Ossi Renkonen

Individual molecular species of different phospholipid classes. Part II. A method of analysis

New analytical possibilities arise when glycerosphophatides are converted into diglyceride acetates or analogous compounds: In this less polar form the phospholipids can be subjected to the usual methods of triglyceride fractionation, including chromatography on silica gel mixed with silver nitrate. This opens a route to subfractionation of various glycerophosphatide classes, and makes analysis of the individual molecular species potentially possible in many cases. The same approach can also be applied to the analysis of sphingomyelins.Two methods are suitable for the conversion of glycerophosphatides into “diglyceride acetates”: 1) Acetolysis in a mixture of acetic anhydride and acetic acid, and 2) treatment with phospholipase C (E.C. 3.1.4.3.) followed by acetylation. Acetolysis was used successfully with phosphatidyl choline, phosphatidyl ethanolamine and corresponding alkoxy phosphatide (native cephalin B), phosphatidyl serine, phosphatidyl inositol, phosphatidyl glycerol, phosphatidic acid and cardiolipin. Phospholipase C fromClostridium welchii was used for native choline and ethanolamine plasmalogens as well as for sphingomyelins. Although this enzyme does not attack pure ethanolamine phosphatides it did so in the presence of added (dipalmitoyl) phosphatidyl choline or serum sphingomyelin. The mixed substrates containing sphingomyelin proved particularly valuable, since the plasmalogenic diglyceride acetates were easily separated from the usual diglyceride acetates and ceramide diacetates on silicic acid.The diglyceride acetates obtained from lecithins of hens’ eggs, ox-brain and human serum were subjected to preparative TLC on Kieselgel G mixed with silver nitrate. The resulting subfractions were characterized by GLC analysis of the fatty acid methyl esters. When these data were combined with enzymic analysis of the acids occupying the alpha and beta positions of the molecules a rather detailed description of the lecithins became possible.The use of diglyceride acetates provides a route to analysis of fatty acids in many phosphatidyl compounds which have been obtained previously only as mixtures with the alkoxy analogs.

Individual molecular species of phospholipids: III. Molecular species of ox-brain lecithins☆

Abstract 1. 1. A mixture of native choline plasmalogens and alkoxy lecithins was isolated from ox brain by partial alkaline methanolysis of the total lecithin; the preparation appeared to be free from phosphatidyl cholines. 2. 2. Hydrolysis of total ox-brain lecithins with phospholipase C (EC 3.1.4.3) from Clostridium welchii converted all three subclasses of the glycerylphosphorylcholine lipids into diglycerides; these were subsequently acetylated. The resulting mixture of diglyceride acetates was separated by chromatography on kieselgel G into ordinary diglyceride acetates (95.2%), plasmalogenic diglyceride acetates (1.9%) and corresponding alkoxy compounds (2.2%). Chromatographic evidence and observations on partial hydrolysis with pancreatic lipase (EC 3.1.1.3) proved that the fraction of the ordinary diglyceride acetates contained about 99% 1,2-diglyceride acetates and about 1% 1,3-diglyceride acetates. Hydrolysis of the plasmalogenic diglyceride acetates and the alkoxy analogs with pancreatic lipase suggested that their long-chain acyl ester groups were located on C-2, and the acetyl group on C-3, of glycerol. 3. 3. All three subclasses of diglyceride acetates were fractionated on silver nitratecontaining kieselgel G into four subfractions which contained 0, 1, 2 and “many” double bonds, respectively, in their long fatty acid chains proper. Furthermore, the dienoic subfractions of the type 1-oleoyl-2-oleoyl-3-acetate and 1-stearoyl-2-linoleyl-3-acetate could be separated from each other. The plasmalogen diglyceride acetates appeared to form silver complexes even though they had vinyl ether double bonds. 4. 4. The fatty acid chains at the C-1 and C-2 positions in the twelve main subfractions of diglyceride acetates were analyzed by gas-liquid chromatography. The composition of the major molecular species could be predicted from the results by assuming that the number of double bonds is constant within each subfraction. 5. 5. The ordinary 1,2-diglyceride acetates were also subjected in intact form to gasliquid chromatography whereby 3–4 fractions of different molecular sizes were revealed in each of the main subfractions. The results confirmed the predictions made by argentation, fractionation and chain analysis. 6. 6. All three subclasses of ox-brain lecithins, the phosphatidyl cholines, the plasmalogens and the alkoxy lecithins, appeared to consist of remarkably similar populations of molecular species. 7. 7. The partial hydrolysis of five different types of diglyceride acetates with pancreatic lipase is described. The primary ester groups appeared to be cleaved faster than the secondary ones in 1,2-diglyceride acetates as well as 1,3-diglyceride acetates, and also in the corresponding two types of plasmalogen compounds having vinyl ether chains on C-1; even the alkoxy analogs of 1,2-diglyceride acetates were deacetylated.

Isolation and characterization of the membrane proteins of Semliki Forest virus

Abstract Three proteins, E 1 , E 2 , and E 3 , were isolated by SDS-hydroxylapatite chromatography from the membrane of Semliki Forest virus (SFV) grown in BHK cells. All the proteins contained covalently linked carbohydrates including glucosamine, mannose, galactose, fucose, and sialic acid. The molecular weights of E 1 and E 2 were estimated to 49,000 and 52,000, respectively, by SDS-polyacrylamide gel electrophoresis. These values were independent of the acrylamide concentration of the gels. E 3 contained 45% carbohydrate and migrated anomalously in SDS gels. Its molecular weight was calculated from its chemical composition to be 10,000. The structural proteins in SFV were found to be present in about equimolar ratios. Using peptide mapping E 3 was shown to be the other cleavage product of a nonviral protein (NVP 68), earlier shown to be the precursor of E 2 in SFV-infected cells.

The lipid class composition of Semliki Forest virus and of plasma membranes of the host cells

Abstract The lipid class composition of Semliki Forest virus grown in two clones of BHK21 cells was determined. The virus contained 0.41 mg of lipid per milligram of protein; of this 31% was neutral lipid, 61% phospholipid, and 8% glycolipid. The major neutral lipid was free cholesterol; the principal phospholipids were phosphatidylcholine, phosphatidylethanolamine, phosphatidylserine, and sphingomyelin. The major glycolipids were tentatively identified as sialolactosyl ceramides. The molar ratio of cholesterol to phospholipid was about 1. The lipids of Semliki Forest virus were similar to those of plasma membrane isolated from either infected or uninfected host cells. However, the virus contained much less free fatty acids and a little more cholesterol than the plasma membrane. The lipid composition of whole BHK21 cells and their endoplasmic reticulum differed clearly from that of the virus. In the virus there are about 10,000 cholesterol-phospholipid pairs, which can be arranged in a bilayer type of structure in the space available in the envelope.

Mono- and dimethyl phosphatidates from different subtypes of choline and ethanolamine glycerophosphatides

Abstract Preparation and some properties of phosphatidic acids and their mono- and dimethyl esters from natural phosphatides were studied. 1. 1. Hydrolysis with cabbage phospholipase D (EC 3.1.4.4) converted all three subclasses of ox heart lecithin, viz. phosphatidyl choline, choline plasmalogen and alkyl acyl lecithin, into phosphatidic acids. Phospholipase D catalysed methanolysis converted phosphatidyl choline to phosphatidyl methanol. The same reaction appeared to take place also with choline plasmalogens and alkyl acyl lecithins. The hydrolysis, and quite likely also the methanolysis, was a fast reaction with phosphatidyl cholines and alkyl acyl lecithins but very slow with the plasmalogens. 2. 2. The three subclasses of phosphatidic acid, the diacyl, the plasmalogenic, and the alkyl acyl forms, were completely separated from each other by thin-layer chromatography of the dimethyl esters. 3. 3. The Chromatographie properties of phosphatidic acid were shown to depend on the cations present ; the Ca2+ salt was not retained by DEAE-cellulose or alumina from neutral solvents, but the Na+ salt was retained appreciably. Also on silicic acid columns these two salts gave remarkably different elution patterns. The behaviour of phosphatidyl methanol on DEAE-cellulose was affected much less than that of phosphatidic acid by the presence of Ca2+. 4. 4. Diazomethanolysis converted all three subclasses of ox heart lecithin into the corresponding dimethyl phosphatidates. Phosphatidyl cholines and alkyl acyl lecithins seemed to react without complication, but choline plasmalogens gave an unidentified product in addition to the dimethyl phosphatidate. Diazomethanolysis of the same three subtypes present in ox brain ethanolamine phosphatides also gave the different dimethyl phosphatidates. 5. 5. The more polar products formed in the diazomethanolysis of the lecithins and ethanolamine lipids appeared to contain phosphatidyl methanol and O-methylated lecithins and chephalins, which are probably intermediate products of the reaction. 6. 6. The dimethyl phosphatidates could be effectively separated into different molecular species by argentation chromatography. The positional analysis of fatty acids in these lipids could be carried out with pancreatic lipase which attacks preferentially the ester groups on C-1 of glycerol. The plasmalogenic dimethyl phosphatidates and their alkyl analogs were virtually stable towards this enzyme. 7. 7. NMR spectra of phosphatidic acid and its mono- and dimethyl esters were recorded.

The lipids of the plasma membranes and endoplasmic reticulum from cultured baby hamster kidney cells (BHK21)

Abstract Plasma membranes and endoplasmic reticulum of two clones of cultured baby hamster kidney cells (BHK21) were prepared by the method of Wallach and Kamat (D. F. H. Wallach and V. B. Kamat , Methods Enzymol., 8 (1966) 164). The two membranes differed significantly from each other in their neutral, phospho- and sphingoglycolipid compositions.

Novel stereoconfiguration in lyso-bis-phosphatidic acid of cultured BHK-cells

Abstract Lyso-bis-phosphatidic acid purified from cultured hamster kidney fibroblast cells (BHK-cells) was subjected to strong alkaline hydrolysis. The hydrolysate contained phosphorus, free glycerol, total glycerol, α-glycerophosphate, β-glycerophosphate and sn-glycerol-3-phosphate in mole ratios of 1.0:1.0:1.9:0.4:0.6:0.02. The absence of sn-glycerol-3-phosphate indicates that the backbone of this lipid has the uncommon structure of 1-sn-glycerophosphoryl-1′-sn-glycerol. Consequently, the biosynthesis and the degradation of this lipid must differ from the other known mammalian glycerophospholipids.

Chemical Composition of Semliki Forest Virus

Chemical analysis of Semliki Forest virus grown in BHK21 cells revealed 6.3 % RNA, 12.2 % nucleocapsid protein, 44.4 % envelope polypeptides, 30.8 % lipid and 6.3 % proteinbound carbohydrate. The mono

Lipids of cultured mosquito cells (Aedes albopictus). Comparison with cultured mammalian fibroblasts (BHK 21 cells).

Abstract The lipids of an established cell line of a mosquito, Aedes albopictus were analyzed by Chromatographie and degradative methods. The lipid classes and their hydro-carbon chains resembled those of several Diptera species that have been analyzed from whole insects. The mosquito cells differed profoundly from cultured baby hamster kidney cells (BHK 21 cells) in their membrane lipids. As these two cells are suitable hosts for Semliki Forest virus, they provide us with good model systems for the study of lipid-protein interactions in the virus.

The stereochemical configuration of lysobisphosphatidic acid from rat liver, rabbit lung and pig lung

Lysobisphosphatidic acid known also as bis(monoacyl-glycerol)phosphate, was isolated from liver of rats treated with Triton WR1339, and from rabbit and pig lung. Alkaline hydrolysates of all these samples of lysobisphosphatidic acid were essentially similar and contained phosphorus, total glycerol, free glycerol, total glycerophosphates, beta-glycerophosphate, total alpha-glycerophosphates, sn-glycero-1-phosphate and sn-glycero-3-phosphate in a molar ratio of 1.0 : 2.0 : 1.0 : 1.0 :0.6 : 0.4 : 0.38 : 0.04. This proves that the backbone of the principal lysobisphosphatidic acid from all three sources has the structure of 1-sn-glycerophospho-1-sn-glycerol.