of amphoteric biomolecules. In this study, a cIEF-WCID method was developed to characterize the phospholipids-protein interactions by monitoring the protein cIEF profiles. Seven proteins with different pI and molecular mass , and a zwitterionic phosphatidylcholine (PC) with zwitterionic properties, were used to evaluate the feasibility of the cIEF-WCID approach in the study of phospholipid-protein interactions. The cIEF profiles changed in response to the changes in protein conformation, clearly exhibiting interactions between the PC vesicles and the targeted proteins. The formation of PC-protein complex was observed in the cIEF electropherograms. It was demonstrated that seven proteins displayed distinct interactions with the PC vesicles due to their different chemical and physical properties. The influences of the PC concentration, incubation time, and incubation temperature on the phospholipids-protein interactions were investigated. This study validated a novel analytical approach for the characterization of phospholipid-protein interactions.

Animals↗
PubMed2006

Regulation of phospholipid synthesis in Saccharomyces cerevisiae by zinc depletion.

The synthesis of phospholipids in the yeast Saccharomyces cerevisiae is regulated by zinc, an essential mineral required for growth and metabolism. Cells depleted of zinc contain increased levels of phosphatidylinositol and decreased levels of phosphatidylethanolamine. In addition to the major phospholipids, the levels of the minor phospholipids phosphatidate and diacylglycerol pyrophosphate decrease in the vacuole membrane of zinc-depleted cells. Alterations in phosphatidylinositol and phosphatidylethanolamine can be ascribed to an increase in PIS1-encoded phosphatidylinositol synthase activity and to decreases in the activities of CDP-diacylglycerol pathway enzymes including the CHO1-encoded phosphatidylserine synthase, respectively. Alterations in the minor vacuole membrane phospholipids are due to the induction of the DPP1-encoded diacylglycerol pyrophosphate phosphatase. These changes in the activities of phospholipid biosynthetic enzymes result from differential regulation of gene expression at the level of transcription. Under zinc-deplete conditions, the positive transcription factor Zap1p stimulates the expression of the DPP1 and PIS1 genes through the cis-acting element UAS(ZRE). In contrast, the negative regulatory protein Opi1p, which is involved in inositol-mediated regulation of phospholipid synthesis, represses the expression of the CHO1 gene through the cis-acting element UAS(INO). Regulation of phospholipid synthesis may provide an important mechanism by which cells cope with the stress of zinc depletion, given the roles that phospholipids play in the structure and function of cellular membranes.

CDPdiacylglycerol-Serine O-Phosphatidyltransferase↗
PubMed2005

Phospholipid mass is increased in fibroblasts bearing the Swedish amyloid precursor mutation.

Phospholipid changes occur in brain regions affected by Alzheimer disease (AD), including a marked reduction in plasmalogens, which could diminish brain function either by directly altering signaling events or by bulk membrane effects. However, model systems for studying the dynamics of lipid biosynthesis in AD are lacking. To determine if fibroblasts bearing the Swedish amyloid precursor protein (swAPP) mutation are a useful model to study the mechanism(s) associated with altered phospholipid biosynthesis in AD, we examined the steady-state phospholipid mass and composition of fibroblasts, including plasmalogens. We found a 15% increase in total phospholipid mass, accounted for by a 24% increase in the combined total of phosphatidylethanolamine and plasmanylethanolamine mass and a 19% increase in the combined total of phosphatidylcholine (PtdCho) and plasmanycholine (PakCho) mass in the swAPP mutant bearing fibroblasts. Cholesterol mass was unchanged in these cells. The changes in phospholipid mass did not alter the cellular molar composition of the phospholipids nor the cholesterol to phospholipid ratio. While plasmalogen mass was not altered, the ratio of choline plasmalogen (PlsCho) mass to PtdCho+PakCho mass was decreased 16% and there was a 14% reduction in the proportion of PlsCho as a percent of total phospholipids in the swAPP mutant bearing fibroblasts. This change in choline plasmalogen is consistent with the reported decreases in plasmalogen proportions in affected regions of AD brain, suggesting that these cells may serve as a useful model to determine the mechanism underlying changes in plasmalogen biosynthesis in AD brain.

Alzheimer Disease↗
PubMed2005

Cerebroside Langmuir monolayers originated from the echinoderms I. Binary systems of cerebrosides and phospholipids.

The surface pressure (pi)-area (A), the surface potential (DeltaV)-A and the dipole moment (mu( perpendicular))-A isotherms were obtained for two-component monolayers of two different cerebrosides (LMC-1 and LMC-2) with phospholipids of dipalmitoylphosphatidylcholine (DPPC) and with dipalmitoylphosphatidylethanolamine (DPPE) on a subphase of 0.5 M sodium chloride solution as a function of phospholipid compositions by employing the Langmuir method, the ionizing electrode method, and the fluorescence microscopy. Surface potentials (DeltaV) of pure components were analyzed using the three-layer model proposed by Demchak and Fort. The contributions of the hydrophilic saccharide group and the head group to the vertical component of the dipole moment (mu( perpendicular)) were estimated. The miscibility of cerebroside and phospholipid in the two-component monolayers was examined by plotting the variation of the molecular area and the surface potential as a function of the phospholipid molar fraction (X(phospholipid)), using the additivity rule. From the A-X(phospholipid) and DeltaV(m)-X(phospholipid) plots, partial molecular surface area (PMA) and apparent partial molecular surface potential (APSP) were determined at the discrete surface pressure. The PMA and APSP with the mole fraction were extensively discussed for the miscible system. Judging from the two-dimensional phase diagrams, these can be classified into two types. The first is a positive azeotropic type; the combinations of cerebrosides with DPPC are miscible with each other. The second is a completely immiscible type: the combination of cerebrosides with DPPE. Furthermore, a regular surface mixture, for which the Joos equation was used for the analysis of the collapse pressure of two-component monolayers, allowed calculation of the interaction parameter (xi) and the interaction energy (-Delta epsilon) between the cerebrosides and DPPC component. The miscibility of cerebroside and phospholipid components in the monolayer state was also supported by fluorescence microscopy.

1,2-Dipalmitoylphosphatidylcholine↗
PubMed2006

Drug permeability across a phospholipid vesicle based barrier: 3. Characterization of drug-membrane interactions and the effect of agitation on the barrier integrity and on the permeability.

Recently, we reported on the development and structural characterization of a phospholipid vesicle based barrier useful for medium throughput screening of passive drug permeability. Here, we investigate the physical and functional integrity of the phospholipid vesicle based barriers to agitation by stirring or shaking, and whether agitation affects drug permeability of sulpiride, metoprolol and testosterone. In addition, three drugs (caffeine, naproxen and sulphasalazine) which were shown in a previous study to affect the electrical resistance of the barriers, were investigated for their influence on the permeability of a simultaneously applied hydrophilic marker (calcein), and on the thermotropic phase transition of the phospholipid bilayers using differential scanning calorimetry (DSC). Electrical resistance measurements indicated that the barriers should withstand shaking speeds up to 150rpm without losing their integrity, but significant release of phospholipids from the membrane barriers to the donor and acceptor chambers was observed under agitation >or=150rpm. When using agitation up to 100rpm no increase in permeability was observed for sulpiride, metoprolol and testosterone. The phospholipid vesicle-based barrier thus differ from other permeability models in that agitation does not lead to an increase in permeability, not even for highly lipophilic drugs such as testosterone. This is explained by the different morphology of the vesicle-based barrier which is containing a 100microm thick layer of mostly aqueous compartments immobilised within a matrix of phospholipids vesicles. Sulphasalazine and naproxen were shown to decrease the electrical resistance and increase the permeability of the hydrophilic marker calcein. The DSC experiments showed that these two drugs probably interact with the head groups of the phospholipids. In contrast, caffeine gave an increase in electrical resistance and a decrease in permeability of calcein. From the DSC experiments no signs of interaction of caffeine with the phospholipid bilayer could be observed.

Anti-Inflammatory Agents, Non-Steroidal↗
PubMed2002

Saturated anionic phospholipids enhance transdermal transport by electroporation.

Anionic phospholipids, but not cationic or neutral phospholipids, were found to enhance the transdermal transport of molecules by electroporation. When added as liposomes to the milieus of water-soluble molecules to be delivered through the epidermis of porcine skin by electroporation, these phospholipids enhance, by one to two orders of magnitude, the transdermal flux. Encapsulation of molecules in liposomes is not necessary. Dimyristoylphosphatidylserine (DMPS), phosphatidylserine from bovine brain (brain-PS), dioleoylphosphatidylserine (DOPS), and dioleoylphosphatidylglycerol (DOPG) were used to test factors affecting the potency of anionic lipid transport enhancers. DMPS with saturated acyl chains was found to be a much more potent transport enhancer than those with unsaturated acyl chains (DOPS and DOPG). There was no headgroup preference. Saturated DMPS was also more effective in delaying resistance recovery after pulsing, and with a greater affinity in the epidermis after pulsing. Using fluorescent carboxyl fluorescein and fluorescein isothiocyanate (FITC)-labeled Dextrans as test water-soluble molecules for transport, and rhodamine-labeled phospholipids to track anionic phospholipids, we found, by conventional and confocal fluorescence microscopy, that transport of water-soluble molecules was localized in local transport spots or regions (LTRs) created by the electroporation pulses. Anionic phospholipids, especially DMPS, were located at the center of the LTRs and spanned the entire thickness of the stratum corneum (SC). The degree of saturation of anionic phospholipids made no difference in the densities of LTRs created. We deduce that, after being driven into the epidermis by negative electric pulses, saturated anionic phospholipids mix and are retained better by the SC lipids. Anionic lipids prefer loose layers or vesicular rather than multilamellar forms, thereby prolonging the structural recovery of SC lipids to the native multilamellar form. In the presence of 1 mg/ml DMPS in the transport milieu, the flux of FITC-Dextran-4k was enhanced by 80-fold and reached 175 microg/cm(2)/min. Thus, the use of proper lipid enhancers greatly extends the upper size limit of transportable chemicals. Understanding the mechanism of lipid enhancers enables one to rationally design better enhancers for transdermal drug and vaccine delivery by electroporation.

Animals↗
PubMed1982

31P nuclear magnetic resonance studies of the phospholipid-protein interface in cell membranes.

Both native and recombined membrane systems from the human erythrocyte membrane and the rabbit sarcoplasmic reticulum have been studied with 31P Nuclear Magnetic Resonance (NMR). We compare intensities of the anisotropic 31P resonance exhibited by these membranes with the intensity expected from the known phospholipid content of the membranous sample. In a recombinant with human erythrocyte glycophorin, a component of the phospholipid is "missing" from the 31P NMR resonance, apparently due to a severe broadening of the resonance of that component. Approximately 29 phospholipid molecules were found immobilized per glycophorin molecule in the membrane, regardless of the phospholipid:protein ratio. Cholesterol may inhibit the immobilization of phospholipids by glycophorin. Recombinants with band three from the human erythrocyte membrane contain an immobilized phospholipid component, analogous to the results with glycophorin. 31P NMR data from the native sarcoplasmic reticulum membrane also revealed an immobilized phospholipid component whose magnitude is independent of temperature between 30 degrees C and 45 degrees C. Extensive papain proteolysis of the membrane completely digests the Ca++ Mg++ ATPase and removes the immobilization of phospholipids noted in the intact membrane. Limited trypsin cleavage, however, does not completely remove the immobilized component; salt reduces the immobilized component.

Animals↗
PubMed1996

7-nitrobenz-2-oxa-1,3-diazole-4-yl-labeled phospholipids in lipid membranes: differences in fluorescence behavior.

Steady-state and time-resolved fluorescence properties of the 7-nitrobenz-2-oxa-1, 3-diazole-4-yl (NBD) fluorophore attached either to the sn-2 acyl chain of various phospholipids (phosphatidylcholine, phosphatidylethanolamine, phosphatidylserine, and phosphatidic acid) or to the polar headgroup of phosphatidylethanolamine were studied after insertion of these NBD-labeled lipid probes into unilamellar vesicles of phosphatidylcholine, phosphatidylglycerol, phosphatidic acid, and phosphatidylserine. The fluorescence response of the NBD group was observed to strongly depend on the chemical structure and physical state of the host phospholipids and on the chemical structure of the lipid probe itself. Among the various fluorescence parameters studied, i.e., Stokes' shifts, lifetimes, and quantum yields, the quantum yields were by far the most affected by these structural and environmental factors, whereas the Stokes' shifts were practically unaffected. Thus, depending on the phospholipid probe and the host phospholipid, the fluorescence emission of the NBD group was found to vary by a factor of up to 5. Careful analysis of the data shows that for the various couples of probe and host lipid molecules studied, deexcitation of the fluorophore was dominated by nonradiative deactivation processes. This great sensitivity of the NBD group to environmental factors originates from its well-known solvatochromic properties, and comparison of these knr values with those obtained for n-propylamino-NBD in a set of organic solvents covering a large scale of polarity indicates that in phospholipids, the NBD fluorophore experiences a dielectric constant of around 27-41, corresponding to a medium of relatively high polarity. From these epsilon values and on the basis of models of the dielectric transition that characterizes any water-phospholipid interface, it can be inferred that for all of the phospholipid probes and host phospholipids tested, the NBD group is located in the region of the polar headgroups, near the phosphoglycerol moiety of the lipids.

1,2-Dipalmitoylphosphatidylcholine↗
PubMed2000

Further studies on the reconstitution of glucosylceramidase activity by Sap C and anionic phospholipids.

The reconstitution of the activity of the lysosomal enzyme glucosylceramidase requires anionic phospholipids and, at least, a protein factor, saposin C (Sap C). We have previously proposed a mechanism for the glucosylceramidase activation [Vaccaro et al. (1993) FEBS Lett. 336, 159-162] which implies that Sap C promotes the association of the enzyme with anionic phospholipid-containing membranes, thus favoring the contact between the enzyme and its lipid substrate, glucosylceramide. We have further investigated the properties of Sap C using a fluorescent hydrophobic probe such as 4, 4'-dianilino-1,1'-binaphthyl-5,5'-disulfonic acid (bis-ANS). The binding between bis-ANS and Sap C was pH-dependent, indicating that protonation leads to increased exposure of hydrophobic surfaces of Sap C. The interaction of Sap C with membranes, triggered by the development of hydrophobic properties at low pH values, was affected by the content of anionic phospholipids, such as phosphatidylserine or phosphatidylinositol, suggesting that anionic phospholipids have the potential to modulate the insertion of Sap C in the hydrophobic environment of lysosomal membranes. We previously showed that Sap C and anionic phospholipids are both required for the binding of glucosylceramidase to large vesicles. We have presently observed that Sap C is able to promote the association of glucosylceramidase with the lipid surface only when anionic phospholipids exceed a concentration of 5-10%. This level can be reached by summing lower amounts of individual anionic phospholipids, since they have additive effects. The present data extend and refine our model of the mechanism of glucosylceramidase activation and stress the key role of pH, Sap C and anionic phospholipids in promoting the interaction of the enzyme with membranes.

Anilino Naphthalenesulfonates↗
PubMed2002

Additional binding sites for anionic phospholipids and calcium ions in the crystal structures of complexes of the C2 domain of protein kinase calpha.

The C2 domain of protein kinase Calpha (PKCalpha) corresponds to the regulatory sequence motif, found in a large variety of membrane trafficking and signal transduction proteins, that mediates the recruitment of proteins by phospholipid membranes. In the PKCalpha isoenzyme, the Ca2+-dependent binding to membranes is highly specific to 1,2-sn-phosphatidyl-l-serine. Intrinsic Ca2+ binding tends to be of low affinity and non-cooperative, while phospholipid membranes enhance the overall affinity of Ca2+ and convert it into cooperative binding. The crystal structure of a ternary complex of the PKCalpha-C2 domain showed the binding of two calcium ions and of one 1,2-dicaproyl-sn-phosphatidyl-l-serine (DCPS) molecule that was coordinated directly to one of the calcium ions. The structures of the C2 domain of PKCalpha crystallised in the presence of Ca2+ with either 1,2-diacetyl-sn-phosphatidyl-l-serine (DAPS) or 1,2-dicaproyl-sn-phosphatidic acid (DCPA) have now been determined and refined at 1.9 A and at 2.0 A, respectively. DAPS, a phospholipid with short hydrocarbon chains, was expected to facilitate the accommodation of the phospholipid ligand inside the Ca2+-binding pocket. DCPA, with a phosphatidic acid (PA) head group, was used to investigate the preference for phospholipids with phosphatidyl-l-serine (PS) head groups. The two structures determined show the presence of an additional binding site for anionic phospholipids in the vicinity of the conserved lysine-rich cluster. Site-directed mutagenesis, on the lysine residues from this cluster that interact directly with the phospholipid, revealed a substantial decrease in C2 domain binding to vesicles when concentrations of either PS or PA were increased in the absence of Ca2+. In the complex of the C2 domain with DAPS a third Ca2+, which binds an extra phosphate group, was identified in the calcium-binding regions (CBRs). The interplay between calcium ions and phosphate groups or phospholipid molecules in the C2 domain of PKCalpha is supported by the specificity and spatial organisation of the binding sites in the domain and by the variable occupancies of ligands found in the different crystal structures. Implications for PKCalpha activity of these structural results, in particular at the level of the binding affinity of the C2 domain to membranes, are discussed.

Animals↗
PubMed1989

Characterization and localization of in vivo phospholipid methylation in the hamster testis.

Although previous studies have demonstrated that phospholipid methylation occurs in the testis and may be involved in Leydig cell function, phospholipid methylation in spermatogenic cells has not been characterized. In this study we describe the occurrence, time course, and localization of phospholipid methylation in the hamster testis following intratesticular injection of radioactive methyl precursor. Adult and pubertal (seven day old) hamsters were injected intratesticularly with [3H-methyl]-methionine and sacrificed 10 min. to 31 hours thereafter. The testes were then removed and homogenized or dispersed into cell suspensions. Spermatogenic cell and Leydig cell enriched preparations were isolated from the dispersed cell preparations using elutriation and Percoll gradient centrifugation and assayed for methylated phospholipids and proteins. These experiments demonstrated that 1) phospholipid methylation occurs in the hamster testis at a level seven-fold greater than protein methylation, 2) the incorporation of radioactivity associated with phospholipid methylation is progressive over time, and 3) in vivo, spermatogenic cell preparations enriched with pachytene spermatocytes have an almost four-fold higher level of measurable phospholipid methylation when compared to whole testis preparations. Taken together, these results suggest that phospholipid methylation may play an important stage-specific role in spermatogenesis.

Animals↗