Reevaluation of the pathways for the biosynthesis of polyunsaturated fatty acids.

Recent studies refute the commonly accepted, but untested, hypothesis that 7,10,13,16-22:4 and 7,10,13,16,19-22:5 are desaturated at position 4 by a microsomal acyl-CoA-dependent desaturase. The synthesis of 4,7,10,13,16,19-22:6 occurs via the following reaction sequence: 7,10,13,16,19-22:5-->9,12,15,18,21-24:5-->6,9,12,15,18,21-24:6 4,7,10,13,16,19-22:6. The synthesis of 4,7,10,13,16-22:5 from 7,10,13,16-22:4 takes place via an analogous pathway. According to these pathways the 24-carbon acids that are made in the endoplasmic reticulum move to a site for partial beta-oxidation, which is most likely peroxisomes. The products of partial beta-oxidation, 4,7,10,13,16-22:5 and 4,7,10,13,16,19-22:6, then move back to the endoplasmic reticulum where they are used as substrates for membrane lipid biosynthesis. The ability of a fatty acid to serve as a substrate for continued peroxisomal beta-oxidation, versus its transfer out of peroxisomes for subsequent endoplasmic reticulum-associated esterification reactions, may be an important control for regulating membrane lipid fatty acid composition. Indeed, the revised pathways of polyunsaturated fatty acid biosynthesis imply that there is considerable intracellular movement and recycling of fatty acids between peroxisomes and the endoplasmic reticulum. In addition, these revised pathways require that two 18-carbon and two 24-carbon acids are substrates for desaturation at position 6. Also, as linoleate and linolenate are metabolized, respectively, to 6,9,12,15,18-24:5 and 6,9,12,15,18,21-24:6, three n-6 acids and three n-3 acids are substrates for malonyl-CoA dependent chain elongation. It remains to be determined how many microsomal enzymes are required to carry out these reactions and whether other ancillary enzymes are expressed in tissues whose membrane lipids accumulate very long-chain polyunsaturated acids with up to 36 carbon atoms.

Peroxisomal-microsomal communication in unsaturated fatty acid metabolism.

The addition of 1-acyl-sn-glycero-3-phosphocholine (1-acyl-GPC) to peroxisomes decreased the production of acid-soluble radioactivity formed by beta-oxidation of [1-(14)C]arachidonate due to substrate removal by esterification into the acceptor. This peroxisomal-associated acyl-CoA:1-acyl-GPC acyltransferase activity was due to microsomal contamination. The production of acid-soluble radioactivity from [1-(14)C]7,10,13,16-22:4, but not from [3-(14)C]7,10,13,16-22:4 was independent of 1-acyl-GPC, with and without microsomes. By comparing rates of peroxisomal beta-oxidation with those for microsomal acylation, it was shown that the preferred metabolic fate of arachidonate, when added directly to incubations, or generated via beta-oxidation, was esterification by microsomal 1-acyl-GPC acyltransferase, rather than continued peroxisomal beta-oxidation.

Double bond removal from odd-numbered carbons during peroxisomal beta-oxidation of arachidonic acid requires both 2,4-dienoyl-CoA reductase and delta 3,5,delta 2,4-dienoyl-CoA isomerase.

The pathway for the peroxisomal beta-oxidation of arachidonic acid (5,8,11,14-20:4) was elucidated by comparing its metabolism with 4,7,10-hexadecatrienoic acid (4,7,10-16:3) and 5,8-tetradecadienoic acid (5,8-14:2) which are formed, respectively, after two and three cycles of arachidonic acid degradation. When [1-14C]4,7,10-16:3 was incubated with peroxisomes in the presence of NAD+ and NADPH, it resulted in a time-dependent increase in the production of acid-soluble radioactivity which was accompanied by the synthesis of 2-trans-4,7,10-hexadecatetraenoic acid and two 3,5,7,10-hexadecatetraenoic acid isomers. The formation of conjugated trienoic acids suggests that peroxisomes contain delta 3,5,delta 2,4-dienoyl-CoA isomerase with the ability to convert 2-trans-4,7,10-hexadecatetraenoic acid to 3,5,7,10-hexadecatetraenoic acid. When 1-14C-labeled 6,9,12-octadecatrienoic acid or 7,10,13,16-docosatetraenoic acid was incubated without nucleotides, the 3-hydroxy metabolites accumulated, since further degradation requires NAD(+)-dependent 3-hydroxyacyl-CoA dehydrogenase. When [1-14C]5,8,11,14-20:4 was incubated under identical conditions, no polar metabolite was detected, but 2-trans-4,8,11,14-eicosapentaenoic acid accumulated. When NADPH was added to incubations, 3-hydroxy-8,11,14-eicosatrienoic, 2-trans-4,8,11,14-eicosapentaenoic, 2-trans-8,11,14-eicosatetraenoic, and 8,11,14-eicosatrienoic acids were produced. Analogous compounds were formed from [1-14C]5,8-14:2. Our results show that the removal of double bonds from odd-numbered carbons in arachidonic acid thus requires both NADPH-dependent 2,4-dienoyl-CoA reductase and delta 3,5,delta 2,4-dienoyl-CoA isomerase. One complete cycle of 5,8-14:2 and 5,8,11,14-20:4 beta-oxidation yields, respectively, 6-dodecenoic and 6,9,12-octadecatrienoic acids.

Arachidonic acid formed by peroxisomal beta-oxidation of 7,10,13,16-docosatetraenoic acid is esterified into 1-acyl-sn-glycero-3-phosphocholine by microsomes.

Peroxisomal beta-oxidation of linoleic acid and arachidonic acid was depressed when 1-palmitoyl-sn-glycero-3-phosphocholine and microsomes were included in incubations. This reduction was due to the esterification of the substrate into the acceptor by microsomal 1-acyl-sn-glycero-3- phosphocholine acyltransferase. The first cycle of the beta-oxidation of 7,10,13,16-docosatetraenoic acid was independent of 1-acyl-sn-glycero-3-phosphocholine and microsomes. However, when arachidonate was produced it was esterified rather than serving as a substrate for continued beta-oxidation. When arachidonate and linoleate were incubated with peroxisomes alone, 2-trans-4,7,10-hexadecatetraenoic acid and 2-trans-4-decadienoic acid were the respective end products of beta-oxidation. 2-Oxo-8,11-heptadecadienone, a catabolite produced from linoleate, was most likely a nonenzymatic decarboxylation product of 3-oxo-9,12-octadecadienoic acid. In addition to the termination of beta-oxidation by microsomal-peroxisomal communication, our results with linoleate and arachidonate suggest that the reaction catalyzed by 2-trans-4-cis-dienoyl-CoA reductase is the control step in double bond removal. In addition, the beta-ketothiolase step may play a regulatory role in the peroxisomal beta-oxidation of linoleate but not arachidonate or 7,10,13,16-docosatetraenoic acid.

Metabolism of [1-14C]linoleic acid in human promyelocytic leukemia HL-60 cells grown and differentiated in serum-free medium.

The metabolism of [1-14C]linoleic acid (LA) was studied in human promyelocytic leukemia cell line HL-60, grown and differentiated in serum-free medium. Both undifferentiated and dibutyryl cyclic AMP-differentiated HL-60 cells exhibited similar patterns of conversion of LA to four other major fatty acids (i.e. 18:3, 20:3, 20:4, and 22:4).

Secretion of proteins by Bacillus subtilis 168 grown in the presence of membrane active agents (alcohols).

Sodium dodecyl sulphate-polyacrylamide gel electrophoresis was used to study the composition of proteins secreted by the Gram-positive microorganism Bacillus subtilis strain 168 when the latter was grown in the presence of primary alcohols (methanol, ethanol, 1-propanol and 1-butanol). These membrane-active agents had different effects on the pattern of proteins exported by B. subtilis. The secretion of some proteins was inhibited by the alkanols while that of others was stimulated, depending on the type of drug used. All alcohols were found to decrease the activity of extracellular enzymes such as alpha-amylase and serine protease without affecting significantly the activity of these enzymes when tested in vitro. The observed effects might be due to the ability of these agents to perturb the structure of biological membranes, thus interfering with the process of protein translocation through the lipid bilayers.

Ultracytochemical localization of ATP-hydrolysing activity in vegetative cells, spores and isolated cytoplasmic membranes of Bacillus subtilis 168.

The localization of ATP-hydrolysing activity in vegetative cells, spores and isolated membranes of Bacillus subtilis 168 was studied by a cytochemical method combined with electron microscopy. The activity was located mainly in the cytoplasmic membrane and the mesosomes, and was also found in the inner layer of the cell wall facing the cytoplasmic membrane. Activity was also detected in the cross-membranes of dividing cells and in spore coats. The product of the reaction was observed either as fine electron-dense granules incorporated into the membranes, or as high-contrast lead precipitates on the surfaces of the membranes.

Fatty acid composition of lipids of Escherichia coli W 1655 F+ and its stable protoplast type L-form.

The comparative fatty acid analysis of extractable and non-extractable lipids of Escherichia coli W 1655 F+ and its stable protoplast type L-form shows quantitative as well as qualitative differences. From 10 different fatty acids obtained 16:0, 17:0 and 18:0 are present at about the same quantities in the lipid fractions of the bacterial and L-form. The absence of larger amounts of 12:0, 14:0, and 14:beta OH fatty acids in non-extractable L-form lipids reflects the loss of the cell wall in L-form cells. 16:1 fatty acid was found in L-form lipids only. This qualitative difference and the 2-3 times higher content of 18:1 in L-form lipids and the 7 times lower content of cyc 19:0 in extractable lipids of the L-form may be interpreted as alterations characteristic for the changed composition of the cytoplasmic membrane in L-form cells.

Studies on the ultrastructure of Yersinia pseudotuberculosis after treatment with some detergents and solvents.

The ultrastructural changes in 3 strains Yersinia pseudotuberculosis with different virulence after treatment with sodium lauryl sulfate (SLS) and petroleum ether were studied. The ultrafine sections after treatment with SLS show heavy destructive changes, concerning the cell wall, the cytoplasmic membrane and the inner structure of the cell. It was established that the same cells Y. pseudotuberculosis after cultivation on a medium with glycerol show a tendency to recover their ultrastructure. The cells treated with petroleum ether did not exhibit any notable ultrastructural changes.