Properties of microsomal acyl-CoA: lysophosphatidylcholine acyltransferase from liver of thermally-acclimated rainbow trout,Salmo gairdneri

Summary Acyl-CoA: lysophosphatidylcholine acyltransferase (LPCAT) (EC 2.3.1.23) activity was assayed in liver microsomes from rainbow trout,Salmo gairdneri, acclimated to 5°C and 20°C to assess its contribution to the temperature-induced restructuring of phospholipid acyl chain composition. The synthesis of phosphatidylcholine (PC) (from lyso-PC) was threefold the synthesis of phosphatidylethanolamine (PE) (from lyso-PE) under similar assay conditions. LPCAT activity (i) displayed an absolute requirement for lysophosphatidylcholine (LPC) and was enhanced by the presence of ATP, MgCl₂ and CoA (which reduced the impact of endogenous acyl-CoA hydrolase activity by regenerating the acyl-CoA substrate) in the assay medium; (ii) remained linear with time up to 30 min; and (iii) increased linearly with microsomal protein concentration up to 0.2 mg/ml for the 20°C assay and 0.4 mg/ml for the 5°C assay. There was no difference in K_m or V_max values due to the acclimation history of the fish, but there were obvious differences due to assay temperature. The apparent K_m values for LPC were 58.54±7.24 M and 12.26±2.14 M when assayed at 5°C and 20°C respectively; values for oleoyl-CoA were 9.11±0.78 M and 1.23±0.25 M under the same assay conditions. Activity was 1.99±0.31 nmol min^–1 mg protein^–1 when assayed at 5°C, and 3.8±0.45 nmol min^–1 mg protein^–1 when assayed at 20°C. These findings indicate that adjustments in the activity of LPCAT play no significant role in the temperature-induced restructuring of PC molecular species composition. However, the marked temperature dependence of the K_m values for LPC and oleoyl CoA suggest that patterns of fatty acid incorporation (i.e. substrate preference) may vary with assay temperature, and in this way LPCAT could contribute to the restructuring response.Abbreviations PC phosphatidylcholine - PE phosphatidylethanolamine - LPCAT acyl-CoA: lysophosphatidylcholine acyltransferase - LPEAT acyl-CoA: lysophosphatidylethanolamine acyltransferase - LPC 1-palmitoyl,2-lysophosphatidylcholine     

Molecular species of phosphatidylcholine and phosphatidylethanolamine in subcellular membranes from rat liver.

Four subfractions of phosphatidycholine and phosphyatidylethanolamine according to the degree of unsaturation of their fatty acids have been separated from lipid extracts of microsomes, and inner and outer mitochondrial membranes. The predominant species found in the three membranes contained one saturated and one unsaturated fatty acid. In microsomes completely saturated species of both phosphatidylcholine and phosphatideylethanolamine were practically nonexistent. In outer mitochondrial membranes species with two unsaturated fatty acids were absent. In the inner mitochondrial membranes, however, disaturated species and those with two unsaturated fatty acids were found.     

Selective changes to phosphatidylcholine and phosphatidylethanolamine molecular species in the developing fetal guinea pig liver and plasma

The molecular species composition of membrane phospholipids influences the activities of integral proteins and cell signalling pathways. We determined the effect of increasing gestational age on fetal guinea pig liver phosphatidylcholine (PC) and phosphatidylethanolamine (PE), and plasma PC molecular species composition. The livers were collected from fetuses (n = 5/time point) at 5 day intervals between 40 and 65 days of gestation, and at term (68 days). Hepatic PC and PE molecular species composition was determined by electrospray ionisation mass spectrometry. An increasing gestational age was accompanied by selective changes in individual molecular species. The proportion of the sn-1 18:0 species increased relative to the sn-1 16:0 species in liver PC, but not PE, with an increasing gestational age. 1-O-alkyl-2-acyl PC species concentrations decreased significantly between 40 and 45 days of gestation (40%), and 65 and 68 days (54%). Total 1-O-alkenyl-2-acyl PE species concentration increased between days 60 and 65, due to a rise in 1-O-16:0 alkyl/20:4 content, and then decreased until term. Between day 40 and term, PC and PE sn-2 18:2n-6 species concentrations increased 3-fold. PC16:0/18:2 increased gradually throughout gestation, while PC18:0/18:2 content only increased after day 65. The overall increase in PE18:2n-6 content was due to PE18:0/18:2 alone. The composition of plasma PC essentially reflected hepatic PC. Overall, these data suggest differential regulation of hepatic PC and PE molecular species composition during development which is essentially independent of the maternal fatty acid supply.     

Changes in desaturase activity and the fatty acid composition of microsomal membranes from liver tissue of thermally-acclimating rainbow trout

Summary Rainbow trout (Salmo gairdneri) were acclimated to either 5 or 20°C, and then transferred to the opposite temperature, and changes in the fatty acid composition of liver microsomal membranes and the activities of the hepatic Δ9, Δ6, and Δ5 desaturases were measured at intervals of up to one month post-transfer. Inital changes (days 0–3) in fatty acid composition were: (1) an increase in the proportion of saturates and a decrease in the proportion of polyunsaturates during warm acclimation, and (2) a decrease in the proportion of saturates during cold acclimation. The activity of the Δ6 desaturase approximately doubled immediately following the changes in temperature, but alterations in Δ9 and Δ5 desaturase activities required at least 3 days to occur. The results indicate that desaturase enzymes do not play a major role in the initial adaptation of membrane fatty acid composition to changes in temperature. However, the desaturase enzymes may be involved in the later stages (3–28 days) of the acclimatory process. The proportion of monoenes was well correlated with Δ9 desaturase activity during both transfers, and appeared to be adjusted as required to offset changes in the proportion of polyunsaturates. Supported by National Science Foundation Grant PCM-8301757 to J.R.H.     

Differential methylation patterns in molecular species of phosphatidylethanolamine derivatives in rat liver membranes

The appearance of individual molecular species of phospholipids in the complete sequence of the transmethylation of phosphatidylethanolamine (PE) was examined in rat liver microsomes incubated with S-adenosyl-L-[methyl-14C]methionine. Reverse-phase HPLC analysis of phosphatidylcholine (PC), phosphatidyl-N,N-dimethylethanolamine (dimethyl-PE), or phosphatidyl-N-monomethylethanolamine (monomethyl-PE) showed that radioactivity was present in the same six principal molecules; a first group is constituted by 16:0/22:6, 16:0/20:4 and 16:0/18:2 and a second one by the homologous molecules with 18:0 instead of 16:0 at the sn-1 position of glycerol. In PC, 16:0/22:6 (23% of total radioactivity) was preponderant, and 18:0/20:4 was the lowest. The ratios cpm in PC/nmol in PE were in the order: 16:0/22:6 greater than 16:0/18:2 greater than 16:0/20:4 followed by the corresponding 18:0 molecules. On the other hand, in intermediate phospholipids, incorporation of methyl groups was most marked in 18:0/20:4 (24-27% of total). 16:0/22:6 and 16:0/18:2 were low in comparison to their relative values in PC. The ratio (18:0/20:4)/(16:0/22:6) was 4.5-5.6-times higher in monomethyl-PE and dimethyl-PE than in PC. These differences were found consistently, regardless of incubation time of microsomes (2.5-60 min) and of S-adenosyl-L-methionine (AdoMet) concentration (3 or 100 microM). In liver membranes, it would therefore seem that there is a different selectivity in methyl group transfer, depending upon whether the first two steps or the third step of the reaction are considered. Side reactions, such as deacylation/reacylation, are unlikely to account for this difference, which could rather be related to the enzyme itself.     

Endogenous phospholipids of swine liver: effect of fat deprivation on molecular species of phosphatidylcholine and phosphatidylethanolamine

Three 1-yr-old swine and two 2.5-wk-old swine were fed a fat-free diet for 1 month and 5 months, respectively. The hepatic phosphatidylcholine and phosphatidylethanolamine were fractionated by silver ion thin-layer chromatography. A distinctive feature of the chromatographic procedure was the development of the chromatograms at low temperatures: -10 degrees C for phosphatidylcholine and 4 degrees C for phosphatidylethanolamine. The chromatographic fractions were hydrolyzed with phospholipase A(2), and the fatty acids were characterized. Significant concentrations of odd-chain saturated and unsaturated fatty acids were found in the swine deprived of fat for 5 months. The major molecular species of phosphatidylcholine in both groups contained monoenoic fatty acids: 16:0/18:1(n - 9), 18:0/18:1(n - 9), and 18:1(n - 9)/18:1(n - 9). Their concentrations changed only slightly with the diet. The molecular species of phosphatidylethanolamine were more sensitive to dietary changes. In the swine deprived of fat for 1 month, about 50% of the molecular species of phosphatidylethanolamine contained tetraenoic fatty acids: 16:0/20:4(n - 6), 18:0/20:4(n - 6), and 18:1(n - 9)/20:4(n - 6). The phosphatidylethanolamine of animals deprived of fat for 5 months contained only 3% molecular species with tetraenoic acids, 18:0/20:4(n - 6), but 36% molecular species with trienoic acids: 18:0/20:3(n - 9), 18:1(n - 9)/20:3(n - 9), 18:0/19:3(n - 8), 16:0/20:3(n - 9), and 17:0/20:3(n - 9). Doubly unsaturated species, such as 18:1(n - 9)/18:1(n - 9), 18:1(n - 9)/20:3(n - 9), and 18:1(n - 9)/20:4(n - 6), were found in both groups of swine, although their total concentrations were higher in the group deprived of fat for a longer period.     

Synthesis of molecular species of phosphatidylcholine and phosphatidylethanolamine by germinating soya bean

The composition and metabolism of phosphatidylcholines and phosphatidylethanomines of germinating soya bean Glycine max have been examined. Both phospholipids have a very similar fatty acid composition and distribution, with saturated acids located at the 1- position. The fatty acid composition and relative amounts of individual molecular species of the two phospholipids were also very similar. The relative amounts of the species were in the order tetraenoic pentaenoic trienoic = dienoic = monoenoic. In contrast, the labelling of the molecular species from choline Me[¹⁴C] or ethanolamine [2-¹⁴C] showed considerable differences. Phosphatidylethanolamine-[¹⁴C] showed 58% label in trienoic, 17% in tetraenoic, 18% in pentaneoic and 5% in dienoic species 48 hr after germination. The equivalent figures for phosphatidylcholine-[¹⁴C] were 37, 34, 13 and 15% respectively. An increase in labelling of the more unsaturated species was seen with time.     

Molecular species of mitochondrial phosphatidylcholine in rat liver and lung

The acyl species of mitochondrial phosphatidylcholine from rat liver and lung were analysed by HPLC separation of the 1,2-diacyl-3-naphthylurethane derivatives. Comparison of phosphatidylcholine species patterns in microsomal, mitochondrial and submitochondrial fractions revealed only minor differences, whereas mitochondria from liver and lung differed markedly in the molecular composition of their respective phosphatidylcholine species.     

Contribution of different pathways to the supply of phosphatidylethanolamine and phosphatidylcholine to mitochondrial membranes of the yeast Saccharomyces cerevisiae

In the yeast, three biosynthetic pathways lead to the formation of phosphatidylethanolamine (PtdEtn): (i) decarboxylation of phosphatidylserine (PtdSer) by phosphatidylserine decarboxylase 1 (Psd1p) in mitochondria; (ii) decarboxylation of PtdSer by Psd2p in a Golgi/vacuolar compartment; and (iii) the CDP-ethanolamine (CDP-Etn) branch of the Kennedy pathway. The major phospholipid of the yeast, phosphatidylcholine (PtdCho), is formed either by methylation of PtdEtn or via the CDP-choline branch of the Kennedy pathway. To study the contribution of these pathways to the supply of PtdEtn and PtdCho to mitochondrial membranes, labeling experiments in vivo with [(3)H]serine and [(14)C]ethanolamine, or with [(3)H]serine and [(14)C]choline, respectively, and subsequent cell fractionation were performed with psd1Delta and psd2Delta mutants. As shown by comparison of the labeling patterns of the different strains, the major source of cellular and mitochondrial PtdEtn is Psd1p. PtdEtn formed by Psd2p or the CDP-Etn pathway, however, can be imported into mitochondria, although with moderate efficiency. In contrast to mitochondria, microsomal PtdEtn is mainly derived from the CDP-Etn pathway. PtdEtn formed by Psd2p is the preferred substrate for PtdCho synthesis. PtdCho derived from the different pathways appears to be supplied to subcellular membranes from a single PtdCho pool. Thus, the different pathways of PtdEtn biosynthesis play different roles in the assembly of PtdEtn into cellular membranes.     

Individual molecular species of phosphatidylcholine and phosphatidylethanolamine in myelin turn over at different rates.

Phosphatidylcholine (PC) and phosphatidylethanolamine (PE) of the myelin membrane exhibit heterogeneity with respect to metabolic turnover rate (Miller, S. L., Benjamins, J. A., and Morell, P. (1977) J. Biol. Chem. 252, 4025-4037). To test the hypothesis that this is due to differential turnover of individual molecular species (which differ in acyl chain composition), we have examined the relative turnover of individual molecular species of myelin PC and PE. Phospholipids were labeled by injection of [2-3H]glycerol into the brains of young rats. Myelin was isolated at 1, 15, and 30 days post-injection, lipids were extracted, and phospholipid classes were separated by thin-layer chromatography. The PC and PE fractions were hydrolyzed with phospholipase C, and the resulting diacylglycerols were dinitrobenzoylated and fractionated by reverse-phase high performance liquid chromatography. The distribution of radioactivity among individual molecular species was determined. The labeled molecular species of myelin PC were 16:0-16:0, 16:0-18:0, 16:0-18:1, and 18:0-18:1, with most of the label present in 16:0-18:1 and 18:0-18:1. Changes in distribution of label with time after injection indicated that 16:0-18:1 turned over more rapidly than 18:0-18:1. The labeled molecular species of myelin PE were 18:0-20:4, 18:1-18:1, 16:0-18:1, 18:0-18:2, and 18:0-18:1. As with myelin PC, 16:0-18:1 (and 18:1-18:1) turned over more rapidly than 18:0-18:1. The relative turnover of individual molecular species of PC in the microsomal fraction from forebrain was also examined. The molecular species profile was different from myelin PC, but again, 16:0-18:1 turned over more rapidly than the other molecular species. Thus, within the same membrane, individual molecular species of a phospholipid class are metabolized at different rates. Comparison of our results with previous studies of turnover of molecular classes of phospholipids indicates that in addition to polar head group composition (Miller et al., 1977), fatty acid composition is very important in determining the metabolic fate of a phospholipid.     

Plasmodium knowlesi induces alterations in phosphatidylcholine and phosphatidylethanolamine molecular species composition of parasitized monkey erythrocytes

Using high performance liquid chromatography and gas-liquid chromatography, we have characterized the phosphatidylcholine and phosphatidylethanolamine molecular species composition of trophozoite and schizont forms of Plasmodium knowlesi parasitized erythrocytes. Similarly, we determined these parameters in the erythrocyte membranes of trophozoite parasitized cells, unparasitized erythrocytes from infected monkeys before and after a chloroquine treatment and erythrocytes from monkeys that had never been infected. Plasma phosphatidylcholine molecular species composition was also studied. P. knowlesi parasitized erythrocytes presented higher amounts of 16:0/18:2-phosphatidylcholine than the various control cells, which appeared to be compensated for by a decrease in 18:0/20:4-, 16:0/20:3-, 16:0/18:1-, 18:0/18:2-, 18:0/20:3-, 16:0/16:0- and 16:0/18:0-phosphatidylcholines. In the case of phosphatidylethanolamine, the alterations were quantitatively of greater importance and consisted of an increase in, again, 16:0/18:2-phosphatidylethanolamine and a decrease in several species containing 20:4, namely 16:0/20:4-, 18:0/20:4- and 18:1/20:4-phosphatidylethanolamine; also the levels of alkoxy-phosphatidylethanolamines were markedly decreased. P. knowlesi development within monkey erythrocytes therefore appears to be associated with changes in phosphatidylcholine and phosphatidylethanolamine molecular species in the whole parasitized cell. These alterations are also exhibited by the host cell membrane, which provides the first experimental evidence that the parasite is able to manipulate the erythrocyte membrane lipid species composition. The consequences of these alterations on membrane physiology are discussed, as well as the implications that these data may have on the trafficking of phosphatidylcholine and phosphatidylethanolamine in the erythrocytes of P. knowlesi infected monkeys.     

Purification and comparative properties of NADPH-cytochrome P-450 reductase from rat and rainbow trout: Differences in temperature optima between reconstituted and microsomal trout enzymes

NADPH-cytochrome P-450 reductase has been purified to apparent homogeneity from liver microsomes of β-naphthoflavone-treated rats and rainbow trout. The apparent monomeric molecular weights were 75,000 and 77,000 for the rat and trout, respectively. Differences in amino acid composition were observed, particularly for lysine, glycine, threonine, and tyrosine. Analysis of the flavin composition showed that there were 0.97 mol of FAD and 0.92 mol of FMN per mol of rat reductase, whereas the values for the trout enzyme were 1.06 and 0.76 for FAD and FMN, respectively. Trout NADPH-cytochrome c reductase was inhibited by anti-rat antibody, but not to the same extent as was the rat enzyme. No precipitin lines between the trout reductase and rat antibody were observed on Ouchterlony plates. Peptide patterns, on sodium dodecyl sulfate-polyacrylamide gel electrophoresis, following limited proteolysis were also markedly different. The trout enzyme was as effective, catalytically, as the rat enzyme in a reconstituted system that contained purified rat cytochrome P-448 and lipid. Comparison of ethoxyresorufin-O-deethylase temperature profiles with various combinations of purified trout and rat P-448, reductase, and lipid, in membranous and nonmembranous reconstitution systems, demonstrated that the lower temperature optimum in trout microsomes could only be reproduced when all three trout components were incorporated into liposomes. These results suggest that it is the structural organization of the mixed-function oxidase enzymes and lipid within trout microsomes which were responsible for the lower temperature optimum compared to rat.     

Import of phosphatidylserine to and export of phosphatidylethanolamine molecular species from mitochondria

Heavy isotope-labeled ethanolamine and serine as well as exogenous PE and PS species were used to study trafficking of phosphatidylethanolamine (PE) and -serine (PS) molecular species between the endoplasmic reticulum (ER) and mitochondria in HeLa cells. Import of both endogenous and exogenous PS to IMM was a relatively slow process (T1/2 = several hours), but depended on the acyl chains. In particular, the 38:4 and 38:5 species were imported more efficiently compared to the other PS species. Knock-down of Mitofusin 2 or Mitostatin had no detectable effect on PS import to mitochondria, suggesting that the ER–mitochondria contacts regulated by these proteins are not essential. Knock-down of PS synthase 1 inhibited PS decarboxylation, suggesting that import of PS to mitochondria is coupled to its synthesis. Also the export of PE from IMM to microsomes is a relatively slow process, but again depends markedly on the acyl chain structure. Most notably, the polyunsaturated 38:4 and 38:5 PE species were less efficiently exported, which together with rapid import of the PS precursors most probably explains their enrichment in IMM. PE synthesized via the CDP-ethanolamine was also imported to IMM, but most of the PE in this membrane derives from imported PS. In contrast to PS, all PC species made in Golgi/ER translocated similarly and rapidly to IMM. In conclusion, selective translocation of PS species and PS-derived PE species between ER and mitochondria plays a major role in phospholipid homeostasis of these organelles.     

A large-scale purification of phosphatidylethanolamine, lysophosphatidylethanolamine, and phosphatidylethanolamine, and phosphatidylcholine by high performance liquid chromatography: a partial resolution of molecular species

Egg yolk phospholipids, on a 10 g scale, were resolved by high-performance liquid chromatography on an 8-m silica column with elution by a stepwise chloroformmethanol gradient into homogenous phosphatidylcholine, phosphatidylinositol, phosphatidylethanolamine, and lysophosphatidylethanolamine fractions. Within these fractions, partial resolution on the basis of fatty acyl side chain composition was achieved.     

Cytosol protein-independent translocation of isomeric spin-labelled radioactive lipids from isolated guinea pig liver microsomal to mitochondrial membranes

Intermembranous translocation of membrane-bound radioactive lipids covalently labelled with 5-, 12, and 16-doxyl stearic acid was studied. Guinea pig liver microsomal membranes containing known amounts of isomeric spin-labelled radioactive phosphatidic acid, phosphatidylcholine, and diglycerides were incubated with unlabelled mitochondria; reisolated mitochondria contained around 28-31% of microsomal labelled lipids above the microsomal contamination. The effect of adding crude or 'pH 5.1' 105 000 X g cytosol supernatant on the amount and composition of translocated labelled lipids was studied. While the translocation of labelled phosphatidylcholine was slightly stimulated by the addition of these cytosol supernatants, no significant increase of the amount of translocated labelled phosphatidic acic and diglycerides was observed by this addition. In view of these results, a probable mechanism for the cytosol protein-independent translocation of lipids between biological membranes is proposed.