Acyl and alk-1-enyl group compositions of the alk-1'-enyl acyl and the diacyl glycerophosphoryl ethanolamines of mouse and ox brain

The ethanolamine phosphoglycerides were prepared from lipid extracts of ox and mouse brains by preparative thin-layer chromatography. The cyclic acetal derivatives of the alk-1-enyl groups were made by treating the ethanolamine phosphoglycerides with 1,3-propanediol. The resulting monoacyl glycerophosphoryl ethanolamines were separated from the unchanged ethanolamine phosphoglycerides by preparative thin-layer chromatography. Methyl ester derivatives of the acyl groups from both of these fractions were prepared by alkaline methanolysis. The cyclic acetal and methyl ester derivatives were analyzed by gas-liquid chromatography. Substantial differences were found in the composition of the side chains when the combined alk-1-enyl and acyl side chains of the alk-1'-enyl acyl glycerophosphoryl ethanolamines were compared with the side chains of the diacyl glycerophosphoryl ethanolamines. The side chains from the 1-position of these two ethanolamine phosphoglycerides are different in chain length and unsaturation as well as in chemical bonding. The acyl groups from the 2-position of the alk-1'-enyl acyl glycerophosphoryl ethanolamines were predominantly unsaturated. Therefore, acyl group compositions of the total ethanolamine phosphoglyceride from brain are of limited value and individual types should be analyzed.     

Replacement of acyl and alk-1-enyl groups in Clostridium butyricum phospholipids by exogenous fatty acids.

The effect of exogenous unsaturated fatty acids on the acyl and alk-1-enyl group composition of the phospholipids of Clostridium butyricum has been examined. Unsaturated fatty acids support the growth of this organism in the absence of biotin. When cells were grown at 37 degrees in media containing oleate or linoleate and a Casamino acid mixture containing traces of biotin, the exogenous fatty acids were found mainly in the alk-1-enyl chains of the plasmalogens with less pronounced incorporation into the acyl chains. However, at 25 degrees in this medium, both the acyl and alk-1-enyl chains contained substantial amounts of the 18:1 supplement plus the C19-cyclopropane chains derived from it. Ak-1-enyl chains in all the major phosphatide classes showed a uniformly high substitution by the oleate supplement in cells grown at 37 degrees. The oleate and C19-cyclopropane content of the acyl chains was more variable among the phosphatide classes. At 37 degrees, trans-9-octadecenoic acid (elaidic acid) also supported growth and was incorporated into both acyl and alk-1-enyl chains at a high level. When cells were grown on oleate at 37 degrees in media containing biotin-free Casamino acids, both the acyl and alk-1-enyl chains had a high level of 18:1 plus C19-cyclopropane chains. In the cells grown at 37 degrees with oleate substantial changes were seen in the phospholipid class composition. There was a large decrease in the ethanolamine plus N-methylethanolamine plasmalogens with a corresponding increase in the glycerol acetals of these plasmalogens. The glycerol phosphoglycerides were also significantly lower with the appearance of an unknown, relatively nonpolar phospholipid fraction.     

Evidence for the existence of cholesteryl alk-1-enyl ethers in bovine and porcine cardiac muscle

Evidence is presented for the existence of cholesteryl alk-1-enyl ethers in bovine and porcine cardiac muscle. Several different fatty chains are present in the cholesteryl ethers, the major species having 16 and 18 carbon atoms. The cholesteryl alk-1-enyl ether concentration was found to be 0.08 and 0.01 micro moles/100 mg of neutral lipid in bovine and porcine cardiac muscle, respectively.     

The alkyl and alk-1-enyl glycerols in the liver of rats fed long-chain alcohols or alkyl glycerols

Dietary long-chain alcohols and alkyl glycerols, including polyunsaturated compounds, are incorporated into the alkyl and alk-1-enyl moieties of the ionic alkoxylipids of rat liver, whereas polyunsaturated fatty acids are not.     

Reductive and oxidative biosynthesis of plasmalogens in myelinating brain

Palmitic acid-1-(14)C and hexadecanol-1-(14)C were administered intracerebrally to 18-day-old rats. Incorporation of radioactivity into the constituent alkyl, alk-1-enyl, and 1-acyl moieties, as well as into the 2-acyl moieties, of the ethanolamine phosphatides of brain was determined after 1, 2, 3, 6, and 22 hr. Incorporation of radioactivity from hexadecanol into both alkyl ethers and alk-1-enyl ethers proceeded at a rate more than 10 times higher than from palmitic acid. Hexadecanol was rapidly oxidized to fatty acids which were incorporated into the acyl moieties of the ethanolamine phosphatides. When palmitic acid was used as a precursor, labeled long-chain alcohols could be isolated from the lipid extract. As labeled long-chain aldehydes could not be detected in any of the lipid extracts, alcohols appear to be key intermediates for the biosynthesis of both alkyl and alk-1-enyl glycerophosphatides.     

Conversion of fatty aldehyde dimethyl acetals to the corresponding alk-1-enyl methyl ethers (substituted vinyl ethers) during gas-liquid chromatography

The behavior of palmitaldehyde and linolealdehyde and of their dimethyl acetals during gas-liquid chromatography on beta-cyclodextrin acetate (beta-CDX acetate) and ethylene glycol succinate polyester-phosphoric acid (EGSP) columns was studied. The aldehydes were well separated from their dimethyl acetals on the beta-CDX acetate column. However, on the EGSP column the retention times of palmitaldehyde and its dimethyl acetal were identical; a mixture of linolealdehyde and its dimethyl acetal gave a split peak. The aldehydes were recovered unchanged in 80-85% yield by preparative GLC from both columns, but the dimethyl acetals were quantitatively converted to the corresponding alk-1-enyl methyl ethers. The structure of these compounds was elucidated by infrared spectroscopy, mass spectrometry, and chemical means. Upon hydrolysis at low temperatures with 100% H(2)SO(4) they yielded the corresponding aldehydes, which were identified as 2,4-dinitrophenylhydrazones.     

Mitochondrial adenosine triphosphatase. Location of sulfhydryl groups and disulfide bonds in soluble enzyme from beef heart.

The soluble beef heart mitochondrial ATPase (F1) contains eight sulfhydryl groups and two disulfide bonds. N-Ethylmaleimide has been used to radioactively label the sulfhydryl groups before and after cleavage of the disulfide bonds by dithiothreitol. After subjecting the labeled protein to polyacrylamide gel electrophoresis in sodium dodecyl sulfate and measuring radioactivity in each of the separated subunits the location of all the sulfhydryl groups and the disulfide bonds may be specified. The conclusions are supported by direct examination of depolymerized, unreduced, enzyme by polyacrylamide gel electrophoresis. The results also indicate that current ideas regarding the overall subunit structure of this enzyme may be incorrect, and this is discussed in light of new data presented here.     

The position of the disulfide bonds in human plasma alpha 2 HS-glycoprotein and the repeating double disulfide bonds in the domain structure

The positions of the inter- and intra-chain disulfide bonds of human plasma alpha 2 HS-glycoprotein were determined. alpha 2 HS-glycoprotein was digested with acid proteinase and then with thermolysin. The disulfide bonds containing peptides were separated by reversed-phase HPLC and detected by SBD-F (7-fluorobenzo-2-oxa-1,3-diasole-4-sulfonic acid ammonium salt) method. One inter-disulfide bond containing peptide and five intra-disulfide bond containing peptides (A-chain) were purified and identified as Cys-18 (B-chain)--Cys-14 (A-chain), Cys-71--Cys-82, Cys-96--Cys-114, Cys-128--Cys-131, Cys-190--Cys-201 and Cys-212--Cys-229, respectively. The location of the intra-disulfide bonds revealed that the A-chain of alpha 2 HS-glycoprotein is composed of three domains. Two domains were shown to possess intramolecular homology judging from the total chain length of the domains, size of the loops formed by the S--S bonds, the location of two disulfide loops near the C-terminal end of domains A and B, the distance between two S--S bonds of each domain, the amino acid sequence homology between these two domains (22.6%), number of amino acid residues between the second S--S loops and the end of domains A and B, and the positions of the ordered structures.     

Formation of disulfide bonds between glutathione and membrane sh groups in human erythrocytes

In erythrocytes treated with the SH-oxidizing agent, diamide, mixed disulfide bonds between membrane proteins and GSH are formed involving 20% of the membrane SH groups. To study the distribution of these mixed disulfides over the membrane protein fractions, intracellular GSH was labelled biosynthetically with [2-³H]glycine prior to diamide treatment of the cells and the radioactivity of defined membrane peptide fractions determined. Mixed disulfides preferentially occur in the extrinsic protein, spectrin (six SH groups), in addition to the formation of peptide disulfides. Intrinsic proteins are much less reactive: only one SH group of the major intrinsic protein (band 3) reacts with GSH, which accounts for previously observed impossibility to dimerize band 3 via disulfide bonds in intact cells. The labelling method described offers a promising strategy to label and map exposed endofacial SH groups of membrane proteins with a physiological, impermeable marker, GSH.In ghosts treated with diamide and GSH the number of mixed disulfides formed is greater than in erythrocytes. Polymerization of spectrin via intermolecular disulfide bridges is suppressed, while intramolecular disulfides are still formed, providing a means for the analysis of spectrin structure.The diamide-induced mixed membrane-GSH disulfides are readily reduced by GSH. This suggests, that GSH may also be able to reduce mixed disulfides formed in the erythrocyte membrane under oxidative stress in vivo. The reversible formation of mixed disulfides may serve to protect sensitive membrane structures against irreversible oxidative damage.     

Phospholipids of Clostridium butyricum. IV. Analysis of the positional isomers of monounsaturated and cyclopropane fatty acids and alk-1'-enyl ethers by capillary column chromatography

The positional isomers of the cyclopropane fatty acids of Clostridium butyricum phospholipids have been analyzed by capillary column gas-liquid chromatography. Greater than 95% of the methylenehexadecanoic acids was the 9,10 isomer. On the other hand, 60-70% of the hexadecenoic acid precursors was the Delta(7) isomer, and the remainder was the Delta(9) isomer. Of the methyleneoctadecanoic acids 75-80% was the 11,12 isomer, with the remainder being the 9,10 isomer. There were approximately equal amounts of the Delta(9)- and Delta(11)-octadecenoic acids in the phospholipids. This study reveals a surprisingly strong specificity of the cyclopropane synthetase for the (n-7) series of monoenoic fatty acids. An analysis by capillary column chromatography of the monoenoic and cyclopropane aldehyde dimethylacetals derived from the plasmalogens (1-alk-1'-enyl-2-acyl-glycero-phosphatides) of C. butyricum revealed the presence of the same positional isomeric mixtures of the 16- and 18-carbon monoenoic residues in approximately the same ratios as were found in the fatty acids. In the formation of the cyclopropane alk-1'-enyl ethers there was also specificity for the (n-7) series, but it was not as strong as that seen in the fatty acids. The ratio of the 7,8 isomer to the 9,10 isomer was higher in the methyl-enehexadecanals than in the corresponding fatty acids. This paper extends the use of Golay capillary columns to the analysis of the positional isomers of plasmalogen aldehydes as their dimethylacetal derivatives.     

Positions of disulfide bonds and N-glycosylation site in juvenile hormone binding protein

The juvenile hormone binding protein (JHBP) from Galleria mellonella hemolymph is a glycoprotein composed of 225 amino acid residues. It contains four Cys residues forming two disulfide bridges. In this study, the topography of the disulfide bonds as well as the site of glycan attachment in the JHBP molecule from G. mellonella was determined, using electrospray mass spectrometry. The MS analysis was performed on tryptic digests of JHBP. Our results show that the disulfide bridges link Cys10 and Cys17, and Cys151 and Cys195. Of the two potential N-glycosylation sites in JHBP, Asn4, and Asn94, only Asn94 is glycosylated. This site of glycosylation is also found in the fully biologically active recombinant JHBP expressed in the yeast Pichia pastoris.     

Oxidative damage in brain from human mutant APP/PS-1 double knock-in mice as a function of age

Oxidative stress is strongly implicated in the progressive decline of cognition associated with aging and neurodegenerative disorders. In the brain, free radical-mediated oxidative stress plays a critical role in the age-related decline of cellular function as a result of the oxidation of proteins, lipids, and nucleic acids. A number of studies indicate that an increase in protein oxidation and lipid peroxidation is associated with age-related neurodegenerative diseases and cellular dysfunction observed in aging brains. Oxidative stress is one of the important factors contributing to Alzheimer's disease (AD), one of whose major hallmarks includes brain depositions of amyloid beta-peptide (Abeta) derived from amyloid precursor protein (APP). Mutation in APP and PS-1 genes, which increases production of the highly amyloidogenic amyloid beta-peptide (Abeta42), is the major cause of familial AD. In the present study, protein oxidation and lipid peroxidation in the brain from knock-in mice expressing human mutant APP and PS-1 were compared with brain from wild type, as a function of age. The results suggest that there is an increased oxidative stress in the brain of wild-type mice as a function of age. In APP/PS-1 mouse brain, there is a basal increase (at 1 month) in oxidative stress compared to the wild type (1 month), as measured by protein oxidation and lipid peroxidation. In addition, age-related elevation of oxidative damage was observed in APP/PS-1 mice brain compared to that of wild-type mice brain. These results are discussed with reference to the importance of Abeta42-associated oxidative stress in the pathogenesis of AD.