Molecular species of extracellular phosphatidylethanolamine from Escherichia coli

1. (1)|Phosphatidylethanolamine was hydrolysed by phospholipase C from Bacillus cereus (E.C. 3.1.4.3) and the resultant diglycerides were separated into five subfractions on thin-layer plates of silica impregnated with silver nitrate. 2. (2)|The positional distribution of the fatty acids in these diglycerides was determined by means of hydrolysis with pancreatic lipase (E.C. 3.1.1.3). The results were in good agreement with those obtained by hydrolysis of the phosphatidylethanolamine with snake-venom phospholipase A (E.C. 3.1.1.4). 3. (3)|The following molecular species accounting for 97.3% of the phosphatidylethanolamine could be calculated: (1-octadecenoyl-2-hexadecenoyl)-, (di-octadecenoyl)-, (1-palmitoyl-2-hexadecenoyl)-, (1-hexadecenoyl-2-palmitoyl), (1-palmitoyl-2-octadecenoyl)-, (1-octadecenoyl-2-palmitoyl)-, (1-palmitoyl-2-cis-3, 10-methylene-hexadecanoyl)-, and (di-palmitoyl)-phosphatidylethanolamine

Asymmetry in the renewal of molecular classes of phosphatidylcholine in the rat-erythrocyte membrane

1. 1. Rat-blood phospholipids were labeled in vivo with [32P]phosphate. The erythrocytes were treated with phospholipase A2 plus sphingomyelinase to discriminate between the labeling patterns of the phospholipids from the inner and outer layer of the membrane. 2. 2. The specific activities of the more unsaturated classes of phosphatidylcholine were higher in the outer layer of the erythrocyte membrane than in the inner layer. The disaturated class, however, had the highest specific activity in the inner layer. 3. 3. After incubating 32P-labeled erythrocytes in unlabeled plasma, the labeling pattern recovered in the molecular classes of plasma phosphatidylcholine was very similar to that of the phosphatidylcholines in the outer layer of the erythrocyte membrane. 4. 4. It is proposed that the exchange of phosphatidylcholines between plasma and the outer layer of the erythrocyte is mainly responsible for the renewal of the unsaturated phosphatidylcholines of the erythrocyte, and that the acylation activity of the erythrocyte is directed towards the formation of disaturated phosphatidylcholines at the inside of the membrane

Asymmetry in the renewal of molecular classes of phosphatidylcholine in the rat-erythrocyte membrane

1. 1. Rat-blood phospholipids were labeled in vivo with [32P]phosphate. The erythrocytes were treated with phospholipase A2 plus sphingomyelinase to discriminate between the labeling patterns of the phospholipids from the inner and outer layer of the membrane. 2. 2. The specific activities of the more unsaturated classes of phosphatidylcholine were higher in the outer layer of the erythrocyte membrane than in the inner layer. The disaturated class, however, had the highest specific activity in the inner layer. 3. 3. After incubating 32P-labeled erythrocytes in unlabeled plasma, the labeling pattern recovered in the molecular classes of plasma phosphatidylcholine was very similar to that of the phosphatidylcholines in the outer layer of the erythrocyte membrane. 4. 4. It is proposed that the exchange of phosphatidylcholines between plasma and the outer layer of the erythrocyte is mainly responsible for the renewal of the unsaturated phosphatidylcholines of the erythrocyte, and that the acylation activity of the erythrocyte is directed towards the formation of disaturated phosphatidylcholines at the inside of the membrane

The effect of dietary fat on the molecular species of lecithin from rat liver

1. 1. Lecithins from the liver of rats maintained on diets devoid of essential fatty acids or supplemented with coconut oil or corn oil revealed significant differences in fatty acid composition, whilst monomolecular films of these lecithin samples exhibited only limited differences in force-area characteristics. 2. 2. The individual molecular species in the three lecithin samples were determined by means of the following techniques: (a) fractionation of the lecithin on silica impregnated with silver nitrate, followed by determination of the positional distribution of the fatty acids with snake-venom phospholipase A (EC 3.1.1.4); (b) hydrolysis of the lecithin with phospholipase C (EC 3.1.4.3) from Bacillus cereus and fractionation of the resultant diglycerides on silica impregnated with silver nitrate. Subsequently, the positions of the fatty acid constituents in the diglyceride fractions were determined by hydrolysis with pancreatic lipase (EC 3.1.1.3). The second approach gave the most detailed information and made it possible to recognize between 14 and 23 species. Quantitative determination of the major species accounted for 78.5 to 90.5% of the molecular composition. 3. 3. Differences in diet induced significant variations in the proportions of the various lecithin species and also brought about qualitative differences. These shifts in the molecular composition of the lecithin samples may contribute to preserving the liquid-crystalline nature of these lipids in membrane structures

The effect of dietary fat on the molecular species of lecithin from rat liver

1. 1. Lecithins from the liver of rats maintained on diets devoid of essential fatty acids or supplemented with coconut oil or corn oil revealed significant differences in fatty acid composition, whilst monomolecular films of these lecithin samples exhibited only limited differences in force-area characteristics. 2. 2. The individual molecular species in the three lecithin samples were determined by means of the following techniques: (a) fractionation of the lecithin on silica impregnated with silver nitrate, followed by determination of the positional distribution of the fatty acids with snake-venom phospholipase A (EC 3.1.1.4); (b) hydrolysis of the lecithin with phospholipase C (EC 3.1.4.3) from Bacillus cereus and fractionation of the resultant diglycerides on silica impregnated with silver nitrate. Subsequently, the positions of the fatty acid constituents in the diglyceride fractions were determined by hydrolysis with pancreatic lipase (EC 3.1.1.3). The second approach gave the most detailed information and made it possible to recognize between 14 and 23 species. Quantitative determination of the major species accounted for 78.5 to 90.5% of the molecular composition. 3. 3. Differences in diet induced significant variations in the proportions of the various lecithin species and also brought about qualitative differences. These shifts in the molecular composition of the lecithin samples may contribute to preserving the liquid-crystalline nature of these lipids in membrane structures

Some studies on the metabolism of phospholipids in Golgi complex from bovine and rat liver in comparison to other subcellular fractions

1. 1. Golgi complex, rough and smooth microsomes, plasma membranes, mitochondria, and nuclei from bovine liver were isolated and the purity assessed using specific marker enzymes. 2. 2. Cholinephosphotransferase and acyl-CoA:1,2-diacyl-sn-glycerol acyltransferases were found to be absent in the Golgi complex, mitochondria, plasma membrane, and nuclei. The de novo synthesis of lecithin and triacylglycerols is apparently localized exclusively in the smooth and rough endoplasmic reticulum. 3. 3. Acyl-CoA:1-acyl-sn-glycero-3-phosphorylcholine acyltransferase was found to be localized mainly in rough and smooth microsomes from bovine and rat liver; though also significant activity was found in the Golgi complex of bovine liver. On the other hand, no significant acylation of 1-acyl-sn-glycero-3-phosphorylcholine could be shown in the Golgi system from rat liver. 4. 4. Phosphatidylglycerol synthesis was found to take place predominantly in rat liver mitochondria. Significant synthesis of this phospholipid was, however, also found in rough and smooth microsomes and in Golgi of rat liver. 5. 5. The Golgi complex of rat liver was shown to contain both phospholipase A1 and A2 activities acting on exogenous phosphatidylethanolamine, the activity of the former being somewhat larger

Composition and metabolism of phospholipids of Fasciola hepatica, the common liver fluk

1. 1. The phospholipid composition of Fasciola hepatica, the common liver fluke, was compared to that of the liver of the host animals (rats and cattle). Considerable differences were found: monoacyl-sn-glycero-3-phosphorylcholine, hardly detectable in the liver, was found in significant amounts in the parasite. On the other hand, sphingomyelin, a normal constituent in the liver, appears to be absent in the liver fluke. Fasciola hepatica isolated from rat and cow liver had a strikingly similar phospholipid composition. 2. 2. Qualitative and quantitative differences were also found between the fatty acyl constituents of the phospholipids of the parasite and the liver. The major difference was the presence of eicosaenoic and eicosadienoic acids in the parasite, whereas these acids were not detected in the liver. 3. 3. In vitro incubations of Fasciola hepatica in the presence of [32 P] phosphate and [2-3H]glycerol resulted in the labelling of all phospholipids of the parasite, except that the [2-3H] label did not incorporate into ethanolamine plasmalogen. This is in agreement with the concept that in animals, glycerol is introduced into plasmalogens via dihydroxyacetonephosphate. 4. 4. Homogenates of liver flukes were found to catalyze the synthesis of phosphatidylcholine from 1,2-diacyl-sn-glycerols and CDPcholine. 5. 5. These results strongly suggest that Fasciola hepatica is capable of synthesizing at least part of its fatty acids and phospholipids

Phospholipase A2 in rat-lung microsomes: Substrate specificity towards endogenous phosphatidylcholines

1. 1. Isolated rat lungs were perfused with a variety of radioactive precursors to label the phosphatidylcholines of the microsomal and lamellar body fractions. These endogenously labelled phosphatidylcholines were used as substrates in experiments to identify and characterize phospholipase A activity in lung subcellular fractions. 2. 2. The microsomal fraction was found to contain a phospholipase A specific for the 2-position of endogenous phosphatidylcholines. The enzyme operated optimally at pH 8.5 and required 10 mM Ca2+ for maximal activity. 3. 3. No evidence was found for the existence of phospholipase A activity in lamellar bodies. 4. 4. The microsomal phospholipase A2 was more active towards phosphatidyl-cholines containing an unsaturated fatty acid at the 2-position than towards the disaturated phosphatidylcholines. 5. 5. Microsomal disaturated phosphatidylcholines labelled with [1-14C]acetate (endogenously synthesized palmitate) were hydrolysed by the microsomal phospholipase A2; however, no hydrolysis occurred when [9,10-3H2]palmitate was used as a precursor notwithstanding the fact that the label from [1-14C]-acetate is mainly incorporated into the 1-position and that from [9,10-3H2]-palmitate almost exclusively into the 2-position of the disaturated phosphatidylcholines of rat-lung microsomes. 6. 6. These results suggest the existence of two pools of disaturated phosphatidylcholines in rat-lung microsomes. They are consistent with the concept that dipalmitoylphosphatidylcholine synthesized by remodeling of unsaturated phosphatidylcholines with exogenously supplied palmitic acid, is not hydrolysed by phospholipase A2 of lung microsomes

Determination of molecular species of lecithin from erythrocytes and plasma

The molecular species of lecithin from erythrocyte and plasma of man and rabbit were determined after conversion of the lecithins into diglycerides by means of hydrolysis with phospholipase C. The resultant diglycerides were separated by thin-layer chromatography on silica impregnated with silver nitrate into 6 or 7 fractions differing with respect to their degree of unsaturation. The positional distribution of the fatty acids in these fractions was determined by hydrolysis with pancreatic lipase and was found to be in agreement with the positional distribution of the fatty acids in the lecithin as ascertained by means of phospholipase A hydrolysis. Using these techniques about 20 molecular species accounting for about 90% of the total lecithin, could be evaluated in the erythrocyte and plasma of man and rabbit. It became clear that qualitatively the molecular species of lecithin in the red cell and the plasma are similar. Quantitatively, however, there were some striking differences to be noted: in man the amount of (dipalmitoyl)- and (di-oleoyl)-lecithin was higher in the corpuscles when compared with plasma. On the other hand (1-palmitoyl-2-linoleoyl)- and (1-palmitoyl-2-arachidonoyl)-lecithin were more abundant in plasma. In rabbit similar differences were found in the make-up of the molecular species of lecithin between the erythrocyte membrane and the surrounding plasma