Effects of unsaturated fatty acids on the peroxisomal enzyme activities of Tetrahymena pyriformis

The effects of unsaturated fatty acids on the activities of peroxisomal enzymes of Tetrahymena pyriformis were investigated. When saturated fatty acids and the corresponding unsaturated fatty acids (C18) were added to the culture medium at 0.05%, the activities of peroxisomal enzymes [fatty acyl-CoA oxidase (FAO), carnitine acetyltransferase (CAT), isocitrate lyase (ICL), and malate synthase (MS)] were significantly increased. The order of effectiveness was linoleic acid greater than oleic acid greater than stearic acid. However, alpha-linolenic acid and gamma-linolenic acid at the same concentration were lethal to the cells. The inhibitory effect on growth disappeared upon addition of an antioxidant, alpha-tocopherol. Lipid peroxides derived from unsaturated fatty acids induced marked cell lysis. In the presence of a low concentration (0.005%) of linolenic acid the production of lipid peroxide was lower and no inhibitory effect on the growth was observed, while the activities of peroxisomal enzymes participating in lipid metabolism and that of catalase were significantly increased. These results indicate that the peroxisomal enzyme systems related to the beta-oxidations of fatty acids and the glyoxylate cycle are regulated by unsaturated long-chain fatty acids, including linolenic acid, at low concentrations, as well as by saturated fatty acid in the medium.     

Developmental changes and regional distribution of phospholipase D and base exchange enzyme activities in rat brain

Phospholipase D (PL-D) activity per mg protein of whole homogenate increased 5.1 fold between Embryonic (E) day 17 and Postpartum (P) day 14 and slightly decreased by P 30 days. This was due to the decrease of PL-D activity of the P₂ fraction. The PL-D activity of P₂ and P₃ fractions increased 11.2 and 6.1 fold respectively between E 17 and P 14. The 3 base exchange enzyme (BEE) activities per mg protein of whole homogenate increased up to P 14 or P 21 and then decreased. This decrease was greater in the P₂ fraction and the P₃ fraction increased after P14. Brains from 1 day to 25 month old rats were dissected into 7 separate regions and both PL-D and BEE activities were measured. In adult rats, the hippocampus and hypothalamus had the highest PL-D activities while medulla+pons and cerebellum had the lowest PL-D activities. The developmental patterns of 5 regions except for hippocampus and hypothalamus were similar. PL-D activity in the hippocampus was maximum at P 7 followed by a steep decrease till P30 suggesting that the PL-D activity of the hypothalamus develops later and that of the hippocampus develops earlier than any other region. The distributions of BEE activities were quite different from those of PL-D activities. In adult rats, the cerebellum had the highest activity while the striatum and medulla+pons had the lowest. The BEE activities of cerebellum were lowest at P 1 and showed steep increase during the next 2 weeks.To whom to address reprint request are to be sent.     

Purification and properties of an ethanolamine-serine base exchange enzyme of rat brain microsomes

The activity of an ethanolamine and serine base exchange enzyme of rat brain microsomes was copurified to near homogeneity. The purification sequence involved detergent solubilization, Sepharose 4B column chromatography, phenyl-Sepharose 4B column chromatography, glycerol gradient sedimentation, and agarose-polyacrylamide gel electrophoresis under non-denaturing conditions. The ratio of the ethanolamine and serine base exchange activities remained almost constant during purification, and both enzyme activities were enriched 25-fold over the initial microsomal suspension. The final enzyme preparation which contained both enzyme activities showed a single protein band on sodium dodecyl sulfate-polyacrylamide gel, having an apparent molecular mass of about 100 kDa. Serine inhibited the ethanolamine incorporation by this preparation and ethanolamine inhibited the serine incorporation. The competitive nature of this inhibition was apparent from Lineweaver-Burk plots, suggesting that the enzyme catalyzes the incorporation of both ethanolamine and serine into their corresponding phospholipids. The Km and Ki values for ethanolamine were quite similar, being 0.02 and 0.025 mM, respectively. The Km and Ki values for serine were also quite similar being 0.11 and 0.12 mM, respectively. The pH optimum was the same at 7.0 with both substrates. The optimum Ca2+ concentration was 8 mM for serine incorporation.     

Modulation of rat myometrial guanylate cyclase activities by sodium nitroprusside and unsaturated fatty acids.

Guanylate cyclase activities are present in both soluble and particulate fractions of rat myometrial extract. Triton, slightly stimulated the soluble (50%) while markedly increasing (1000%) the particulate activity. Both fractions appear to be regulated independently. Predominantly, the soluble form was activated by sodium nitroprusside, involving interactions with SH-groups. On the other hand, the particulate form was stimulated by a series of unsaturated fatty acids and their hydroperoxides. The latter activation appears to result from direct hydrophobic effects rather than peroxide or free radical generation.     

Synthesis of unsaturated fatty acids

This article reviews synthetic routes leading to polyunsaturated fatty acids having “skipped” double bonds. Emphasis is placed on the “acetylenic approach”.The suitability of building blocks, their condensation reactions as well as the controlled reduction of triple bonds to cis double bonds are discussed. In addition, the application of the various methods to the preparation of polyunsaturated fatty acids labelled with ³H and/or ¹⁴C at distinct positions of the molecules is reviewed.     

Three distinct forms of rat brain protein kinase C: differential response to unsaturated fatty acids

Although the three distinct forms of protein kinase C isolated from rat brain soluble fraction are structurally very similar, they respond differently to free unsaturated fatty acids such as arachidonic acid to exhibit their catalytic activity. Type I enzyme encoded by gamma-sequence, as predicted by cDNA clone analysis, responds to these fatty acids only slightly, whereas Type III enzyme determined by alpha-sequence is activated by free unsaturated fatty acids in the presence of Ca2+ in a comparable manner to phosphatidylserine plus diacylglycerol. Type II, a mixture of two enzymes encoded by beta I- and beta II-sequence, resulting from alternative splicing, shows properties in between those of Type I and Type III. Some of these forms of protein kinase C may function at a relatively later phase of cellular responses when large amounts of unsaturated fatty acids and Ca2+ are mobilized.     

The effect of three triterpene acids and sporidesmin on the enzyme activities of rat liver plasma membranes.

Vinyl chloride and vinyl benzene (styrene) are mutagenic in microbial tests, in Drosophila, in yeast, and in mammalian cells. Reports from various countries have shown an excess of chromosomal aberrations in the lymphocytes of workers exposed to vinyl chloride monomer when the workers were compared with controls. Workers occupationally exposed to styrene also revealed a clear increase in the rate of chromosome aberrations in their lymphocytes. Both chloroethylene oxide and styrene oxide, the primary biotransformation products of vinyl chloirde and styrene respectively, bind covalently to cellular macromolecules. Vinyl chloride is a carcinogen in both animals and man. Styrene is currently being tested in animals. These findings, the demonstration of mutagenic response via microbial and other test systems and with observations of significant excesses of chromosomal aberrations among workers exposed to these agents, raise scientific and health oriented concern about the possible genetic risks of vinyl chloride and styrene to man.     

Reversible intramitochondrial release of protein related to unsaturated fatty acids of membranes.

The influence of temperature on intramitochondrial protein and enzyme release was studied in control and “lipid-deficient” rat liver mitochondria and in synaptosomal and “cell body” mitochondria of rat brain. (i) The fatty acid composition of the phospolipid fraction was shown to be different in control and lipid-deficient preparations. (ii) Arrhenius curves for temperature-dependent release of protein showed breaks. (iii) When comparing control to lipid-free rat liver mitochondria and cell body to synaptosomal rat brain mitochondria, shifts in the breaks in the Arrhenius plots were observed for release of aspartate aminotransferase, protein and malate dehydrogenase. (iv) Intramitochondrial temperature-dependent, succinate-induced protein release was also studied in rat liver mitochondria which had previously undergone a succinate-induced release and rebinding cycle. These mitochondria showed a temperature-dependent protein release identical to that of untreated mitochondria.     

Regional differences in lipid composition and incorporation of saturated and unsaturated fatty acids into microsomal membranes of rat small intestine

Incorporation of [1-14C]palmitic (16:0) and [1-14C]linoleic (18:2 omega 6) acids into microsomal membranes of proximal (jejunum) and distal (ileum) regions of rat small intestine was investigated, and the lipid composition, including fatty acid profiles of membrane phospholipids, was determined. Jejunal microsomes contained significantly higher amounts of total phospholipids, phosphatidylcholine, and phosphatidylinositol, and lower amounts of cholesterol and sphingomyelin when compared with ileal microsomes. Jejunal microsomal phospholipids contained higher levels of stearic (18:0), 18:2 omega 6, and eicosapentaenoic (20:5 omega 3) acids followed by reduced levels of oleic (18:1 omega 9), arachidonic (20:4 omega 6), and docosahexaenoic (22:6 omega 3) acids when compared with those from the ileum, except for phosphatidylinositol where no significant difference between 20:4 omega 6 content of each site was observed. In both jejunal and ileal microsomes, incorporation of [1-14C]18:2 omega 6 was significantly higher than that of [1-14C]16:0. Incorporation of both [1-14C]16:0 and [1-14C]18:2 omega 6 was significantly higher in jejunal microsomal lipid fractions (phospholipids, diacylglycerols, triacylglycerols) when compared with the ileal microsomal fraction. These data suggest that (1) jejunal and ileal microsomal membranes differ from each other in terms of lipid composition and lipid synthesis, (2) site variations in the specificity of acyltransferases for different fatty acids exist, and (3) higher delta 9-, delta 6-, delta 5-, and delta 4-desaturase activities exist in ileal compared with jejunal enterocytes.     

Ca2+ translocation across liposomal membranes enhanced by unsaturated long-chain fatty acids

The putative ionophoretic action of phosphatidic acid or arachidonic acid metabolites for Ca2+ has offered an attractive explanation for stimulation-coupled mobilization of cytoplasmic Ca2+. We have examined the effects of Ca2+ ionophore and long-chain unsaturated fatty acids on the translocation of Ca2+ across the liposomal membrane by using Quin II-entrapped liposomes, a sensitive assay system for ionophoresis of Ca2+. A23187 increased Quin II fluorescence intensity corresponding to the translocation of Ca2+ into liposomes. Similar translocation was observed with unsaturated long-chain fatty acids but not with saturated fatty acids. Thus, when phospholipases of cell membrane are activated by certain stimuli, unsaturated long-chain fatty acids are liberated and might mediate the mobilization of cytoplasmic Ca2+.     

Phospholipid base exchange enzyme activity in sarcolemmal membranes from the heart of cardiomyopathic hamsters

The activity of phospholipid base exchange enzymes has been evaluated in cardiac sarcolemmal membranes from Syrian Golden hamsters and from a hamster strain (UM-X7.1) characterized by a genetic form of hypertrophic cardiomyopathy. No choline base exchange activity and only a little serine base exchange activity were detected, whereas the ethanolamine base exchange enzyme was found highly active in membranes from both strains. For this reason, the present study is focussed on the ethanolamine base exchange enzyme. The apparent Km for ethanolamine of ethanolamine base exchange enzyme from Syrian Golden membranes and from UM-X7.1 strain membranes are 18 and 32 μM, respectively. The specific activity of the sarcolemmal ethanolamine base exchange enzyme is lower in the UM-X7.1 strain than in Syrian Golden hamsters. The calcium-dependence of the enzyme appears different when the membranes from the two strains are compared. Indeed, after removal of the membrane-bound divalent cations, comparable activities are found in both membrane preparations, whereas, upon addition of Ca²⁺ to the incubation mixtures, the activity of the enzyme is enhanced in the membranes from Syrian Golden strain more than in those from UM-X7.1 strain. The cholesterol content of sarcolemmal membranes is higher in the cardiomyopathic strain than in the Syrian Golden hamsters. A possible relation between changes of the membrane lipid composition and of the ethanolamine base exchange activity is discussed.     

Inhibition of rat alpha-fetoprotein-estrogen interaction by adult rat ovarian extracts--role of unsaturated fatty acids

Thin layer chromatography with four different solvent systems enabled us to show that ovarian extracts contained nonesterified unsaturated fatty acids able to compete with estradiol on the alpha-fetoprotein binding site. Gas chromatography demonstrated a high level of oleic and linoleic acids and the presence of arachidonic acid, a strong competitor of the rat alpha-fetoprotein-estrogen interaction. Arachidonic acid is a precursor of prostaglandins thus we suggest that its binding to AFP might play a role in the AFP-mediated ovarian regulation previously demonstrated.