Inadequacy of myelin phospholipids for restoration of succinoxidase activity in lipid-depleted mitochondria

Succinoxidase activity in lipid-depleted mitochondria was not restored efficiently by mixed myelin phospholipids at difference with the natural mitochondrial phospholipids, yeast phospholipids, and Asolectin. Since similar differences in activity were present between pure phosphatidyl-ethanolamine fractions separated from myelin phospholipids and Asolectin, they should be due to the different fatty acid composition of the phospholipids. In contrast with the differentability in restoration of succionoxidase, all the phospholipids studied were bound to the lipid-depleted membranes to similar extents.     

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

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

Inhibition of the phosphatase activity of the red cell membrane Ca2+ pump by acidic phospholipids.

The effect of phospholipids was tested on the p-nitrophenylphosphatase activity of the Ca2+ pump. Acidic phospholipids like phosphatidylserine and phosphatidylinositol inhibited the phosphatase activity, while neutral phospholipids like phosphatidylcholine did not. This result contrasts sharply with the known activating effect of acidic phospholipids on the Ca2(+)-ATPase activity of the pump. It is known that the phosphatase activity of the Ca2+ pump can be elicited either by calmodulin and Ca2+ or by ATP and Ca2+. Unlike calmodulin, acidic phospholipids failed to stimulate the phosphatase activity. Furthermore, calmodulin-activated phosphatase was completely inhibited by acidic phospholipids. Maximal inhibition of the ATP-activated phosphatase was only 70%. Inhibition by acidic phospholipids was non-competitive regarding to calmodulin, suggesting that acidic phospholipids and calmodulin do not bind to the same domain of the pump. The presence of Ca2+ was essential for the inhibition, and the apparent affinity for Ca2+ for this effect was increased by acidic phospholipids. Results are consistent with the idea that acidic phospholipids stabilize an enzyme-Ca complex lacking phosphatase activity.     

Inhibition of peroxyl radical-mediated lipid oxidation by plasmalogen phospholipids and alpha-tocopherol

The recently discovered peroxyl radical scavenging properties of plasmalogen phospholipids led us to evaluate their potential interactions with alpha-tocopherol. The oxidative decay of plasmalogen phospholipids and of polyunsaturated fatty acids as induced by peroxyl radicals (generated from 2,2'-azobis-2-amidinopropane hydrochloride; AAPH) was studied in micelles using 1H-NMR and chemical analyses. In comparison with alpha-tocopherol, a 20- to 25-fold higher concentration of plasmalogen phospholipids was needed to induce a similar inhibition of peroxyl radical-mediated oxidation of polyunsaturated fatty acids. Plasmalogen phospholipids and alpha-tocopherol protected each other from oxidative degradation. In low-density lipoproteins (LDL) and micelles supplemented with plasmalogen phospholipids plus alpha-tocopherol, the peroxyl radical-promoted oxidation was additively diminished. The differences in the capacities to inhibit oxidation processes induced by peroxyl radicals between the plasmalogen phospholipids and alpha-tocopherol were less pronounced in the LDL particles than in the micelles. In conclusion, plasmalogen phospholipids and alpha-tocopherol apparently compete for the interaction with the peroxyl radicals. Oxidation processes induced by peroxyl radicals are inhibited in an additive manner in the presence of the two radical scavengers. The contribution of the plasmalogen phospholipids to the protection against peroxyl radical promoted oxidation in vivo is expected to be at least as important as that of alpha-tocopherol.     

Hydrolysis of N-acylated glycerophospholipids by phospholipases A2 and D: a method of identification and analysis

We have previously identified N-acylethanolamine phospholipids in infarcted dog heart and in normal fish brain by chemical and enzymatic degradation. We now report that hydrolysis with phospholipase D from Streptomyces chromofuscus removes N-acylethanolamine from N-acylethanolamine phospholipids and lyso N-acylethanolamine phospholipids, or N-acylserine from lyso N-acylserine phospholipids. At acidic pH, a phosphatase present in the phospholipase D preparation further hydrolyzes the resulting phosphatidic acid (PA) or lyso-PA to diacyl- or monoacylglycerol. Because N-acylserine phospholipids are a poor substrate for the phospholipase D, pretreatment with phospholipase A2 (Trimeresurus flavoviridis venom) is used to remove the 2-O-acyl group. Thus, both types of N-acylated phospholipids can be analyzed by consecutive phospholipase A2 and phospholipase D treatment. Reaction products, i.e., free fatty acids, monoacylglycerols and N-acylethanolamine or N-acylserine, are separable by thin-layer chromatography. Both N-acyl components can be further characterized by conversion to the t-butyldimethylsilyl derivatives. The method was used to identify and analyze the N-acylserine phospholipids of bovine brain.     

Synthesis of phospholipids containing photoactivatable carbene precursors in the headgroups and their crosslinking with membrane proteins

Many integral membrane enzymes require for their activity interactions with the polar headgroups of phospholipids, in addition to the hydrophobic interactions within the lipid bilayer. The interactions with the polar headgroups may have preferential or absolute specificity. To study such interactions, phospholipids have been synthesized which carry photoactivable moieties in their headgroups. Three types of phospholipids, PL-I, PL-II and PL-III, were synthesized. The synthetic phospholipids, PL-I and PL-II were able to reconstitute enzymatic activity of the membrane proteins which were studied. Covalent crosslinking between these phospholipids and the membrane proteins was demonstrated after photolysis of the reconstituted phospholipid-protein complexes.     

Phospholipids and inositol phosphates linked to the epigenome

Even though the majority of knowledge about phospholipids comes from their cytoplasmic functions, in the last decade, it has been shown that nuclear phospholipids and their building blocks, inositol phosphates, have many important roles in the cell nucleus. There are clear connections of phospholipids with the regulation of gene expression and chromatin biology, however, this review focuses on less known functions of nuclear phospholipids in connection with the epigenome regulation. In particular, we highlight the roles of nuclear phospholipids and inositol phosphates that involve histone modifications, such as acetylation or methylation, tightly connected with the cell physiology. This demonstrates the importance of nuclear phospholipids in the regulation of cellular processes, and should encourage further research of nuclear phospholipids and inositol phosphates.     

Immunomodulating activity of polyunsaturated phospholipids and regulators of energy metabolism in toxic anemia]

Possible potentiation of the immunomodulating effects of carbohydrate and lipid metabolism regulators by their use in combination with polyunsaturated phospholipids was studied. The polyunsaturated phospholipids in toxic anemia icreased the immunomodulating effects of thiamine and inosine which activated glucose catabolism in erythrocytes. The combined use of the polyunsaturated phospholipids and thiamine normalized the oxidation--energy status and lowered manifestation of the immunosuppressing properties of light erythrocytes in laboratory rats exposed to hemolytic poison. The use of the combination of the polyunsaturated phospholipids and inosine normalized the oxidation--energy status and induced manifestation of the immunomodulating properties in heavy erythrocytes of the poisoned rats. The globulin fraction of the rat serum containing antibodies to erythrocytes of the poisoned rats exposed to the polyunsaturated phospholipids and inosine increased the immunity status of the poisoned rats treated with the above mentioned agents. Carnitine and biotin in combination with the polyunsatured phospholipids showed no effect on the phagocytic and metabolic activity of leukocytes and the immunity status of the rats exposed to hemolytic poison.     

Extensive segregation of acidic phospholipids in membranes induced by protein kinase C and related proteins

Protein kinase C and two other proteins with molecular masses of 64 and 32 kDa, purified from bovine brain, constitute a type of protein that binds a large number of calcium ions in a phospholipid-dependent manner. This study suggested that these proteins also induced extensive clustering of acidic phospholipids in the membranes. Clustering of acidic phospholipids was detected by the self-quenching of a fluorescence probe that was attached to acidic phospholipids (phosphatidic acid or phosphatidylglycerol). Addition of these proteins to phospholipid vesicles containing 15% fluorescently labeled phosphatidic acid dispersed in neutral phosphatidylcholine resulted in extensive, rapid, and calcium-dependent quenching of the fluorescence signal. Fluorescence-quenching requirements coincided with protein-membrane binding characteristics. As expected, the addition of these proteins to phospholipid vesicles containing fluorescent phospholipids dispersed with large excess of acidic phospholipids produced only small fluorescence changes. In addition, association of these proteins with vesicles composed of 100% fluorescent phospholipids resulted in no fluorescence quenching. Protein binding to vesicles containing 5-50% fluorescent phospholipid showed different levels of fluorescence quenching that closely resemble the behavior expected for extensive segregation of the acidic phospholipids in the outer layer of the vesicles. Thus, the fluorescence quenching appeared to result from self-quenching of the fluorophores that become clustered upon protein-membrane binding. These results were consistent with protein-membrane binding that was maintained by calcium bridges between the proteins and acidic phospholipids in the membrane. Since each protein bound eight or more calcium ions in the presence of phospholipid, they may each induce clustering of a related number of acidic phospholipids.(ABSTRACT TRUNCATED AT 250 WORDS)     

Pulmonary surfactant protein D specifically binds to phosphatidylinositol.

Alveolar type II cells produce and secrete a complex mixture of lipids and proteins called pulmonary surfactant of which phospholipids are the major components. Surfactant proteins (SP) A, B, and C interact with phospholipids and are believed to play important roles in alveolar spaces. However, whether surfactant protein D (SP-D) interacts with phospholipids is unknown. In the present study, we examined whether SP-D binds to phospholipids and investigated phospholipid specificities of SP-D binding and the structural requirements of phospholipids for that binding using 125I-SP-D as a probe. 125I-SP-D bound exclusively to phosphatidylinositol (PI) in various phospholipids or a fraction containing phospholipids extracted from surfactant, which were developed on thin layer chromatography. 125I-SP-D also bound to PI coated on microtiter wells in a manner dependent upon the SP-D concentration. Unlabeled SP-D competed well with 125I-SP-D for PI binding and the antibody against SP-D abolished 125I-SP-D binding to PI. PI liposome also attenuated 125I-SP-D binding to the solid phase PI. Ca2+ is absolutely required for the binding of SP-D to PI. SP-D failed to bind to lyso-PI, fatty acids derived from PI digested with phospholipase A2, or diacylglycerol obtained after phospholipase C treatment of PI. SP-D bound to neither phosphatidylinositol 4-monophosphate nor phosphatidylinositol 4,5-diphosphate. We conclude that SP-D specifically binds to PI. This is the first report that demonstrates that SP-D interacts with surfactant phospholipids.     

The role of oxidized phospholipids in mediating lipoprotein(a) atherogenicity

PURPOSE OF REVIEW: To review emerging data on the relationship between lipoprotein(a) and oxidized phospholipids. RECENT FINDINGS: We have recently proposed that a unique physiological role of lipoprotein(a) may be to bind and transport proinflammatory oxidized phospholipids and that this interaction may mediate a common biological influence on cardiovascular disease. In a large series of clinical studies performed to date, a very strong correlation was found between plasma levels of lipoprotein(a) and the content of oxidized phospholipids on apolipoprotein B-100 particles (OxPL/apoB), measured by monoclonal antibody E06, which binds the phosphocholine head group of oxidized phospholipids but not native phospholipids. The correlation of OxPL/apoB to lipoprotein(a) is very strong in individuals with small apolipoprotein(a) isoforms (r = approximately 0.95) and modest in individuals with large isoforms (r = approximately 0.60). In-vitro studies have demonstrated that the vast majority of oxidized phospholipids detected by E06 are bound to lipoprotein(a) in human plasma. A similarly strong association with oxidized phospholipids was also documented in transgenic mice overexpressing lipoprotein(a), even in mice not fed atherogenic diets or with overt atherosclerosis. SUMMARY: A better understanding of the ability of human lipoprotein(a) to bind oxidized phospholipids may allow clinically important insights into the role of oxidized phospholipids and lipoprotein(a) in human atherogenesis and cardiovascular disease and may provide novel diagnostic tools and therapeutic interventions aimed at measuring and treating elevated levels of OxPL/apoB and lipoprotein(a).     

Interaction of europium(III) with phospholipid vesicles as monitored by laser-excited europium(III) luminescence

The technique of laser-excited Eu(III) luminescence was applied to monitor Eu(III) binding to a variety of phospholipids. Eu(III) excitation spectra were similar with and without the presence of neutral phospholipids, while acidic phospholipids changed the spectrum in a concentration-dependent manner. Eu(III) appears to bind to the phosphate moiety with at least a 2:1 phospholipid:metal ion stoichiometry. Analysis of luminescence lifetimes reveals that only one or two waters of hydration are removed from Eu(III) by addition of neutral phospholipids, whereas acidic phospholipids and inorganic phosphate strip off all but one or two waters. Implications with regard to fusion and use of lanthanides as probes in membrane preparations are discussed.     

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

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

KINETICS OF THE BIOSYNTHESIS OF PHOSPHOLIPIDS IN NEURONS AND GLIAL CELLS ISOLATED FROM RAT BRAIN CORTEX

—³²P incorporation into different rat-brain cortex neuronal and glial phospholipids was investigated. The half life of each compound was measured. Neuronal phospholipids had a faster turnover than glial phospholipids. Phosphatidyl-inositol and choline plasmalogen had the fastest, diphosphatidylglycerol the lowest turnover in both cell-types. Phosphatidylcholine, ethanolamine phospholipids and serine phospholipids had turnover intermediate with that of the previously described compounds. Turnover of neuronal sphingomyelin was similar to that of phosphatidylcholine, whereas in glial cells it was much lower.     

The influence of polyunsaturated fatty acids on spermine inhibition of lipoperoxidation. Studies on liposomes prepared with microsomal and mitochondrial phospholipids of sea bass (Dicentrarchus labrax L.) and rat liver

1. Composition of phospholipids extracted from different organelles of European sea bass liver was determined and compared with that of phospholipids extracted from the same organelles of rat liver. 2. Spermine binding to the vesicles prepared from microsomal and mitochondrial phospholipids and their aggregation was studied: these parameters indicate that only the presence of acidic phospholipids and not their unsaturation was essential for polyamine action. 3. No correlation exists between polyunsaturated fatty acid and spermine inhibition of lipid peroxidation. In fact microsomal phospholipids, which have a low content of acidic phospholipids, and a prevalent presence of phosphatidylinositol, are not protected by spermine. 4. Mitochondrial phospholipids, which have high content of cardiolipin, elicit the capability of spermine to inhibit lipid peroxidation.     

Radioiodination of naturally occurring phospholipids

A simple and rapid nonenzymatic method for radioiodination of phospholipids is described. It involves oxidation of Na125I with TlCl3 (or chloramine-T) in an aqueous medium, with subsequent exposure of the phospholipids, dissolved in chloroform/methanol, to the action of the oxidizing mixture. Purification of the radiolabelled phospholipids was effected by washing with sodium thiosulphate followed by thin-layer chromatography on silica gel. Specific radioactivity of 125I-labelled phosphatidylcholine was estimated to be about 10 muCi/mg phospholipid. The method is designed for radioiodination of various naturally occurring phospholipids.     

Enzymatic modification of phospholipids for functional applications and human nutrition

Rapid progress in biochemistry of phospholipids and evolution of modern bioengineering has brought forth a number of novel concepts and technical advancements in the modification of phospholipids for industrial applications and human nutrition. Highlights cover preparation of novel phospholipid analogs based on the latest understanding of pivotal role of phospholipids in manifold biological processes, exploration of remarkable application potentials of phospholipids in meliorating human health, as well as development of new chemical and biotechnological approaches applied to the modification of phospholipids. This work reviews the natural occurrence and structural characteristics of phospholipids, their updated knowledge on manifold biological and nutritional functions, traditional and novel physical and chemical approaches to modify phospholipids as well as their applications to obtain novel phospholipids, and brief introduction of the efforts focusing on de novo syntheses of phospholipids. Special attention is given to the summary of molecular structural characteristics and catalytic properties of multiple phospholipases, which helps to interpret experimental phenomena and to improve reaction design. This will of course provide fundamental bases also for the development of enzymatic technology to produce structured or modified phospholipids.     

Phospholipids as Plant Growth Regulators

In this paper the potential to use phospholipids and lysophospholipids as plant growth regulators is discussed. Recent evidence shows that phospholipids and phospholipases play an important signalling role in the normal course of plant development and in the response of plants to abiotic and biotic stress. It is apparent that phospholipase A (PLA), C (PLC) and D (PLD), lysophospholipids, and phosphatidic acid (PA) are key components of plant lipid signalling pathways. By comparison, there is very little information available on the effect of exogenously applied phospholipids on plant growth and development. This paper serves to introduce phospholipids as a novel class of plant growth regulator for use in commercial plant production. The biochemistry and physiology of phospholipids is discussed in relation to studies in which phospholipids and lysophospholipids have been applied to plants and plant parts. Implicit in the observations is that phospholipids impact the hypersensitive response and systemic acquired resistance in plants to improve crop performance and product quality. Based on published data, a scheme outlining a possible mode of action of exogenously applied phospholipids is proposed.     

Adrenocorticotropic hormone-mediated changes in rat adrenal mitochondrial phospholipids

We examined the subcellular localization of ACTH (adrenocorticotropic hormone)-induced changes in adrenal phospholipids using dexamethasone-treated rats. In adrenal mitochondrial fraction, ACTH significantly enhanced both concentrations and contents of phosphatidylinositol (37%), phosphatidylcholine (22%), and phosphatidylethanolamine (20%). Other mitochondrial phospholipids including cardiolipin did not change upon administration of ACTH. In adrenal plasma membrane, endoplasmic reticulum, and peroxisomes, no increase in phospholipids was observed. The ACTH-induced increases in mitochondrial phosphatidylinositol, phosphatidylcholine, and phosphatidylethanolamine were specific to adrenal among tissues tested. These changes were observed specifically in cortical cells rather than medulla. Nonsteroidogenic ACTH fragments and related peptides were unable to induce the change in adrenal mitochondrial phospholipids. From the dose-response profile with ACTH, the changes in mitochondrial phospholipids were closely related to ACTH-dependent stimulation of steroidogenesis. Furthermore, in vitro treatment with cyclic AMP enhanced both concentrations and contents of mitochondrial phosphatidylinositol, phosphatidylcholine, and phosphatidylethanolamine similar to those by the in vivo administration of ACTH. Both in vivo and in vitro experiments revealed that the hormone-induced changes in mitochondrial phospholipids were sensitive to a protein-synthesis inhibitor, cycloheximide. However, aminoglutethimide and cytochalasin B, which strongly inhibited the hormone-induced formation of corticosterone, did not affect the increases in mitochondrial phospholipids. These results suggest that the hormone-induced increases in these phospholipids occur between ACTH-mediated ribosomal protein synthesis and corticosterone formation.