Substrate specificity of lysophospholipase-transacylase from rat lung and its action on various physical forms of lysophosphatidylcholine

Lysophospholipase-transacylase (lysolecithin acylhydrolase, EC 3.1.1.5) from rat lung catalyzes the transfer of acyl groups from lysophosphatidylcholine to either water or another molecule of lysophosphatidylcholine. Studies on the substrate specificity of the purified enzyme showed that a phosphate group in the substrate is essential for enzymatic activity; monoacylglycerol is not hydrolyzed, nor does it serve as an acceptor of acyl groups. The influence of the acyl chain in lysophosphatidylcholine was investigated by using mixtures of differently labelled lysophosphatidylcholine species, or by studying the transfer of [1-¹⁴C]Palmitate from [1-¹⁴C]palmitoylpropane (1,3)diol-phosphocholine to various 1-acyl-sn-glycero-3-phosphocholines. Lysophosphatidylcholines with acyl chains comprised of ten or more C-atoms were found to serve as acyl acceptors. This finding was used to determine the action of the enzyme on 1-[1-¹⁴C]lauroyl- and 1[1-¹⁴C]myristoyl-sn-glycero-3-phosphocholine both below and above the critical micelle concentration of the substrate. Monomeric substrate was effectively hydrolyzed, but the transacylase activity of the enzyme was only expressed when substrate micelles were present. Likewise, no transacylase activity was found when lysophosphatidylcholine was embedded in liposomal membranes prepared from lung total lipids. These findings, which persist with crude enzyme preparations (100 000 × g supernatant), are discussed in relation to the putative function of the lysophospholipase-transacylase in the synthesis of disaturated phosphatidylcholine in lung.     

Comparison of albumin-mediated release of lysophosphatidylcholine and lysophosphatidylethanolamine from cultured rat hepatocytes.

We have investigated the albumin-stimulated release from cultured rat hepatocytes of lysophosphatidylcholine derived from methylation of phosphatidylethanolamine and of lysophosphatidylethanolamine. In the absence [corrected] of albumin, neither lysophosphatidylethanolamine nor lysophosphatidylcholine was released into the culture medium. Albumin stimulated the accumulation of both phospholipids in the medium. After 2 h, 14.1 nmol of lysophosphatidylcholine and 2.0 nmol of lysophosphatidylethanolamine per 3 x 10(6) cells had accumulated in the medium. The rate of release of [3H]ethanolamine-labelled lysophosphatidylethanolamine was rapid in the first 2 h and then was decreased, whereas there was a 1 h lag in the release of [3H]ethanolamine-labelled lysophosphatidylcholine. This apparent lag probably reflected the time necessary for the synthesis of phosphatidylcholine from phosphatidylethanolamine in the cells. Albumin caused a decrease in labelled cellular lysophosphatidylethanolamine and lysophosphatidylcholine which only partially accounted for the accumulation of the labelled phospholipids in the medium. Albumin also stimulated the release of labelled phosphatidylethanolamine (almost 3-fold) and phosphatidylcholine (2-fold) into the medium. There was no detectable change in the labelling of the cellular pools of these phospholipids, most likely owing to the large amounts in the cells compared with the medium. The labelled lysophospholipids did not arise from catabolism of the parent phospholipid in the medium. Analysis of the fatty acids of the secreted lysophospholipids showed a preferential release of unsaturated fatty acyl species of lysophosphatidylcholine, whereas lysophosphatidylethanolamine contained similar amounts of saturated and unsaturated fatty acids.     

Localization of lysophosphatidylcholine in bovine chromaffin granules.

One of the unique features of the chromaffin granule membrane is the presence of about 17 mol% lysophosphatidylcholine. Lysophosphatidylcholine isolated from the granules could be degraded by approx. 94% by lysophospholipase. This result is consistent with chemical analyses data showing that about 9% of this lysophospholipid is 1'-alkenyl glycerophosphocholine. The localization of the acylglycerophosphocholine in the chromaffin granule membrane was studied by using pure bovine liver lysophospholipases. In intact granules only about 10% of the total lysophosphatidylcholine was directly available for enzymic hydrolysis. In contrast, when granule membranes (ghosts) were treated with lysophospholipases approx. 60% of the lysophosphatidylcholine was deacylated. These values did not increase after pre-treatment of intact granules or ghosts with trypsin. Added 1-[1-14C]palmitoyl-sn-glycero-3-phosphocholine did not mix with the endogenous lysophosphatidylcholine pool(s) and remained completely accessible to added lysophospholipases.     

The uptake and metabolism of plasma lysophosphatidylcholine in vivo by the brain of squirrel monkeys

1. Adult squirrel monkeys were injected intravenously with doubly labelled lysophosphatidylcholine (a mixture of 1-[1-(14)C]palmitoyl-sn-glycero-3-phosphorylcholine and 1-acyl-sn-glycero-3-phosphoryl[Me-(3)H]choline; (3)H:(14)Cratio 3.75) complexed to albumin, and the incorporation into the brain was studied at times up to 3h. 2. After 20min, 1% of the radioactivity injected as lysophosphatidylcholine had been taken up by the brain. 3. Approx. 70% of the doubly labelled lysophosphatidylcholine taken up by both grey and white matter was converted into phosphatidylcholine, whereas about 30% was hydrolysed. 4. The absence of significant radioactivity in the phosphatidylcholine, free fatty acid and water-soluble fractions of plasma up to 30min after injection of doubly labelled lysophosphatidylcholine rules out the possibility that the rapid labelling of these compounds in brain could be due to uptake from or exchange with their counterparts in plasma. 5. The similarity between the (3)H:(14)C ratios of brain phosphatidylcholine and injected lysophosphatidylcholine demonstrates that formation of the former occurred predominantly via direct acylation. 6. Analysis of the water-soluble products from lysophosphatidylcholine catabolism revealed that appreciable glycerophosphoryl-[Me-(3)H]choline did not accumulate in the brain and that radioactivity was incorporated into choline, acetylcholine, phosphorylcholine and betaine. 7. The role of plasma lysophosphatidylcholine as both a precursor of brain phosphatidylcholine and a source of free choline for the brain is discussed.     

Acylation of lysophosphatidylcholine in bovine heart muscle microsomes: purification and kinetic properties of acyl-CoA:1-acyl-sn-glycero-3-phosphocholine O-acyltransferase

Bovine heart muscle microsomes rapidly convert lysophosphatidylcholine (LPC) into phosphatidylcholine (PC) in the presence of oleoyl-CoA. Both substrates are incorporated into the product, although the rate of incorporation of radiolabel into PC from 1-[14C]palmitoyl-LPC was approximately threefold higher than the rate of incorporation from [14C]oleoyl-CoA. Furthermore, the rate of incorporation of radiolabel from [14C]LPC was stimulated fivefold by the presence of oleoyl-CoA. These results demonstrate the presence of both acyl-CoA:1-acyl-sn-glycero-3-phosphocholine O-acyltransferase (EC 2.3.1.23) and an LPC:LPC transacylase (EC 3.1.1.5) in microsomes. Separation of the two enzymatic activities and purification of the acyltransferase was achieved by a procedure involving extraction with 3-[3-cholamidopropyl)dimethylammonio)-1-propanesulfonate detergent and chromatography on DEAE-cellulose, Reactive blue agarose, and Matrex gel green A. The isolated acyltransferase was a single species of 64,000 Da as judged by polyacrylamide gel electrophoresis in the presence of dodecyl sulfate. The substrate specificity of the enzyme was studied by using a series of lysophospholipids as acyl acceptors and acyl-CoA derivatives as acyl donors. The enzyme was catalytically active with LPC as acyl acceptor but displayed little or no activity with lysophosphatidylethanolamine, lysophosphatidylinositol, or lysophosphatidylserine. Of the LPC derivatives tested, the highest activity was obtained with 1-palmitoyl-LPC. Wider specificity was exhibited for the nature of the acyl donor, for which arachidonoyl-CoA, linoleoyl-CoA, and oleoyl-CoA were highly active substrates. These properties of the acyltransferase are in accord with a role of the enzyme in determining the composition of PC in myocardium.     

Lipid metabolism by the gall-bladder. I. The in situ uptake and metabolism of lysophosphatidylcholine

Accumulation of lysophosphatidylcholine in gall-bladder bile is involved in the pathogenesis of acute cholecystitis. [1-14C]oleoyl- or [1-14C]palmitoyl-lysophosphatidylcholine was thus instilled in the in situ guinea pig gall-bladder and the absorption and metabolism of the lipid were determined. We found that, after 6 h instillation, 53% of the oleoyl derivative was adsorbed by the gall-bladder, whereasee only 37% of the palmitoyl derivative was absorbed. Although some differences in the metabolism of these two lipids were observed, a major portion of the absorbed radioactivity was found in the gall-bladder wall as phosphatidylcholine. To determine the mechanism of phosphatidylcholine formation from lysophosphatidylcholine by the gall-bladder mucosa, we used lysophosphatidylcholine which was labelled in the fatty acid moiety with 14C and in the choline moiety with 3H. Our data suggest that the mechanism of phosphatidylcholine formation from lysophosphatidylcholine involved acylation with an acyl donor other than a second molecule of lysophosphatidylcholine. We hypothesize that this mechanism as well as others described serve to prevent accumulation of lysophosphatidylcholine within the gall-bladder lumen and thus prevent damage to the gall-bladder mucosa.     

Coenzyme A-dependent and -independent acyl transfer between dog heart microsomal phospholipids

We have recently shown that dog heart microsomes catalyze the transfer of acyl groups from the sn-2 position of exogenous phosphatidylcholine to lysophosphatidylethanolamine with strong preference for arachidonate over linoleate (Biochem. Biophys. Res. Commun. 129, 381-388 (1985)). We now report that the addition of 0.5 mM CoA enhances the acyl transfer activity 3-4-fold but reduces the selectivity for arachidonate. Acyl transfer in the absence of CoA exhibits a pH optimum of 7.5-8.5, whereas two pH optima (7.5 and 4.5) are observed in the presence of CoA with transfer activity at pH 4.5 exceeding that of pH 7.5 by 4-5-fold. The plasmalogen (alkenyl) analog of lysophosphatidylethanolamine is an equally effective acyl acceptor in the absence of CoA but less effective in its presence. The microsomal acyl-CoA/lysophosphatidylethanolamine acyltransferase does not favor arachidonate over linoleate. Therefore, transacylation from phosphatidylcholine may account for the high arachidonate content of dog heart microsomal phosphatidylethanolamine and its plasmalogen analog. In fact, acyl transfer from endogenous lipids to 1-[1'-14C]palmitoyl-2-lyso-sn-glycerophosphoethanolamine results in the generation of mostly (over 80%) tetraunsaturated phosphatidylethanolamine. This proportion is reduced by the addition of CoA and, even more, by CoA plus acyl-CoA-generating cofactors. We conclude that in dog heart microsomes, lysophosphatidylethanolamine can be acylated by different mechanisms, of which the CoA-independent transacylase exhibits the greatest acyl selectivity.     

Purified Rat Brain Myelin Contains Measurable Acyl-CoA:Lysophospholipid Acyltransferase(s) but Little, if Any, Glycerol-3-Phosphate Acyltransferase

Previous reports from several laboratories have demonstrated the presence of many lipid-metabolizing enzymes in myelin, including all the enzymes needed to convert diacylglycerol to phosphatidylcholine and phosphatidylethanolamine. Axonal transport studies had suggested the presence of additional enzymes which incorporate acyl chains into specific phospholipids of myelin. We report here evidence for one such group of enzymes, the acyl-CoA:lysophospholipid acyltransferases. At the same time, activity of acyl-CoA:sn-glycerol-3-phosphate acyltransferase was negligible in myelin. Oleoyl-CoA and arachidonoyl-CoA were both active substrates for transfer of acyl chains to lysophosphatidylcholine and lysophosphatidylinositol. Activity in myelin varied from 7 to 19% of microsomal activity, values well above the likely level of microsomal contamination as judged by microsomal markers. Additional evidence for a myelin locus came from assays at sequential stages of purification and from mixing experiments. Arachidonoyl-CoA was somewhat more reactive than oleoyl-CoA toward lysophosphatidylcholine; the myelin Km for these two CoA derivatives was 98 microM and 6.6 microM, respectively. Activity with lysophosphatidylinositol as substrate was approximately 40% of that with lysophosphatidylcholine in myelin, whereas activities with lysophosphatidylethanolamine and lysophosphatidylserine were considerably less.     

Structure of 1-acyl lysophosphatidylcholine and fatty acid complex in bilayers

The phase transition characteristics of bilayers formed in a codispersion of 1-acyl lysophosphatidylcholine and a fatty acid depend on the chain length of both the components and on the pH of the aqueous medium. Incorporation of cholesterol as a third component abolishes the transition. It is suggested that acyl chain interactions between fatty acid and 1-acyl lysophosphatidylcholine molecules in their aqueous codispersions are maximized by close-packing such that the acyl chains of both molecules are aligned parallel to each other and the carboxyl group is located in the vicinity of the 2-hydroxyl group of lysophosphatidylcholine. The shape and size of a functional dimer thus formed are similar but not identical to those of 1,2-diacyl phosphatidylcholine. Several predictions arising from this suggestion, including phase separation in codispersions of fatty acid + 1-acyl lysophosphatidylcholine + diacyl phosphatidylcholine, are experimentally confirmed.     

Evidence for the reversibility of the acyl-CoA:lysophosphatidylcholine acyltransferase in microsomal preparations from developing safflower (Carthamus tinctorius L.) cotyledons and rat liver.

Acyl exchange between acyl-CoA and position 2 of sn-phosphatidylcholine occurs in the microsomal preparations of developing safflower cotyledons. Evidence is presented to show that the acyl exchange is catalysed by the combined back and forward reactions of an acyl-CoA:lysophosphatidylcholine acyltransferase (EC 2.3.1.23). The back reaction of the enzyme was demonstrated by the stimulation of the acyl exchange with free CoA and by the observation that the added CoA was acylated with acyl groups from position 2 of sn-phosphatidylcholine. Re-acylation of the, endogenously produced, lysophosphatidylcholine with added acyl-CoA occurred with the same specificity as that observed with added palmitoyl lysophosphatidylcholine. A similar acyl exchange, catalysed by an acyl-CoA:lysophosphatidylcholine acyltransferase, occurred in microsomal preparations of rat liver. The enzyme from safflower had a high specificity for oleate and linoleate, whereas arachidonate was the preferred acyl group in the rat liver microsomal preparations. The rate of the back reaction was 3-5% and 0.2-0.4% of the forward reaction in the microsomal preparations of safflower and rat liver respectively. Previous observations, that the acyl exchange in safflower microsomal preparations was stimulated by bovine serum albumin and sn-glycerol 3-phosphate, can now be explained by the lowered acyl-CoA concentrations in the incubation mixture with albumin and in the increase in free CoA in the presence of sn-glycerol 3-phosphate (by rapid acylation of sn-glycerol 3-phosphate with acyl groups from acyl-CoA to yield phosphatidic acid). Bovine serum albumin and sn-glycerol 3-phosphate, therefore, shift the equilibrium in acyl-CoA:lysophosphatidylcholine acyltransferase-catalysed reactions towards the rate-limiting step in the acyl exchange process, namely the removal of acyl groups from phosphatidylcholine. The possible role of the acyl exchange in the transfer of acyl groups between complex lipids is discussed.     

Lysophosphatidylcholine acyltransferase and lysophosphatidylcholine: lysophosphatidylcholine acyltransferase in alveolar type II cells from fetal rat lung

The specific activity of lysophosphatidylcholine acyltransferase in sonicated fetal rat lung type II cells was found to be an order of magnitude greater than that of lysophosphatidylcholine:lysophosphatidylcholine acyltransferase. The specific activity of lysophosphatidylcholine acyltransferase in sonicated fetal rat lung type II cells increases towards the end of gestation, whereas that of lysophosphatidylcholine:lysophosphatidylcholine acyltransferase does not show a change. While lysophosphatidylcholine acyltransferase in whole fetal lung homogenate is more active towards oleoyl-CoA than towards palmitoyl-CoA, the enzyme in sonicated fetal type II cells is more active towards palmitoyl-CoA. If measured with palmitoyl-CoA as acyl donor, the specific activity of lysophosphatidylcholine acyltransferase in type II cells is higher than that in whole lung during late gestation. In contrast, the specific activity of lysophosphatidylcholine:lysophosphatidylcholine acyltransferase in type II cells is lower than that in whole lung. These observations indicate that in fetal rat type II cells the deacylation-reacylation cycle is more important for the formation of dipalmitoylphosphatidylcholine than the deacylation-transacylation process.     

Albumin stimulates the release of lysophosphatidylcholine from cultured rat hepatocytes.

The effect of albumin on the release of [3H]lysophosphatidylcholine from cultured rat hepatocytes prelabelled with [Me-3H]choline was studied. In the absence of serum and albumin from the medium, the cells released essentially no [3H]lysophosphatidylcholine. Albumin stimulated this process dramatically, and it reached a plateau at 2 mg/ml. After an initial lag of 30 min, the release of [3H]lysophosphatidylcholine was linear for at least 4 h. At low concentrations, albumin slightly stimulated [3H]phosphatidylcholine release. The albumin had no measurable effect on the metabolism of cellular [3H]phosphatidylcholine, [3H]lysophosphatidylcholine or [3H]glycerophosphocholine. In addition, albumin did not alter the release of 3H-labelled water-soluble compounds, including [3H]glycerophosphocholine, into the medium. The possibility that the [3H]lysophosphatidylcholine was arising from catabolism of [3H]phosphatidylcholine in the medium by secreted enzymes was excluded. The effect on [3H]lysophosphatidylcholine secretion was also observed when the cells were incubated with alpha-cyclodextrin, a cyclic polysaccharide that has the ability to bind lysophosphatidylcholine. The albumin-released lysophosphatidylcholine was enriched in unsaturated fatty acids. Alteration of the fatty acid composition of cellular phosphatidylcholine gave rise to parallel changes in phosphatidylcholine and lysophosphatidylcholine in the medium. It is concluded that phosphatidylcholine is constantly being degraded in the rat hepatocyte to lysophosphatidylcholine which is released into the medium only when a suitable acceptor is present.     

A sensitive, radiometric assay for lysophosphatidylcholine

To facilitate investigation of the metabolism of lysophosphatidylcholine and choline lysoplasmalogen in small quantities of tissue, a method for the quantification of these phospholipid species that is capable of accurate and reproducible analysis in samples which contain less than 1 nmol of total choline lysophospholipid was developed. The procedure employs chloroform and methanol extraction of phospholipids from isolated tissue with subsequent separation of the choline lysophospholipid fraction by high-performance liquid chromatography. The choline lysophospholipids are then acetylated with [3H]acetic anhydride and the [3H]acetyl-lysophosphatidylcholine product is isolated by thin-layer chromatography and quantified by liquid scintillation counting. The choline lysophospholipid content in the sample is determined from a standard curve constructed from samples containing a known amount of synthetic lysophosphatidylcholine with correction for recovery based on the inclusion of [14C]lysophosphatidylcholine as an internal standard.     

Interaction of gramicidin with lysophosphatidylcholine as revealed by calorimetry and fluorescence spectroscopy.

The effect of the interaction of gramicidin (GA) with lysophosphatidylcholine (LPC) on the change in lipid structure upon heat incubation was revealed by differential scanning calorimetry (DSC) and fluorescence spectroscopy. DSC showed a large endothermic transition in both pure LPC micelles and GA-containing LPC micelles after prolonged heat incubation at 70 degrees C. To elucidate this behavior, fluorescence spectra of 1-anilinonaphthalene-8-sulfonate embedded in LPC micelles were measured. About 40% of the resultant LPC micelles was found to be transformed into the interdigitated gel structures after prolonged heat incubation. On the other hand, intrinsic fluorescence spectra of GA-containing LPC micelles caused a blue-shift of the emission maxima with incubation time, suggesting that tryptophans near the C-terminus of GA moved into a more apolar environment. In addition, GA-containing LPC micelles caused quenching of fluorescence with incubation time, due to the interaction between GA molecules. To determine the location of GA in LPC membranes, surface pressure was measured using the mixed monolayers composed of GA and LPC. The result suggests that GA molecule is localized by interdigitating the C-terminal part of adjacent to acyl chain of LPC.     

Identification of a 51-kilodalton polypeptide fatty acyl chain acceptor in soluble extracts from mouse cardiac tissue

We have identified a protein in the soluble fraction from mouse cardiac tissue extracts which is rapidly and selectively acylated by myristyl CoA. This protein was partially purified by anion-exchange chromatography and gel filtration, and the acylation reaction was measured using [3H]myristyl CoA as substrate, followed by sodium dodecyl sulfate - polyacrylamide gel electrophoresis to resolve [3H]fatty acyl polypeptides. The [3H]acyl protein migrated as heterogeneous bands corresponding to relative masses (MrS) of 42,000-51,000 under nonreducing conditions or as a single polypeptide of Mr 51,000 in the presence of reducing agents. Fatty acyl chain incorporation into protein was very rapid and already maximum after 30 s of incubation, whereas no acylation was detected using heat-denatured samples or when the reaction was stopped immediately after initiation. Only the acyl CoA served as fatty acyl chain donor. No incorporation into protein occurred when myristyl CoA was substituted by myristic acid, ATP, and CoA. A time-dependent reduction in the level of [3H]fatty acyl polypeptide was observed upon addition of excess unlabeled myristyl CoA, indicating the ability of the labeled acyl moiety of the protein to turn over during incubation. The saturated C10:0, C14:0, and C16:0 acyl CoAs were more effective to chase the label from the [3H]acyl polypeptide than the C18:0 and C18:1 acyl CoAs. These results provide evidence for a 51-kilodalton polypeptide which serves as an acceptor for fatty acyl chains and could represent an important intermediate in fatty acyl chain transfer reactions in cardiac tissue.     

Rapid labeling of lipoproteins in plasma with radioactive cholesterol. Application for measurement of plasma cholesterol esterification

In order to efficiently and rapidly label lipoproteins in plasma with [3H]cholesterol, micelles consisting of lysophosphatidylcholine (lysoPC) and [3H]cholesterol (molar ratio, 50:1) were prepared. When trace amounts of these micelles were injected into plasma, [3H]cholesterol rapidly equilibrated among the plasma lipoproteins, as compared to [3H]cholesterol from an albumin-stabilized emulsion. The distributions of both [3H]cholesterol and unlabeled free cholesterol in plasma lipoproteins were similar in labeled plasma samples. This method of labeling can be used for the measurement of cholesterol esterification, or lecithin:cholesterol acyltransferase activity, in small amounts (20-40 microliters) of plasma samples.     

Localization of lysophosphatidylcholine in bovine chromaffin granules

One of the unique features of the chromaffin granule membrane is the presence of about 17 mol% lysophosphatidylcholine. Lysophosphatidylcholine isolated from the granules could be degraded by approx. 94% by lysophospholipase. This result is consistent with chemical analyses data showing that about 9% of this lysophospholipid is 1′-alkenyl glycerophosphocholine.The localization of the acylglycerophosphocholine in the chromaffin granule membrane was studied by using pure bovine liver lysophospholipases. In intact granules only about 10% of the total lysophosphatidylcholine was directly available for enzymic hydrolysis. In contrast, when granule membranes (ghosts) were treated with lysophospholipases approx. 60% of the lysophosphatidylcholine was deacylated. These values did not increase after pre-treatment of intact granules or ghosts with trypsin. Added $\text{1-[1-}{}^{14}\text{C]palmitoyl}\text{-sn-}\text{glycero-3-phosphocholine}$ did not mix with the endogenous lysophosphatidylcholine pool(s) and remained completely accessible to added lysophospholipases.     

Effects of acyl chain length on the conformation of myelin basic protein bound to lysolipid micelles.

The interactions of myelin basic protein with micelles of lysophosphatidylcholine detergents of different acyl chain lengths were investigated by circular dichroism (CD), small-angle X-ray scattering, Fourier transform infrared spectroscopy (FT-IR), and 1H, 13C and 31P nuclear magnetic resonance spectroscopy (NMR). Circular dichroic, FT-IR, and 1H NMR measurements indicated that the conformational changes induced in the protein molecules by association with micelles depended on the acyl chain length of the detergents. Size is one of the physical properties of micelles which is a function of the length of the acyl chains. The radii of gyration of detergent micelles in complexes with the protein measured by small-angle X-ray scattering indicated that the average size of the micelles was a quadratic function of the acyl chain length. The dependence of the protein conformational changes on micelle size was used to ascertain the order in which different protein segments associate with the detergents. Several procedures were employed to change the fluidity of micelles formed with detergents of given acyl chain lengths. The conformational changes observed on the MBP molecule by varying the micelle properties without changing the length of the chain, suggested that the changes depended on the size and fluidity of the micelles.     

Effects of lysophosphatidylcholine micelle and phosphatidylcholine liposome on photoreduction of methylviologen

Photoinduced reduction of methylviologen (MV2+) by ethylenediaminetetraacetate (EDTA3-), which was sensitized by thiacarbocyanine dyes having long alkyl chains (C+m-n) embedded in palmitoyl lysophosphatidylcholine micelle and dipalmitoyl phosphatidylcholine liposomal membrane, was carried out. The formation rate of reduced methylviologen cation radical (MV+.) decreased with the time of irradiation with visible light, and the deceleration was more pronounced in the micellar solution. In kinetic studies, we found that the sensitizer divalent cation radical (C2+.m-n) is formed through the reaction of photoexcited sensitizer (C+*m-n) with MV2+ as an intermediate in this reaction, and that the reduction of C2+.m-n with EDTA3- inhibits the back reaction of MV+. with C2+.m-n. The inhibition was greater in the liposomal solution than in the micellar solution. This was ascribed to a higher concentration of EDTA3- on the liposomal surfaces through the electrostatic interaction between EDTA3- and the liposomal surfaces, the charge of which is attributed to the univalent cation sensitizer embedded in the liposomal membrane. The difference in the positive charge density of the surface of these lipid aggregates was due to the difference in the curvature of the micelle and the liposome. These results suggest that the dipalmitoyl phosphatidylcholine liposome is a more effective carrier than the palmitoyl lysophosphatidylcholine micelle for the production of MV+. in the photoreduction studied here.     

Lysophospholipase and lysophosphatidylcholine:Lysophosphatidylcholine transacylase from rat lung: Evidence for a single enzyme and some aspects of its specificity

An enzyme preparation was isolated from rat lung cytosol with the capability to transfer the fatty acyl chain from 1-acyl-sn-glycero-3-phosphocholine to water and to another molecule of 1-acyl-sn-glycero-3-phosphocholine. The evidence presented to indicate that a single protein confers both activities includes: (a) both normal and sodium dodecyl sulfate polyacrylamide disc gel electrophoresis showed a single protein band, and (b) heat treatment and preincubation with increasing amounts of diisopropylfluorophosphate resulted in concomitant loss of fatty acid and phosphatidylcholine formation. The enzyme converted 1-[9,10-³H₂]stearoyl-sn-glycero-3-phospho[¹⁴C-methyl]choline into phosphatidylcholine with an isotopic ³H/¹⁴C ratio twice that of the substrate, even when an excess of unlabeled fatty acid was present. The acyl group from palmitoyl-propanediol (1,3)-phosphocholine and palmitoyl-propanediol (1,3)-phosphoethanolamine could be transferred to lysophosphatidylcholine acceptor to yield phosphatidylcholine. Neither acylglycerols and cholesterol nor glycero-3-phosphate and glycero-3-phosphocholine served as acyl acceptors. Lysophosphatidylethanolamine and lysophosphatidyglycerol were converted also into the corresponding diacylphospholipids. Palmitoyllysophosphatidylcholine is preferentially converted into phosphatidylcholine when compared with stearoyllysophosphatidylcholine. The possible involvement of the enzyme in the synthesis of dipalmitoylphosphatidylcholine for the production of lung surfactant is discussed.