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PubMed1997

Trimetazidine increases phospholipid turnover in ventricular myocyte.

Trimetazidine (TMZ) is an anti-ischemic compound devoid of hemodynamic effects. It was recently suggested to induce cardiomyocyte protection by a mechanism involving lipid metabolism. The effects of TMZ were evaluated in rats on cardiac lipid composition, and in cultured rat cardiomyocytes on phospholipid metabolism. Rats were treated with TMZ for 4 weeks, and the fatty acid compositions were determined. Treatment with TMZ induced a significant decrease in phospholipid linoleic acid, balanced by a small increase in oleic and stearic acids. These changes were not correlated to alterations in plasma fatty acid composition. Cultured ventricular myocytes were treated with TMZ, 16 and 1 before experimentation. The time-dependent incorporation of radio labelled precursors of membrane phospholipids (3-inositol, 14C-ethanolamine, 14C-choline, 14C-arachidonic acid, 10 mumol/L) was investigated. The cells were harvested 30, 60, 105 or 150 min after precursor addition. In TMZ-cells, arachidonic acid (AA) incorporation was increased in the phospholipids, but not in other lipid fractions. This increase elicited a net increase in the total AA uptake. The incorporation of 3-inositol in the phospholipids was strongly stimulated by TMZ, although the uptake of inositol was not altered. The difference was significant within 30 min, and after 150 min the phospholipid labelling in TMZ cells was higher by 70%. A similar result was obtained with ethanolamine as precursor, which turnover increased by 50% in TMZ-treated cells. Conversely, the incorporation of choline was not significantly affected by the presence of TMZ. In conclusion TMZ appears to interfere with the metabolism of phospholipids in cardiac myocytes in a manner which could indicate an increase of membrane phospholipid turnover.

Animals↗
PubMed2003

Effect of membrane phospholipid composition and charge of the signal peptide of Escherichia coli alkaline phosphatase on efficiency of its secretion.

The secretion of the Escherichia coli alkaline phosphatase with a different charge of signal peptide due to replacement of positively charged Lys(-20) has been studied depending on the phospholipid composition of the membranes and the activity of the translocational ATPase--protein SecA. Changing the signal peptide charge, along with a change in phospholipid composition, has been shown to reduce the efficiency of secretion. In the absence of phosphatidylethanolamine the membrane contains anionic phospholipids only, and the dependence of secretion on the signal peptide charge decreases. The dependence of secretion on membrane phospholipid composition and the signal peptide charge is also determined by the activity of SecA protein. If SecA is inactivated by sodium azide, then the dependence of secretion on anionic phospholipids increases; on the contrary, higher content of anionic phospholipids (in the absence of phosphatidylethanolamine) decreases the dependence of secretion on the SecA activity. The results suggest a direct interaction of positively charged signal peptide with negatively charged membrane phospholipids under initiation of secretion and also interdependent contribution of the signal peptide charge, anionic phospholipids, and translocational ATPase to secretion.

Adenosine Triphosphatases↗
PubMed1991

31P magnetic resonance phospholipid profiles of neoplastic human breast tissues.

Phospholipids from malignant, benign and noninvolved human breast tissues were extracted by chloroform-methanol (2:1) and analysed by 31P MR spectroscopy at 202.4 MHz. Thirteen phospholipids were identified as constituents of the profiles obtained among the 55 tissue specimens analysed. Observed patterns in phospholipid tissues profiles were distinct, allowing qualitative characterisation of the three tissue groups. Multivariate analysis of lysophosphatidylcholine (LPC) and an uncharacterised phospholipid were shown to be independently significant in predicting benign tissue histology as either fibrocystic disease or fibroadenoma in 92% of cases. Univariate analysis of relative mole-percentage of phosphorus concentrations of individual phospholipids using the Scheffé comparison procedure revealed that in malignant tissues, phosphatidylethanolamine was significantly elevated compared to benign (+ 32%) and noninvolved tissues (+ 22%). Phosphatidylinositol (+ 33%) and phosphatidylcholine plasmalogen (PC plas) (+ 25%) were increased in malignant compared to benign and LPC was decreased (-44%) in malignant compared to noninvolved. LPC was significantly depressed (-39%) in benign tissue compared to normal. Phospholipid indices computed to further characterise the three tissue groups showed PC plas/PC elevated in malignant tissue compared to benign and PE plas/PE depressed in malignant tissue compared to noninvolved. These findings support previous investigations reporting that the alkyl-phospholipid analogues of phosphatidylcholine are released by malignant tissues and that levels of ethanolamine are elevated in malignant tissues. Indices describing the choline-containing phospholipids showed that these lipids are depressed significantly in malignant tissue relative to healthy tissue.

Breast↗
PubMed1969

Incorporation of [32P]orthophosphate into brain-slice phospholipids and their precursors. Effects of electrical stimulation.

1. The incorporation of [(32)P]phosphate into phospholipids was measured in slices cut from the pial surface of guinea-pig cerebral cortex; incorporation into the phosphorus of some water-soluble precursors of phospholipid was measured under similar conditions. 2. Slices subjected to overall electrical stimulation at a frequency of 5pulses/sec. differed from control slices in their pattern of phospholipid labelling. After 1hr. of stimulation, incorporation of [(32)P]phosphate into phosphatidylcholine, ethanolamine phospholipid and cardiolipin was respectively 54, 55 and 58% of the control value, and that into phosphatidylinositol was 186% of control. Phosphatidic acid labelling tended to increase with electrical stimulation, but the statistical significance of this change was marginal. Labelling of phosphatidylglycerol and di- and tri-phosphoinositides was not affected significantly by electrical stimulation. 3. Electrical stimulation of the tissue altered the specific radioactivities of water-soluble precursors of phospholipid. 4. The turnover rates of the phosphate groups of phospholipids were estimated approximately from the specific radioactivities of phospholipids and their precursors. Phosphatidylinositol (and its lipid-soluble precursors) showed the largest change in turnover rate in response to electrical stimulation of the tissue; the turnover rates of other lipids were also affected. Changes in the specific radioactivity of phospholipids did not correspond to changes in turnover in these experiments.

Animals↗
PubMed1969

Phospholipid exchange reactions within the liver cell.

1. Isolated rat liver mitochondria do not synthesize labelled phosphatidylcholine from CDP-[(14)C]choline or any phospholipid other than phosphatidic acid from [(32)P]phosphate. The minimal labelling of phosphatidylcholine and other phosphoglycerides can be attributed to microsomal contamination. However, when mitochondria and microsomes are incubated together with [(32)P]phosphate, the phosphatidylcholine, phosphatidylinositol and phosphatidylethanolamine of the reisolated mitochondria become labelled, suggesting a transfer of phospholipids between the two fractions. 2. When liver microsomes or mitochondria containing labelled phosphatidylcholine are independently incubated with the opposite un-labelled fraction, there is a substantial and rapid exchange of the phospholipid between the two membranes. Exchange of phosphatidylinositol also occurs rapidly, whereas phosphatidylethanolamine and phosphatidic acid exchange only slowly. There is no corresponding transfer of marker enzymes. The transfer of phosphatidylcholine does not occur at 0 degrees , and there is no requirement for added substrate, ATP or Mg(2+), but the omission of a heat-labile supernatant fraction markedly decreases the exchange. 3. After intravenous injection of [(32)P]phosphate, short-period labelling experiments of the individual phospholipids of rat liver microsomes and mitochondria in vivo give no evidence for a similar exchange process. However, the incubation of isolated microsomes and mitochondria with [(32)P]phosphate also fails on reisolation of the fractions to demonstrate a precursor-product relationship between the individual phospholipids of the two membranes. 4. The intraperitoneal injection of [(32)P]phosphate results in a far greater proportion of the dose entering the liver than does intravenous administration. After intraperitoneal administration of [(32)P]phosphate the specific radioactivities of the individual phospholipids are in the order microsomes > outer mitochondrial membrane > inner mitochondrial membrane. 5. The incorporation of (32)P into cardiolipin is very slow both in vivo and in vitro. After labelling in vivo the radioactivity in the cardiolipin persists compared with that of the other phospholipids, whose specific radioactivities in the microsomes and mitochondrial fragments decay at a similar rate to that of the acid-soluble phosphate pool. 6. The possibility of phospholipid exchange processes occurring in the liver cell in vivo is discussed, and it is suggested that only a small but highly labelled part of the endoplasmic-reticulum lipoprotein pool is involved in the transfer.

Animals↗
PubMed1971

The turnover of myelin phospholipids in the adult and developing rat brain.

1. Inorganic [(32)P]phosphate, [U-(14)C]glycerol and [2-(14)C]ethanolamine were injected into the lateral ventricles in the brains of adult rats, and the labelling of individual phospholipids was followed over 2-4 months in both a microsomal and a highly purified myelin fraction. 2. All the phospholipids in myelin became appreciably labelled, although initially the specific radioactivities of the microsomal phospholipids were somewhat higher. Eventually the specific radioactivities in microsomal and myelin phospholipids fell rapidly at a rate corresponding to the decline of radioactivity in the acid-soluble pools. 3. Equivalent experiments carried out in developing rats with [(32)P]phosphate administered at the start of myelination showed some persistence of phospholipid labelling in the myelin, but this could partly be attributed to the greater retention of (32)P in the acid-soluble phosphorus pool and recycling. 4. It is concluded that a substantial part of the phospholipid molecules in adult myelin membranes is readily exchangeable, although a small pool of slowly exchangeable material also exists. 5. A slow incorporation into or loss of labelled precursor from myelin phospholipids does not necessarily give a good indication of the rate of renewal of the molecules in the membrane. As presumably such labelled molecules originate by exchange with those in another membrane site (not necessarily where synthesis occurs) it is only possible to calculate the turnover rate in the myelin membrane if the behaviour of the specific radioactivity with time of the phospholipid molecules in the immediate precursor pool is known.

Amino Alcohols↗
PubMed1972

Exchange of phospholipids between brain membranes in vitro.

1. When unlabelled mitochondria from guinea-pig brain were incubated with a (32)P-labelled microsomal fraction from brain there was a transfer of phospholipid to the mitochondria, which could not be accounted for by an aggregation of microsomes and mitochondria or an exchange with microsomes contaminating the mitochondria. Under similar circumstances there was a transfer of phospholipid from (32)P-labelled mitochondria to microsomes, indicating that the process was one of exchange. 2. The transfer from microsomes was greatly stimulated by a non-dialysable heat-labile macromolecular component in the brain supernatant fraction but not by the concentration of the particulate fractions. 3. Phospholipid-exchange processes occurred most readily between pH7 and 7.5 and were inhibited by the presence of myelin and on the addition of lysophosphatidylcholine. 4. The rates of transfer of individual phospholipids from brain microsomes to mitochondria were similar. 5. (32)P-labelled microsomes could slowly donate phospholipid to the isolated synaptosomal (nerve-ending) fraction but the phospholipids of the myelin fraction did not exchange. 6. Subfractionation of the synaptosomal fraction after [(32)P]phospholipid transfer showed that the mitochondria were most actively labelled during the incubation. All of the isolated individual synaptosomal membranes were capable of acquiring phospholipid on incubation with a (32)P-labelled brain supernatant fraction although a greater percentage was again exchanged by the mitochondrial fraction.

Animals↗
PubMed1993

Binding of phospholipase C delta 1 to phospholipid vesicles.

Binding of phospholipase C delta 1 (PLC delta) to phospholipid vesicles was studied using large, unilamellar phospholipid vesicles (LUVs). PLC delta bound weakly to vesicles composed of phosphatidylserine (PS) or phosphatidylcholine (PC) or phosphatidylethanolamine (PE) + PC, and even more weakly to vesicles composed of phosphatidylinositol. The enzyme bound strongly to LUVs composed of PE + PC and phosphatidylinositol 4,5-bisphosphate (PIP2) or sphingomyelin (SM). Binding of 50% of PLC delta occurred at 0.25 nmol/ml PIP2 when LUVs composed of PE + PC (molar ratio of 80:20), plus various amounts of PIP2, were used at a constant phospholipid concentration of 300 nmol/ml. When LUVs composed of PE + PC + PIP2 (molar ratio of 79:20:1) were tested as a function of increasing phospholipid concentration, 50% binding of PLC delta occurred at 1.2 nmol/ml PIP2 and 120 nmol/ml total phospholipid. Similar measurements were conducted with other phospholipids and PIP2 at a molar ratio of 99:1. These showed that 50% binding of PLC delta occurred at a level of 0.9 nmol/ml PIP2 with 80 nmol/ml PC; at 2.2 nmol/ml PIP2 with 170 nmol/ml PS; at 4.2 nmol/ml PIP2 with 320 nmol/ml PI; and at 0.26 nmol/ml PIP2 with 20 nmol/ml total liver phospholipids. Binding to phosphatidylinositol 4-phosphate was much weaker. When LUVs composed of PE + PC + SM (molar ratio 48:12:40) were tested as a function of increasing phospholipid concentration, 50% binding of PLC delta occurred at a level of 96 nmol/ml SM. This is well below the concentration of SM that can be calculated to face the cytosol. Binding of PLC delta to LUVs decreased as the temperature was lowered from 37 degrees C to 0 degree C. Thus PLC delta shows a high degree of specificity for binding to PIP2 and SM. Under physiological conditions a considerable fraction of PLC delta may be bound to cellular membranes, either in an inactive form if bound to PIP2 at low resting Ca2+ concentrations, or in the inhibited form if bound to SM.

Animals↗
PubMed1997

Interaction of caldesmon with endoplasmic reticulum membrane: effects on the mobility of phospholipids in the membrane and on the phosphatidylserine base-exchange reaction.

We have previously demonstrated by tryptophan fluorescence the interaction of caldesmon with anionic phospholipid vesicles [Czurylo, Zborowski and Dabrowska (1993) Biochem. J. 291, 403-408]. In the present work we investigated the interaction of caldesmon with natural-membrane (rat liver endoplasmic reticulum) phospholipids by co-sedimentation assay. The results indicate that 1 mol of caldesmon binds approx. 170 mol of membrane phospholipids with a binding affinity constant of 7.3 x 10(6) M-1. The caldesmon-membrane phospholipid complex dissociates with increasing salt concentration and in the presence of Ca2+/calmodulin. As indicated by EPR measurements of membrane lipids labelled with 5-doxyl stearate and TEMPO-phosphatidylethanolamine, binding of caldesmon results in an increase in mobility of the acyl chains (in the region of carbon 5) and a decrease in polar headgroup mobility of phospholipids. Interaction of caldesmon with phospholipids is accompanied by inhibition of phosphatidylethanolamine synthesis via a phospholipid base-exchange reaction, with phosphatidylserine as substrate. This shows that, of the endoplasmic reticulum membrane phospholipids, the main target of caldesmon is phosphatidylserine.

Animals↗
PubMed2000

Encapsulated fish oil enriched in alpha-tocopherol alters plasma phospholipid and mononuclear cell fatty acid compositions but not mononuclear cell functions.

BACKGROUND: Several studies have reported that dietary fish oil (FO) supplementation alters cytokine production and other functional activities of peripheral blood mononuclear cells (PBMC). However, few of these studies have been placebo controlled and few have related the functional changes to alterations in PBMC fatty acid composition PATIENTS AND METHODS: Healthy subjects supplemented their diets with 9 g day-1 of encapsulated placebo oil (3 : 1 mix of coconut and soybean oils), olive oil (OO), safflower oil (SO), evening primrose oil (EPO) or FO [providing 2.1 g eicosapentaenoic acid (EPA) plus 1.1 g docosahexaenoic acid (DHA) per day] for 12 weeks; the capsules also provided 205 mg alpha-tocopherol per day. Blood was sampled at 4-weekly intervals and plasma and PBMC prepared. Plasma phospholipid and PBMC fatty acid composition, plasma alpha-tocopherol and thiobarbituric acid-reactive substance concentrations, plasma total antioxidant capacity, the proportions of different PBMC subsets, the proportions of PBMC expressing the adhesion molecules CD2, CD11b and CD54, and PBMC functions (lymphocyte proliferation, natural killer cell activity, cytokine production) were measured. All measurements were repeated after a 'washout' period of 8 weeks. RESULTS: The placebo, OO and SO capsules had no effect on plasma phospholipid or PBMC fatty acid composition. The proportion of dihomo-gamma-linolenic acid in plasma phospholipids was elevated in subjects taking EPO and was decreased in subjects taking FO. There was no appearance of gamma-linolenic acid in the plasma phospholipids or PBMC in subjects taking EPO. There was a marked increase in the proportion of EPA in the plasma phospholipids (10-fold) and PBMC (four-fold) of subjects taking FO supplements; this increase was maximal after 4 weeks of supplementation. There was an increase in the proportion of DHA in plasma phospholipids and PBMC, and an approximately 20% decrease in the proportion of arachidonic acid in plasma phospholipids and PBMC, during FO supplementation. Plasma concentrations of alpha-tocopherol were significantly elevated during supplementation in all subjects and returned to baseline values after the washout period. There were no effects of supplementation with any of the capsules on total plasma antioxidant activity or plasma thiobarbituric acid-reactive substances or on the proportion of different PBMC subsets, on the proportion of PBMC expressing adhesion molecules, on natural killer cell activity, on the proliferation of mitogen-stimulated whole blood cultures or PBMC, or on the ex vivo production of a range of cytokines by whole blood cultures or PBMC cultures stimulated by either concanavalin A or lipopolysaccharide. CONCLUSION: Supplementation of the diet with 3.2 g EPA plus DHA per day markedly alters plasma phospholipid and PBMC fatty acid compositions. The lack of effect of FO upon PBMC functions may relate to the level of alpha-tocopherol included in the supplements.

Adult↗
PubMed2001

Dispensable nature of phosphatidylglycerol in Escherichia coli: dual roles of anionic phospholipids.

The major anionic phospholipids of Escherichia coli, phosphatidylglycerol (PG) and cardiolipin (CL), have been considered to be indispensable for essential cellular functions, such as the initiation of DNA replication and translocation of proteins across the cytoplasmic membrane. However, we successfully constructed a null pgsA mutant of E. coli that had undetectable levels of PG and CL if the major outer membrane lipoprotein was deficient, clearly indicating that these anionic phospholipids are not indispensable. In the null mutant, we observed the accumulation of phosphatidic acid, an acidic biosynthetic precursor. This suggests a functionally substitutable nature of these anionic phospholipids and allows us to formulate a dual role model for the physiological roles of the anionic phospholipids in E. coli. The anionic phospholipids may play dual roles in E. coli as (i) substrates for head group-specific enzyme reactions, albeit the viability of null PG mutants indicates that the products of head group-specific reactions are not essential; and (ii) those that are replaceable, partly or entirely, by other phospholipids bearing net negative charges, because of their rather loose head group specificity. These two aspects of the physiological roles of anionic phospholipids are discussed with special reference to the phospholipids of other bacteria and eukaryotic organelles.

Anions↗
PubMed1999

Phospholipid bound to the flavohemoprotein from Alcaligenes eutrophus.

The structurally characterized flavohemoprotein from Alcaligenes eutrophus (FHP) contains a phospholipid-binding site with 1-16 : 0-2-cyclo-17 : 0-diacyl-glycerophospho-ethanolamine and 1-16 : 0-2-cyclo-17 : 0-diacyl-glycerophospho-glycerol as the major occupying compounds. The structure of the phospholipid is characterized by its compact form, due to the -sc/beta/-sc conformation of the glycerol and the nonlinear arrangement of the sn-1- and sn-2-fatty acid chains. The phospholipid-binding site is located adjacent to the heme molecule at the bottom of a large cavity. The fatty acid chains form a large number of van der Waal's contacts with nonpolar side chains, whereas the glycerophosphate moiety, which points towards the entrance of the channel, is linked to the protein matrix by polar interactions. The thermodynamically stable globin module of FHP, obtained after cleaving off the oxidoreductase module, also contains the phospholipid and can therefore be considered as a phospholipid-binding protein. Single amino acid exchanges designed to decrease the lipid-binding site revealed both the possibility of blocking incorporation of the phospholipid and its capability to evade steric barriers. Conformational changes in the phospholipid can also be induced by binding heme-ligating compounds. Phospholipid binding is not a general feature of flavohemoproteins, because the Escherichia coli and the yeast protein exhibit less and no lipid affinity, respectively.

Alcaligenes↗
PubMed1999

Structure of the Alzheimer beta-amyloid peptide (25-35) and its interaction with negatively charged phospholipid vesicles.

The secondary structure of amyloid betaAP(25-35) peptide was studied in pure form and in the presence of different phospholipid vesicles, by using Fourier transform infrared spectroscopy (FT-IR). Pure peptide aggregated with time, forming fibrils with beta-structure. Phospholipid vesicles formed by negatively charged phospholipids such as 1,2-dimyristoyl-sn-glycerol-3-phospho-L-serine (Myr2PtdSer), 1,2-dimyristoyl-sn-glycerol-3-phospho-rac-1-glycerol (Myr2PtdGro) and 1,2-dimyristoyl-sn-glycerol 3-phosphate (Myr2PtdH), greatly accelerated the aggregation of the peptide. However, the presence of vesicles formed by the zwitterionic phospholipid, 1, 2-dimyristoyl-sn-glycerol-3-phosphocholine (Myr2PtdCho), slowed down the aggregation process. Differential scanning calorimetry (DSC) measurements showed that the effect of betaAP(25-35) on the gel to crystal liquid phase transition was small at neutral pH for negatively charged phospholipids and practically nil for Myr2PtdCho. In the case of Myr2PtdSer the effect was also zero at pH 9 but the effect was large at pH 3. The effect on Myr2PtdH was not, however, very dependent on pH. These results were fully confirmed by the observation through FT-IR of the change with temperature of the CH2 antisymmetric stretching vibration. The case of Myr2PtdGro was special as this phospholipid presents polymorphism giving solid quasicrystalline phases when it is not sufficiently hydrated, and it is remarkable that betaAP(25-35) was able to induce the formation of crystalline phases in samples prepared through a method which ensure a good hydration of phospholipid. These results show that the interaction of amyloid betaAP(25-35) peptide with phospholipids is based on electrostatic interactions, that these interactions favour the aggregation of the peptides, and that the presence of the aggregates may disturb the lipid-water interphase of the membrane.

Alzheimer Disease↗
PubMed1995

Decrease of brain phospholipid synthesis in free-moving n-3 fatty acid deficient rats.

The autoradiographic method with [14C]-docosahexaenoic acid ([14C]22:6 n-3) was used to determine whether a diet deficient in n-3 fatty acids, inducing a decrease in 22:6 n-3 circulating level, was associated with changes in local rates of phospholipid synthesis in the rat brain. As compared with rats fed a normal diet (peanut plus rapeseed oil), a n-3 fatty acid deficiency [peanut oil group (P group)] induced a generalized decrease (-35 to -76%) of 22:6 n-3 incorporation rates into phospholipids in all the regions examined. This effect was confirmed by using [3H]22:6 n-3 infusion by biochemical analysis and quantifications corrected for the contribution of docosahexaenoate derived from lipid store recycling to the unesterified pool, taken as the precursor pool for phospholipid synthesis in the whole brain. In normal or n-3 fatty acid-deficient rats, the values of the brain-to-plasma 22:6 n-3 specific activity ratio (psi) were similar (0.03), indicating that a considerable endogenous source of 22:6 n-3 (97%), likely derived from phospholipid degradation, dilutes the specific activity of the tracer coming from plasma. Using the specific activity of 22:6 n-3 in plasma instead of brain would thus lead to a gross underestimation of the rate of phospholipid synthesis. The results also demonstrate that the pattern of 14C or 3H distribution in brain lipids was not modified by the n-3 fatty acid-deficient diet. The major lipids labeled were phospholipids, particularly phosphatidylethanolamine. Nevertheless, the unesterified 22:6 n-3 concentrations in plasma and brain were significantly reduced (eight-and threefold, respectively) in the P group. In addition, the proportion of 22:6 n-3 in the brain total lipid fraction, total phospholipids, and phosphatidylcholine, -ethanolamine, and -serine was significantly decreased in n-3 fatty acid-deficient rats. This was partially compensated for by an increase in the 22:5 n-6 level. These results are discussed in relation to the limitation of 22:6 n-3 use to quantify, by the quantitative autoradiographic method, changes in local rates of phospholipid synthesis in rat brain.

Animals↗
PubMed1995

Specific activity of brain palmitoyl-CoA pool provides rates of incorporation of palmitate in brain phospholipids in awake rats.

In vivo rates of palmitate incorporation into brain phospholipids were measured in awake rats following programmed intravenous infusion of unesterified [9,10-3H]palmitate to maintain constant plasma specific activity. Animals were killed after 2-10 min of infusion by microwave irradiation and analyzed for tracer distribution in brain phospholipid and phospholipid precursor, i.e., brain unesterified palmitate and palmitoyl-CoA, pools. [9,10-3H]Palmitate incorporation into brain phospholipids was linear with time and rapid, with > 50% of brain tracer in choline-containing glycerophospholipids at 2 min of infusion. However, tracer specific activity in brain phospholipid precursor pools was low and averaged only 1.6-1.8% of plasma unesterified palmitate specific activity. Correction for brain palmitoyl-CoA specific activity increased the calculated rate of palmitate incorporation into brain phospholipids (0.52 nmol/s/g) by approximately 60-fold. The results suggest that palmitate incorporation and turnover in brain phospholipids are far more rapid than generally assumed and that this rapid turnover dilutes tracer specific activity in brain palmitoyl-CoA pool owing to release and recycling of unlabeled fatty acid from phospholipid breakdown.

Animals↗
PubMed1995

Properties of Acanthamoeba myosin I heavy chain kinase bound to phospholipid vesicles.

The actin-activated Mg(2+)-ATPase and in vitro motility activities of the three Acanthamoeba myosin I isozymes depend upon phosphorylation of their single heavy chains by myosin I heavy chain kinase. Previously, the kinase had been shown to be activated by autophosphorylation, which is enhanced by acidic phospholipids, or simply by binding to purified plasma membranes in the absence of significant autophosphorylation. In this paper, we show that the rate of phosphorylation of myosin I by unphosphorylated kinase is approximately 20-fold faster when both the myosin I and the kinase are bound to acidic phospholipid vesicles than when both are soluble. This activation is not due to an increase in the local concentrations of vesicle-bound kinase and myosin I. Thus, acidic phospholipids, like membranes, can activate myosin I heavy chain kinase in the absence of significant autophosphorylation, i.e. membrane proteins are not required. Kinetic studies show that both binding of kinase to phospholipid vesicles and autophosphorylation of kinase in the absence of phospholipid increase the Vmax relative to soluble, unphosphorylated kinase with either an increase in the apparent Km (when myosin I is the substrate) or no significant change in Km (when a synthetic peptide is the substrate). Kinetic data showed that autophosphorylation of phospholipid-bound kinase is both intermolecular and intervesicular, and that phosphorylation of phospholipid-bound myosin I by phospholipid-bound kinase is also intervesicular even when the kinase and myosin are bound to the same vesicles. The relevance of these results to the activation of myosin I heavy chain kinase and phosphorylation of myosin I isozymes in situ are discussed.

Acanthamoeba↗
PubMed1997

Conformational change of cytochrome P450 1A2 induced by phospholipids and detergents.

Recently, it was reported that the activity of rabbit P450 1A2 is markedly increased at elevated salt concentration (Yun, C-H., Song, M., Ahn, T., and Kim, H. (1996) J. Biol. Chem. 271, 31312-31316). The activity increase of P450 1A2 coincides with the raised alpha-helix content and decreased beta-sheet content. The presence of phospholipid magnified this effect. Here, possible structural change of rabbit P450 1A2 accompanying the phospholipid-induced increase in its enzyme activity was investigated by circular dichroism, fluorescence spectroscopy, and absorption spectroscopy. Studies with the reconstituted system supported by cumene hydroperoxide or NADPH showed that the P450 1A2 activities were found to be dependent on the head group and hydrocarbon chain length of phospholipid. Phosphatidylcholines having short hydrocarbon chains with a carbon number of 8-12 were very efficient for reconstitution of the P450-catalyzed reactions supported by both cumene hydroperoxide and NADPH. It was found that the phospholipid increased the alpha-helix content and lowered the beta-sheet content of P450. Intrinsic fluorescence intensity is also increased in the presence of phospholipid. The low spin iron configuration of P450 1A2 shifted toward the high spin configuration by most of the phospholipids in the endoplasmic reticulum. Some synthetic phospholipids having short hydrocarbon chains with a carbon number of 10-12 caused a shift in the spin equilibrium of P450 1A2 toward low spin. The effect of detergents on the activity and conformation of P450 1A2 was also studied. It was found that the addition of detergents to P450 1A2 solution increased the enzyme activity of P450 1A2. Detergents also increased the alpha-helix content and lowered the beta-sheet content of P450 1A2. Intrinsic fluorescence emissions also increased with the presence of detergents. Octyl glucoside and deoxycholate caused a shift toward high spin. On the other hand, cholate caused a shift toward low spin. It was found that the activity increase of rabbit P450 1A2 coincides with the conformational change including raised alpha-helix content. It is proposed that the interaction with the phospholipid molecules surrounding P450 1A2 in the endoplasmic reticulum is important for a functional conformation of P450 1A2 in a monooxygenase system including NADPH-P450 reductase.

Animals↗
PubMed1999

Studies of the role of group VI phospholipase A2 in fatty acid incorporation, phospholipid remodeling, lysophosphatidylcholine generation, and secretagogue-induced arachidonic acid release in pancreatic islets and insulinoma cells.

An 84-kDa group VI phospholipase A2 (iPLA2) that does not require Ca2+ for catalysis has been cloned from Chinese hamster ovary cells, murine P388D1 cells, and pancreatic islet beta-cells. A housekeeping role for iPLA2 in generating lysophosphatidylcholine (LPC) acceptors for arachidonic acid incorporation into phosphatidylcholine (PC) has been proposed because iPLA2 inhibition reduces LPC levels and suppresses arachidonate incorporation and phospholipid remodeling in P388D1 cells. Because islet beta-cell phospholipids are enriched in arachidonate, we have examined the role of iPLA2 in arachidonate incorporation into islets and INS-1 insulinoma cells. Inhibition of iPLA2 with a bromoenol lactone (BEL) suicide substrate did not suppress and generally enhanced [3H]arachidonate incorporation into these cells in the presence or absence of extracellular calcium at varied time points and BEL concentrations. Arachidonate incorporation into islet phospholipids involved deacylation-reacylation and not de novo synthesis, as indicated by experiments with varied extracellular glucose concentrations and by examining [14C]glucose incorporation into phospholipids. BEL also inhibited islet cytosolic phosphatidate phosphohydrolase (PAPH), but the PAPH inhibitor propranolol did not affect arachidonate incorporation into islet or INS-1 cell phospholipids. Inhibition of islet iPLA2 did not alter the phospholipid head-group classes into which [3H]arachidonate was initially incorporated or its subsequent transfer from PC to other lipids. Electrospray ionization mass spectrometric measurements indicated that inhibition of INS-1 cell iPLA2 accelerated arachidonate incorporation into PC and that inhibition of islet iPLA2 reduced LPC levels by 25%, suggesting that LPC mass does not limit arachidonate incorporation into islet PC. Gas chromatography/mass spectrometry measurements indicated that BEL but not propranolol suppressed insulin secretagogue-induced hydrolysis of arachidonate from islet phospholipids. In islets and INS-1 cells, iPLA2 is thus not required for arachidonate incorporation or phospholipid remodeling and may play other roles in these cells.

Animals↗