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PubMed1982

Cholesterol-phospholipid interaction in membranes. 2. Stoichiometry and molecular packing of cholesterol-rich domains.

A model for the molecular interaction between cholesterol and phospholipid in bilayer membranes is presented. We propose that cholesterol forms associations with phospholipids with stoichiometries of both 1:1 and 1:2. A hydrogen bond between the beta-OH of cholesterol and the glycerol ester oxygen of a phospholipid is suggested as a likely mechanism for tight binding in a 1:1 complex. A second phospholipid molecule is loosely associated with the complex to form domains of 1:2 stoichiometry, which may coexist with pure phospholipid domains. Interfacial boundary phospholipid separates these two domains. Under conditions in which interfacial phospholipid is maximal, the perturbed phospholipid assumes a composition of 20 mol % cholesterol. To account for the phase behavior and surface properties of cholesterol-lipid membranes, we propose a molecular packing model for linear arrays within the cholesterol-rich domains. In this arrangement, two rows of 1:1 complex run antiparallel with loosely associated phospholipid intercalated between them. The loosely associated phospholipid can pack in the nearly hexagonal manner in which pure crystalline phospholipid is known to pack. The model provides maximal van der Waals contact in the hydrocarbon region of the bilayer and can maintain phospholipids as cholesterol's nearest neighbors at all concentrations up to 50 mol % cholesterol. The model is compatible with the diverse experimental observations compiled by many investigators over the past decade.

Cholesterol↗
PubMed1982

Factor Va-factor Xa interaction. Effects of phospholipid vesicles of varying composition.

The interaction between factor Xa and factor Va was investigated both in solution and in the presence of phospholipid vesicles with varying contents of phosphatidylserine. The binding parameters were inferred from the kinetics of prothrombin activation. Factor Xa and factor Va form in solution an equimolar complex with a dissociation constant of 3.3 X 10(-9) M. Phospholipid vesicles promote the formation of the factor Xa-Va complex. The Kd of complex formation is dependent on both the phospholipid concentration and the composition of the phospholipid vesicle. For the interaction between factor Xa and factor Va in the presence of phospholipid vesicles containing 40 mol % dioleoylphosphatidylserine (DOPS) and 60 mol % dioleoylphosphatidylcholine (DOPC), the Kd increases linearly with increasing phospholipid concentration. In the presence of 10 microM phospholipid (DOPS/DOPC, 40/60 mol/mol) Kd = 3 X 10(-11) M. When the mole percentage of DOPS in the phospholipid vesicles is lowered from 20 to 5 mol %, there is a gradual increase of the Kd. In the presence of 10 microM phospholipid vesicles containing 5 mol % DOPS and 95 mol % DOPC Kd = 2.8 X 10(-10) M. The Kd measured in the presence of phospholipid vesicles containing 5 mol % DOPS and 95 mol % DOPC is independent of the phospholipid concentration. Two models are discussed that can quantitatively explain the effect of phospholipid vesicles on the complex formation between factor Xa and factor Va. Studies on the effect of the polypeptides with Mr 80 000 and Mr 94000 of which factor Va is composed on the Kd of the factor Xa-Va complex suggest that factor Xa binding to factor Va requires a Ca2+-mediated interaction between the two polypeptides.

Animals↗
PubMed1976

Transbilayer phospholipid asymmetry and its maintenance in the membrane of influenza virus.

Two phospholipid exchange proteins and two phospholipases C have been employed to determine the phospholipid composition of the outer surface of the membrane of influenza virus. These four protein probes have defined the same accessible and inaccessible pool for each viral phospholipid. Phospholipids which are exchangeable or hydrolyzable are located on the outer surface, whereas the inaccessible pool is located at the inner surface of the viral bilayer. The two pools are unequal in size, with ca. 30% of the total phospholipid accessible to the four proteins, and ca. 70% inaccessible. The membrane is thus highly asymmetric with regard to the amount of phospholipid on each side of the membrane. There is also a marked asymmetry of phospholipid composition. Phosphatidylcholine and phosphatidylinositol are enriched in the outer surface, and sphingomyelim is enriched in the inner surface, whereas phosphatidylethanolamine and phosphatidylserine are present in similar proportions in each surface. This distribution is qualitatively different from that previously reported for the human erythrocyte. The close agreement between results obtained with excahnge proteins and phospholipases C demonstrates that the hydrolytic action of these enzymes does not alter phospholipid asymmetry. The nonperturbing nature of the exchange proteins has permitted the rate of transmembrane movement of phospholipids (flip-flop) in the intact virion to be studied. This process could not be detected after 2 days at 37 degrees C. It was estimated that the half-time for flip-flop is indeterminately in excess of 30 days for sphingomyelin and 10 days for phosphatidylcholine at 37 degrees C. These extremely long times provide a simple explanation for the maintenance of transbilayer asymmetry in influenza virions and possibly, other membranes. Since the viral membrane is acquired by budding through the host cell plasma membrane, the transbilayer distribution of phospholipids observed in the virions presumably reflects a similar asymmetric distribution of phospholipids in the host cell surface membrane. Because animal cells in culture do not incorporate extracellular phospholipid, our results demonstrate that individual cells have the capacity to generate asymmetric membranes.

Binding Sites↗
PubMed2007

Diet alters the fatty acid composition of individual phospholipid classes in beef muscle.

The objective of this study was to investigate the effect of diet on the distribution of phospholipid classes and fatty acid profiles of individual phospholipid classes in longissimus muscle of beef. An experiment was established to examine the effect of pasture-based versus concentrate diet offered to two different breeds (German Holstein and German Simmental bulls) to enhance the content of beneficial fatty acids in beef and improve the meat quality for the consumer. High-performance thin-layer chromatography was utilized to separate the phospholipid classes. The fatty acid composition of the individual phospholipid classes was determined by gas chromatography. The main phospholipid classes in the muscle were phosphatidylethanolamine and phosphatidylcholine, representing approximately 60% of the total phospholipids, followed by phosphatidylinositol ranging between 11.8 and 14.8%. The results have shown that the fatty acid profiles in the detected seven phospholipid classes can be affected by different feeding systems. Pasture-based feeding resulted in an enrichment of total and individual n-3 fatty acids in all phospholipid classes of muscle lipids of bulls compared with those fed on concentrate. In contrast, pasture-based diet significantly decreased the proportion of total and individual n-6 fatty acids in phospholipid classes, except in the sphingomyelin fraction. The total saturated fatty acid proportions in the phospholipid classes were different and ranged between 4.5% in the cardiolipin fraction and 50.5% in the sphingomyelin fraction of muscle lipids of bulls. Furthermore, the diet effects on the saturated fatty acid proportion in the different phospholipid classes differ widely. The results have shown that the C18:1 trans and CLA profiles in the detected seven phospholipid classes can be affected by different feeding systems.

Animals↗
PubMed1985

Phospholipid association with the bovine cardiac mitochondrial adenosine triphosphatase.

The association of different phospholipids with a lipid-depleted oligomycin-sensitive ATPase from bovine cardiac mitochondria [Serrano, Kanner & Racker (1976) J. Biol. Chem. 251, 2453-2461] has been examined using three approaches. First, reconstitution of the ATPase with different synthetic diacyl phospholipids resulted in a 2-10-fold stimulation of ATPase specific activity depending upon the particular phospholipid employed. The phospholipid headgroup region displayed the following order of ATPase reactivation potential: dioleoylphosphatidylglycerol greater than dioleoylphosphatidic acid greater than dioleoylphosphatidylcholine. Furthermore, the ATPase showed higher levels of specific activity when reconstituted with dioleoyl phospholipid derivatives compared with dimyristoyl derivatives. Second, examination of the phospholipid remaining associated with the lipid-depleted ATPase upon purification showed that phosphatidylcholine, phosphatidylethanolamine, and diphosphatidylglycerol were present. No relative enrichment of any of these phospholipids (compared with their distribution in submitochondrial particles) was noted. Therefore, no preferential association between the ATPase and any one phospholipid could be found in the mitochondrial ATPase. Third, the sodium cholate-mediated phospholipid exchange procedure was employed for studying the phospholipid requirements of the ATPase. Replacement of about 50% of the mitochondrial phospholipid remaining with the lipid-depleted ATPase could be achieved utilizing either synthetic phosphatidic acid or phosphatidylcholine. Examination of the displaced mitochondrial phospholipid showed that phosphatidylcholine, phosphatidylethanolamine, and diphosphatidylglycerol were replaced with equal facility.

Adenosine Triphosphatases↗
PubMed1986

Phospholipid methylation in starfish spermatozoa is linked to sperm chemoattraction.

The mechanism whereby ovarian peptides cause sperm attraction was studied in the starfish. Phospholipid methylation and protein-O-carboxyl methylation, reactions linked to chemotactic responses in a variety of systems, were studied in starfish sperm. When sperm were preincubated with [methyl-3H]methionine and then exposed to the attractant, a rapid drop in radioactivity occurred in the phospholipid fraction. Methylated phospholipids decreased by 90% in the first 2 sec; however, no change was observed in endogenous methylation of protein carboxyl groups. The effect on phospholipid methylation was dose dependent, with a 40% reduction in radioactive phospholipids in sperm occurring with the minimal amount of attractant necessary to obtain a positive response in a sperm attraction bioassay. Attractants from species of starfish with little or no cross-reactivity in the bioassay had a limited effect on phospholipid methylation. The transmethylase inhibitor, homocysteine, caused a marked decrease in the accumulation of methylated phospholipids under basal conditions, which was correlated with as much as a 50-fold increase in sperm sensitivity to the attractant. The addition of chemoattractant resulted in a reduction in the amount of all individual methylated phospholipids, but the amount of phosphatidylmono[3H]methylethanolamine relative to the other methylated phospholipid decreased by a factor of 4 after stimulation. Homocysteine had the same effect. The reduction in methylated phospholipids by attractants suggests that phospholipid methylation is linked to the mechanism of action of these peptides. Methylation of phospholipids may play a role in the rapid desensitization of sperm cells to the attractant, which would be required for the orientation of the spermatozoa in the gradient of ovarian peptide.

Animals↗
PubMed2000

Scavenger receptor BI transfers major lipoprotein-associated phospholipids into the cells.

The phospholipids of lipoproteins can be transferred to cells by an endocytosis-independent uptake pathway. We analyzed the role of scavenger receptor BI (SR-BI) for the selective cellular phospholipid import. Human monocytes rapidly acquired the pyrene (py)-labeled phospholipids sphingomyelin (SM), phosphatidylcholine, and phosphatidylethanolamine from different donors (low and high density lipoproteins (LDL, HDL), lipid vesicles). The anti-SR-BI antibody directed against the extracellular loop of the membrane protein lowered the cellular import of the phospholipids by 40-80%. The phospholipid transfer from the lipid vesicles into the monocytes was suppressed by LDL, HDL, and apoprotein AI. Transfection of BHK cells with the cDNA for human SR-BI enhanced the cellular import of the vesicle-derived py-phospholipids by 5-6-fold. In the case of the LDL donors, transfer of py-SM to the transfected cells was stimulated to a greater extent than the uptake of the other py-phospholipids. Similar differences were not observed when the vesicles and HDL were used as phospholipid donors. The concentration of LDL required for the half-maximal phospholipid import was close to the previously reported apparent dissociation constant for LDL binding to SR-BI. The low activation energy of the SR-BI-mediated py-phospholipid import indicated that the transfer occurs entirely in a hydrophobic environment. Disruption of cell membrane caveolae by cyclodextrin treatment reduced the SR-BI-catalyzed incorporation of py-SM, suggesting that intact caveolae are necessary for the phospholipid uptake. In conclusion, SR-BI mediates the selective import of the major lipoprotein-associated phospholipids into the cells, the transfer efficiency being dependent on the structure of the donor lipoprotein.

Animals↗
PubMed1959

Phospholipide turnover in microsomal membranes of the pancreas during enzyme secretion.

After incubation of pigeon pancreas slices with P(32) and isolation of various fractions by differential centrifugation the deoxycholate extract of the microsome fraction was found to account for over half of the phospholipide P and over half of the P(32) incorporated into the phospholipides. The remaining phospholipide P and P(32) were fairly evenly distributed in the nuclei, zymogen granules, mitochondria, microsomal ribonucleoprotein particles, and the soluble fraction. When enzyme secretion was stimulated with acetylcholine about two-thirds of the increment in radioactivity in the total phospholipides was found in deoxycholate soluble components of the microsome fraction. The remainder of the increment was distributed in the other fractions. This indicates that the cellular component in which the increase in phospholipide turnover occurs on stimulation of secretion is a membranous structure. Evidence is presented which indicates that the increment in radioactivity in the non-microsomal fractions on stimulation of secretion is due to contamination of these fractions with fragments of the stimulated membranous structure. The distribution of P(32) radioactivity in each of the chromatographically separated phospholipides in the various fractions from unstimulated tissue paralleled the distribution of radioactivity in the total phospholipide fraction, indicating that individual phospholipides are not concentrated in different fractions but are associated together in the membranous structures of the microsome fraction. The major proportion of the stimulation of the turnover of the individual phospholipides also occurred in the microsome fraction. The distribution of radioactivity from glycerol-1-C(14) in the total phospholipides and in the individual phospholipides in the various fractions was similar to the distribution of P(32). In the microsome fraction acetylcholine stimulated the incorporation of glycerol-1-C(14) in each phospholipide which showed a stimulation of P(32) incorporation. The significance of the turnover of phosphatides in microsomal membranes in relation to the mechanism of secretion is discussed.

Acetylcholine↗
PubMed1988

An evaluation of serum high density lipoproteins-phospholipids.

Phospholipids in high density lipoproteins (HDL) is being used as a negative risk indicator of atherosclerosis. Phospholipids in HDL may not demonstrate the actual level of HDL-phospholipids when determined by the precipitation or ultracentrifugal methods, because HDL fractions contain very high density lipoproteins (VHDL) and albumin. In the present study, the true level of phospholipids in HDL was estimated using high performance liquid chromatography (HPLC), and it was compared with the level of phospholipids in HDL determined by the precipitation method. Sera from 18 healthy subjects were used as materials. In the HPLC method, the HDL fraction was extracted making sure that it contained no free albumin, which is albumin not bound to phospholipids. The HDL fraction was separated into subfractions. It was found that phospholipids in the VHDL fraction make a 20.2 +/- 7.3% (mean +/- S.D.) part of the total HDL-phospholipids. A large part of the VHDL fraction was constituted of albumin-bound phospholipids. A significant correlation was observed between HDL-phospholipids determined by the precipitation method, which contain albumin, and the actual HDL fraction phospholipids determined by HPLC, which do not contain VHDL (r = 0.903, p less than 0.01). These results suggest that HDL-phospholipids values determined by the precipitation method give useful clinical data.

Adolescent↗
PubMed1999

Microheterogeneity of beta-2 glycoprotein I: implications for binding to anionic phospholipids.

Considerable interest is currently focused on the interactions of beta-2 glycoprotein I (beta2GPI) and anti-phospholipid antibodies with anionic phospholipids in an attempt to understand the association between these antibodies and clinical diseases such as thrombosis. The interactions of beta2GPI and anionic phospholipids have only been characterized partially, and the physiological role of this glycoprotein remains uncertain. In this study we have explored in detail the physical and phospholipid-binding characteristics of a number of beta2GPI preparations. We have found (i) that perchloric acid-purification methods are damaging to beta2GPI during purification, (ii) that the dissociation constants of the various preparations for phosphatidylserine vary between 0. 1-2 microM and are considerably weaker than previously reported, (iii) that considerable differences in affinity of the various beta2GPI preparations for anionic phospholipids are obtained when comparing anionic phospholipids immobilized to a solid-phase versus phospholipid assembled in unilamellar vesicles, (iv) that the integrity of the fifth domain of beta2GPI is important for binding immobilized anionic phospholipid but not especially important in binding vesicular anionic phospholipid, and (v) that beta2GPI preparations with differing isoelectric species content bind anionic phospholipids differently, suggesting that varying glycosylation and/or protein polymorphisms impact upon phospholipid binding. These results highlight the importance of assessing the determinants of the interaction of beta2GPI with anionic phospholipids assembled in unilamellar vesicles.

Anions↗
PubMed2000

Sterol carrier protein-2 expression alters phospholipid content and fatty acyl composition in L-cell fibroblasts.

The effects sterol carrier protein-2 (SCP-2) expression on L-cell phospholipid levels and fatty acyl composition was assessed using L-cells transfected with the murine cDNA encoding for either the 15 kDa proSCP-2 or 13.2 kDa SCP-2. Expression of these proteins reduced total phospholipid mass (nmol/mg protein) by 24% and reduced the cholesterol to phospholipid ratio 60 and 28%, respectively. In 15 kDa proSCP-2 expressing cells, individual phospholipid class masses, excluding sphingomyelin (CerPCho), were reduced as follows: phosphatidylinositol (PtdIns) and phosphatidylserine (PtdSer) >> ethanolamine glycerophospholipid (EtnGpl) > choline glycerophospholipid (ChoGpl). Furthermore, ethanolamine plasmalogen mass was decreased 25%, while choline plasmalogen mass was elevated 30% in 15 kDa proSCP-2 expressing cells. In 13.2 kDa SCP-2 expressing cells, phospholipid class mass was decreased as follows: PtdIns and PtdSer >> ChoGpl. These changes in phospholipid mass resulted in altered cellular phospholipid composition. Expression of either protein differentially altered the type of fatty acid esterified onto the phospholipids. These effects included a greater proportion of polyunsaturated fatty acids and a reduction in saturated fatty acids, although 15 kDa proSCP-2 expression had a more robust effect on these parameters than did 13.2 kDa SCP-2 expression. In summary, expression of SCP-2 reduced individual phospholipid class mass, except for CerPCho, and altered the fatty acid composition of each phospholipid class examined. These results clearly demonstrate that SCP-2 expression altered basal phospholipid levels, suggesting that SCP-2 can alter the function of endoplasmic reticulum phospholipid synthetic enzymes.

Animals↗
PubMed1991

Synthesis and prostaglandin E2-induced secretion of surfactant phospholipid by isolated gastric mucous cells.

Lipids, particularly surface-active phospholipids, have been proposed to provide an important protective barrier in the gastric mucosa. The predominant surface-active phospholipid in the pulmonary surfactant complex is dipalmitoylphosphatidylcholine. To determine whether the gastric epithelium synthesizes and secretes this phospholipid, primary cultures of canine gastric mucous cells isolated by counterflow elutriation were studied. During the 24-hour period of culture, the gastric mucous cells incorporated 3H-choline into phosphatidylcholine, with dipalmitoylphosphatidylcholine representing 13.8% +/- 0.6% of the phosphatidylcholine synthesized. When mucous cell preparations with greater chief cell contamination were studied, they incorporated significantly less precursor into dipalmitoylphosphatidylcholine. Administration of prostaglandin E2, a cytoprotective agent, to the cultured mucous cells for 1 hour led to a significant increase in phosphatidylcholine release, reaching a maximum of 120.4% +/- 4.2% (P less than 0.001) at 10(-6) mol/L. No significant stimulation of phospholipid release by prostaglandin E2 was seen in the fractions containing a greater proportion of chief cells. To further establish the relationship between mucin and phospholipid secretion, two gastric cancer cell lines, Hs746T and KATO III, were studied. Using immunocytochemical and biochemical techniques, mucin synthesis and secretion were confirmed by these cell lines. The Hs746T cells were significantly more active in the secretion of both mucin and phospholipid than the KATO III cells. The Hs746T line secreted 5.7-fold more mucin and 7.3-fold more phospholipid than KATO III cells during a 24-hour period of culture. The association between mucin and phospholipids in an aqueous solution was also studied. Purified mucin in the concentration of 0.5-2 mg/mL of glycoprotein led to a significant dose-dependent increase in phospholipid solubility, suggesting the formation of a glycoprotein-phospholipid complex. The current studies indicate that the gastric mucous cell is the source of surfactant phospholipids as well as mucin. The synthesis and release of mucin and phospholipid are functions of the mucous cell that play a critical role in the primary defense of gastric epithelium.

1,2-Dipalmitoylphosphatidylcholine↗
PubMed1989

[Effect of insulin on inositol-phospholipid metabolism in isolated rat fat cells].

The effects of insulin on the incorporation of [32P]Pi was studied in isolated rat fat cells. When the cells were incubated with insulin and [32P]Pi, a novel radioactive phospholipid, which migrated slower than phosphatidylinositol 4,5-bisphosphate, was detected on thin layer chromatography. The effect of insulin was both dose- and time-dependent. Pretreatment of insulin with anti-insulin antibody or the cells with anti-insulin receptor sodium vanadate stimulated synthesis of this phospholipid in rat adipocytes. These findings indicate that the synthesis of this phospholipid is stimulated through the insulin receptors. The radioactivity of this phospholipid was recovered in the water phase after deacylation with mild alkaline treatment. When extracted lipid were applied to a silicic acid column, the radioactivity of this phospholipid was eluted with methanol, but not with chloroform. This phospholipid coincided with the product of [32P]-phosphatidylinositol 4-phosphate treated with phospholipase A2 on thin layer chromatography. Quinacrine, a phospholipase A2 inhibitor, suppressed the incorporation of [32P]Pi into this phospholipid on dose dependently. This suggests that this phospholipid is lyso-from. suggests that this phospholipid is lyso-from. Insulin stimulated similarly the incorporation of [3H]myo-inositol into this phospholipid. It indicates that this is an inositol phospholipid. Under the same condition, insulin also stimulated the synthesis of this inositol phospholipid in isolated rat hepatocytes, but not in mouse macrophage. It is possible that this reaction is common in insulin target organ.

Adipose Tissue↗
PubMed1984

Liver ischemia increases the molecular order of microsomal membranes by increasing the cholesterol-to-phospholipid ratio.

An accelerated degradation of phospholipid is the likely basis of irreversible cell injury in ischemia, and the membranes of the endoplasmic reticulum of the liver are a convenient system with which to study the effect of such a disturbance on the structure and function of cellular membranes. In the present report, electron spin resonance spectroscopy has been used to evaluate changes in the molecular ordering of microsomal membrane phospholipids in the attempt to relate the loss of lipid to alterations in membrane structure. The order parameter, S, was calculated from spectra reflecting the anisotropic motion of 12-doxyl stearic acid incorporated into normal and 3-h ischemic microsomal membranes. Over the temperature range 4-40 degrees C, the molecular order (S) of ischemic membranes was increased by 8-10%. This increase was reproduced in the ordering of the phospholipids in liposomes prepared from total lipid extracts of the same membranes. In contrast, after removal of the neutral lipids, liposomes prepared from phospholipids of ischemic and control membranes had the same molecular order. There were no differences in the phospholipid species of control and ischemic membranes or in the fatty acid composition of the phospholipids. In the neutral lipid fraction of ischemic membranes, however, triglycerides and cholesterol were increased compared to control preparations. There were no free fatty acids. The total cholesterol content of the liver was unchanged after 3 h of ischemia. The cholesterol-to-phospholipid ratio of ischemic membranes, however, was increased by 22% from 0.258 to 0.315 as a consequence of the loss of phospholipid. Addition of cholesterol to the control total lipid extracts to give a cholesterol-to-phospholipid ratio the same as in ischemic membranes resulted in liposomes with order parameters similar to those of liposomes prepared from ischemic total lipids. It is concluded that the degradation of the phospholipids of the microsomal membrane results in a relative increase in the cholesterol-to-phospholipid ratio. This is accompanied, in turn, by an increased molecular order of the residual membrane phospholipids.

Animals↗
PubMed1983

Interaction of bovine blood clotting factor Va and its subunits with phospholipid vesicles.

Thrombin-activated factor Va and factor Va subunit binding to large-volume vesicles was investigated by a technique based on the separation by centrifugation of phospholipid-bound protein from the bulk solution. This technique allows the direct measurement of free-protein concentration. It is concluded that the phospholipid binding site on factor Va is located on a basic factor Va subunit with Mr 80 000 (factor Va-LC). The effects of phospholipid vesicle composition, calcium concentration, pH, and ionic strength on the equilibrium constants of factor Va- and factor Va-LC-phospholipid interaction were studied. Factor Va and factor Va-LC binding to phospholipid requires the presence of negatively charged phospholipids. It is further demonstrated that the following occur: (a) Calcium ions compete with factor Va and factor Va-LC for phospholipid-binding sites. (b) The dissociation constant of protein-phospholipid interaction increases with the ionic strength, whereas the maximum protein-binding capacity of the phospholipid vesicle was not affected by ionic strength. (c) The dissociation constant for factor Va-phospholipid interaction depends on pH when the vesicle consists of phosphatidic acid. It is concluded that factor Va-phospholipid interaction is primarily electrostatic in nature, where positively charged groups on the protein directly interact with the phosphate group of net negatively charged phospholipids. The results suggest that factor Va, like factor Xa and prothrombin, has the characteristics of an extrinsic membrane protein.

Animals↗
PubMed1975

Interaction of D-beta-hydroxybutyrate apodehydrogenase with phospholipids.

The interaction of a soluble homogeneous preparation of D-beta-hydroxybutyrate apodehydrogenase with phospholipid was studied in terms of restoration of enzymic activity and complex formation. The purified apoenzyme, which is devoid of lipid, is inactive. It is reactivated specifically by the addition of lecithin or mixtures of phospholipids containing lecithin. Mitochondrial phospholipid, i.e. the mixture of phospholipids in mitochondria, reactivates with the highest specific activity (approximately 100 micromol of DPN reduced/min/mg at 37 degrees and with the greatest efficiency (2.5 to 4 mol of lecithin/mol of enzyme subunit). Each of the lecithins of varying chain length and unsaturation reactivated the enzyme, albeit to differing extents and efficiencies. In general, lecithins containing unsaturated fatty acid moieties reactivated better than those containing the comparable saturated lipid. Optimal reactivation can be obtained for the various lecithins when they are microdispersed together with phosphatidylethanolamine. When the lecithins are added microdispersed together with both phosphatidylethanolamine and cardiolipin, maximal efficiency is obtained. Also, PC6:0 and 8:0 reactivate as soluble molecules, so that a phospholipid bilayer is not necessary to reactivate the enzyme. Complex formation was studied using gel exclusion chromatography. It can be shown that each of the phospholipids which reactivate combines with the apoenzyme. Mitochondrial phospholipid, which reactivates the best, binds most effectively; PC8:0, which reactivates with poor efficiency, can be shown to bind with low affinity, and negligible binding occurs at concentrations which do not reactivate the enzyme. Since the apoenzyme is apparently homogeneous and devoid of phospholipid or detergents, it would appear that reactivation does not involve reversal of inhibition such as by removal of a regulatory subunit or detergent from the catalytic subunit. Rather, we conclude that phospholipid is a necessary and integral portion of this enzyme whose active form is a phospholipid-protein complex. The apoenzyme also forms a complex with phosphatidylethanolamine and/or cardiolipin, which do not reactivate enzymic activity. Salt dissociates such complexes in contrast with the lecithin-apoenzyme complex. Binding of phospholipid is a necessary but not sufficient requisite for enzymic activity. The same energies of activation are obtained from Arrhenius plots for the membrane-bound enzyme and for the purified soluble enzyme reactivated with mitochondrial phospholipid or different lecithins. This observation is compatible with the view that the purified enzyme has not been adversely modified in the isolation. Furthermore, essentially the same energies of activation were obtained for saturated lecithins below their transition temperatures and for unsaturated lecithins above their transition temperatures. Hence, there is no indication that a lipid phase transition occurs to influence the activity of this enzyme.

Animals↗
PubMed2001

Correlation of antiphospholipid antibody recognition with the structure of synthetic oxidized phospholipids. Importance of Schiff base formation and aldol condensation.

The oxidation of low density lipoproteins (LDL) has been correlated with atherogenesis through a variety of pathways. The process involves nonspecific fragmentation, oxidative breakdown, and modification of the lipids and protein of LDL. The process yields a variety of bioactive products, including aldehyde-containing phospholipids, which can cross-react with primary amines (i.e. peptides or phospholipid head groups) to yield Schiff base products. We also demonstrate that such oxidized phospholipid products may further react through a post-oxidation chemical pathway involving aldol condensation. EO6, an IgM monoclonal autoantibody to oxidized phospholipids, blocks the uptake of oxidized LDL (OxLDL) by macrophages. Because the epitope(s) of EO6 also blocks the uptake of OxLDL, a series of oxidized phospholipids, their peptide complexes, and their aldol condensates have been synthesized and characterized, and their antigenicity has been determined. This study defines structural motifs of oxidized phospholipids responsible for antigenicity for EO6. Certain monomeric phospholipids containing short chain fatty acids were antigenic whether oxidized or not in the sn-2 position. However, oxidized phospholipids containing sn-1 long chain fatty acids were not antigenic unless the sn-2 oxidized fatty acid contained an aldehyde that first reacted with a peptide yielding a Schiff base or the sn-2 oxidized fatty acid underwent an aldol type self-condensation. Our data indicate that the phosphorylcholine head group is essential for antigenicity, but its availability depends on the oxidized phospholipid conformation. We suggest that upon oxidation, similar reactions occur in phospholipids on the surface of LDL, generating ligands for macrophage recognition. Synthetic imine adducts of oxidized phospholipids of this type are capable of blocking the uptake of OxLDL.

Antibodies, Antiphospholipid↗
PubMed1987

Alteration of membrane phospholipids in hypertrophied rat salivary glands induced by chronic administration of isoproterenol.

Hypertrophy of parotid and submandibular glands, and elevated phospholipid content per gland in both were induced. However, phospholipid content per tissue weight increased only in the parotid gland. Sublingual gland phospholipids were unaffected with regard to the gland weight and phospholipid content; phospholipid class compositions were altered in each of the three glands, typically an increase of phosphatidylcholine and decreases of sphingomyelin and phosphatidic acid. Concerning fatty-acyl groups in phospholipid, prominent increase of octadecadienoic acid was observed in total and individual phospholipids of both parotid and submandibular glands. However, eicosatetraenoic acid decreased in the total phospholipids of these two glands. In the sublingual gland, phospholipids, fatty-acyl compositions were not changed by isoproterenol. The results suggest that isoproterenol-induced changes of membrane phospholipids are regulated by at least two different effects, one for fatty-acyl groups and another for base moieties of phospholipids.

Animals↗