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PubMed1975

Lipid metabolism during bacterial growth, sporulation, and germination: kinetics of fatty acid and macromolecular synthesis during spore germination and outgrowth of Bacillus thuringiensis.

The timing and kinetics of fatty acid synthesis are delineated for Bacillus thuringiensis spore germination and outgrowth by analyzing [U-14C]acetate and [2-3H]glycerol incorporation into chloroform-methanol-extractable and trichloroacetic acid-precipitable lipids. In addition to measurement of pulsed and continuous labeling of fatty acids, monitoring the incorporation of radioactive phenylalanine, thymidine, and uridine from the onset of germination through first cell division provides a profile of biochemical activities related to membrane differentiation and cellular development. Upon germination, ribonucleic acid synthesis is initiated, immediately followed by rapid and extensive fatty acid synthesis that in turn precedes protein, deoxyribonucleic acid and triglyceride synthesis. Significantly, formation of fatty acids from acetate exhibits further developmental periodicity in which a large transient increase in fatty acid synthetic activity coincides with the approach of cell division. Radiorespirometric analyses indicates only slight oxidative decarboxylation of acetate and corroborates the extreme involvement of acetate in specific fatty acid biosynthetic reactions throughout cellular modification. These findings graphically demonstrate an intimate association of fatty acid metabolism with commitment to spore outgrowth and subsequent cell division.

Acetates↗

PubMed1976

[Study of the biosynthesis of phleic acids, polyunsaturated fatty acids synthesised by Mycobacterium phlei (author's transl)].

Because of their structures, phleic acids (general formula: CH3-(CH2)m-(CH=CH-CH2-CH2)n-CO2H; main component: m = 14, n = 5) cannot be synthesized by the same kinds of enzymatic systems as other natural polyunsaturated fatty acids. By using specifically labelled 14C compounds, we have tested the ability of different molecules to be incorporated in the phleate skeletons by Mycobacterium phlei. The localisation of radioactive carbon atoms has been studied by chemical degradation of labelled phleates, isolation and purification of the degradation products, and determination of their specific radioactivity. When M. phlei cells are incubated with labelled acetate, the unsaturated and saturated parts of the molecules of phleic acids are unequally labelled. The radioactivity of succinate monoester on the one hand and fatty acids (mixture of myristic and palmitic acids) on the other hand, measured after oxidative degradation of phleate esters, shows a constant ratio under definite conditions. Whether [1-14C]acetate or [2-14C]acetate is used for incubation, the same ratio is observed. Therefore acetate is the precursor of the unsaturated part as well as of the saturated part of the phleate molecules. By using labelled fatty acid esters, it has been found that palmitic acid is the precursor of phleates with m = 14, while myristic acid is the precursor of phleates with m = 12. Stearic and eicosanoic acids are not incorporated without degradation. The hypothesis of a condensation of a saturated fatty acid with a preformed polyunsaturated molecule was examined. Search for such a molecule in the lipids of M. phlei gives negative results. Pentaunsaturated phleate arising from palmitate is more abundant than pentaunsaturated phleate arising from myristate, while the reverse is true for hexaunsaturated phleates. These observations make very unlikely such an hypothesis. An elongation process fits well with the observed facts provided that this process involves elongation by two acetate units simultaneously, making elongation by four carbon atoms at a time. Such a requirement would be easily satisfied if two molecules of acetate are condensed together before their utilization in the elongation process. In such a hypothetical process, crotonate would be the most probable substrate of the elongation reaction.

Acetates↗

PubMed1992

Delta 5-3 beta-hydroxysteroid acyl transferase activity in the rat brain.

Pregnenolone and dehydroepiandrosterone accumulate in brain as sulfate and fatty acid esters and unconjugated steroids. The steroid fatty acid ester-synthesizing activity was investigated in rat brain microsomes. Endogenous fatty acids in the microsomal fraction were used for the esterification of steroids. The enzyme system had a pH optimum of 4.5 in acetate buffer with [3H]dehydroepiandrosterone as substrate. The apparent Km was 9.2 +/- 3.1 x 10(-5) M and Vmax was 18.6 +/- 3.4 nmol/h/mg protein (mean +/- SEM). The inhibition constants of pregnenolone and testosterone were 123 and 64 microM, respectively. Results were compatible with a competitive type of inhibition. A high level of synthetic activity was found in the brain of 1- to 3-week-old male rats, which rapidly decreased with aging. Saponification of purified [3H]pregnenolone esters yielded pregnenolone and a mixture of palmitate, oleate, linoleate, stearate, and myristate as the predominant fatty acids. Contrasting with the high rates of esterification of several radioactive delta 5-3 beta-hydroxysteroids or 17 beta-hydroxysteroids, no fatty acid esters of either cholesterol, epitestosterone (with a hydroxyl group at position C-17 alpha), or corticosterone (with hydroxyl groups at C-21 and C-11 beta) were formed in the same incubation conditions.

Acyltransferases↗

PubMed1977

Comparative inactivation of yeast fatty acid synthetase component enzymes by 100 atmospheres of oxygen.

Yeast fatty acid synthetase at 4 degrees C was stable during 1- and 2-h exposures to oxygen at 100 atm, but was 48% and 90% inactivated after 20 h and 40 h, respectively, with fatty acid synthesis measured by both radioactive and optical assays. Incubation with dithiothreitol did not restore activity. Component enzyme activities were compared before and after 40 h in 100 atm of oxygen. Ketoacyl reductase activity was most reduced followed by ketoacyl synthetase and then acetyl transferase while malonyl transferase, enoyl reductase and palmitoyl transferase were not significantly inactivated.

Drug Stability↗

PubMed1982

[Modes of action of a shortening system for erucic acid at the mitochondrial level of rat liver].

In this work, were studied the conditions of erucic acid (cis-docosenoic, n-9) shortening by using Rat liver mitochondrial preparations which were incubated in vitro with [14-14C] erucic acid (22:1), with inhibitors of the respiratory chain (rotenone, cyanide) or not, with activators of either the shortening reaction (NAD+, NADP+), or beta-oxidation (malate, carnitine, cytochrome c) or not. The shortening activity was measured by the amount of 14C radioactivity recovered in the shorter fatty acids formed (20:1, 18:1, 16:1) when beta-oxidation was inhibited. The beta-oxidation activity was measured by the amount of 14C recovered in the acid-soluble products (P A S). The incubations were performed under conditions which were the least favourable to peroxysomal activity. Data showed that, with increasing amounts of albumin, which inhibits peroxysomal activity, increasing amounts of shorter fatty acids (20:1, 18:1, 16:1) were formed from erucic acid. This shortening reaction was strongly stimulated by NAD+, more than by NADP+; it was also stimulated by cytochrome c and much more when both NAD+ and cytochrome c were added. Similar data were observed in beta-oxidation, except that practically NADP+ did not exhibit any stimulating effect. Oxidation of NADH by mitochondria only occurred when cytochrome c was added to the medium and was not modified by the addition of ADP or rotenone. These data show that liver mitochondria are capable of shortening erucic acid, as are peroxysomes. This shortening reaction is highly NAD+-dependent and seems to be localized outside the matrix. This system could constitute a second route for utilization of fatty acids in mitochondria, besides the well-known path of beta-oxidation.

Animals↗

PubMed2003

Glycerolipid synthesis in Chlorella kessleri 11h. I. Existence of a eukaryotic pathway.

The fatty acid distributions at the sn-1 and sn-2 positions in major chloroplast lipids of Chlorella kessleri 11h, monogalactosyl diacylglycerol (MGDG) and digalactosyl diacylglycerol (DGDG), were determined to show the coexistence of both C16 and C18 acids at the sn-2 position, i.e. of prokaryotic and eukaryotic types in these galactolipids. For investigation of the biosynthetic pathway for glycerolipids in C. kessleri 11h, cells were fed with [14C]acetate for 30 min, and then the distribution of the radioactivity among glycerolipids and their constituent fatty acids during the subsequent chase period was determined. MGDG and DGDG were labeled predominantly as the sn-1-C18-sn-2-C16 (C18/C16) species as early as by the start of the chase, which suggested the synthesis of these lipids within chloroplasts via a prokaryotic pathway. On the other hand, the sn-1-C18-sn-2-C18 (C18/C18) species of these galactolipids gradually gained radioactivity at later times, concomitant with a decrease in the radioactivity of the C18/C18 species of phosphatidylcholine (PC). The change at later times can be explained by the conversion of the C18/C18 species of PC into galactolipids through a eukaryotic pathway. The results showed that C. kessleri 11h, distinct from most of other green algal species that were postulated mainly to use a prokaryotic pathway for the synthesis of chloroplast lipids, is similar to a group of higher plants designated as 16:3 plants in terms of the cooperation of prokaryotic and eukaryotic pathways to synthesize chloroplast lipids. We propose that the physiological function of the eukaryotic pathway in C. kessleri 11h is to supply chloroplast membranes with 18:3/18:3-MGDG for their functioning, and that the acquisition of a eukaryotic pathway by green algae was favorable for evolution into land plants.

Acetates↗

PubMed1975

The origin of fatty acids in the hydrocarbon-utilizing microorganism Mycobacterium vaccae.

The fatty acid pattern in Mycobacterium vaccae strain JOB5 was examined after growth on n-alkanes (C14-C18), 1-alkenes (C14-C18), 2- or 3-methyl octadecane, and 8-heptadecene. It was evident that monoterminal oxidation of n-alkanes was followed by beta-oxidation and that both parent fatty acid and products of beta-oxidation were incorporated into cellular lipids. Radioactive experiments demonstrated that there was desaturation of long-chain fatty acids. There was no evidence of chain elongation. Growth on 1-alkenes resulted in the incorporation of fatty acids that were products of two primary modes of oxidation: (1) methyl group attack resulting in omega-unsaturated fatty acids and (2) double-bond attack resulting in the removal of one carbon from the substrate. Cells of strain JOB5 grown on 2- and 3-methyl octadecane contained the corresponding iso- or anteiso-fatty acids in significant quantity. Cells cultured on 8-heptadecene contained 8- and 9-heptadecenoic acids, 6- and 7-pentadecenoic acids, 9- and 10-methyl heptadecanoic acids, and 7- and 8-methyl pentadecanoic acids. Fatty acid composition (C13 to C19) was affected by substrate chain length and was additionally modified by cellular control mechanisms.

Acetates↗

PubMed1983

In vivo incorporation of [3H]palmitic acid into PO protein, the major intrinsic protein of rat sciatic nerve myelin.

Separation of rat sciatic nerve myelin proteins by sodium dodecyl sulfate-slab gel electrophoresis 18 h after injection of [3H]palmitic acid into the nerve demonstrated acylation of the PO protein. When sciatic nerve myelin labeled with [3H]palmitic acid was extracted with acidified chloroform/methanol or chloroform/methanol, the radioactivity associated with PO was retained. These results provided evidence that the radioactivity derived from [3H]palmitic acid in PO protein was not due to labeled phospholipid bound to PO protein by strong physical interaction. Furthermore, the radioactivity associated with purified preparations of PO remained after dialysis and re-electrophoresis, providing additional evidence that [3H]palmitic acid was firmly bound to PO. Treatment of myelin proteins with hydroxylamine at pH 6.6 released most of the radioactivity, indicating that [3H]palmitic acid was covalently bound by ester linkage to PO. Cleavage of purified acylated PO with methanolic NaOH released 85% of the protein-bound radioactivity. Gas-liquid chromatography of the fatty acids released from PO showed labeling of methyl esters of palmitate, stearate, and oleate (56, 16, and 5%, respectively). In addition, a small unlabeled peak with retention time identical with methyl linoleate was also observed. Intraneural injection of a mixture of [3H]palmitic acid and [14C]fucose or [3H]palmitic acid and [35S]sulfate in vivo followed by disc gel electrophoresis and determination of incorporated radioactivity clearly showed that PO was glycosylated, sulfated, and acylated. The potential significance of fatty acids linked to PO is discussed.

Acylation↗

PubMed1972

The uptake and metabolism of plasma lysophosphatidylcholine in vivo by the brain of squirrel monkeys.

1. Adult squirrel monkeys were injected intravenously with doubly labelled lysophosphatidylcholine (a mixture of 1-[1-(14)C]palmitoyl-sn-glycero-3-phosphorylcholine and 1-acyl-sn-glycero-3-phosphoryl[Me-(3)H]choline; (3)H:(14)Cratio 3.75) complexed to albumin, and the incorporation into the brain was studied at times up to 3h. 2. After 20min, 1% of the radioactivity injected as lysophosphatidylcholine had been taken up by the brain. 3. Approx. 70% of the doubly labelled lysophosphatidylcholine taken up by both grey and white matter was converted into phosphatidylcholine, whereas about 30% was hydrolysed. 4. The absence of significant radioactivity in the phosphatidylcholine, free fatty acid and water-soluble fractions of plasma up to 30min after injection of doubly labelled lysophosphatidylcholine rules out the possibility that the rapid labelling of these compounds in brain could be due to uptake from or exchange with their counterparts in plasma. 5. The similarity between the (3)H:(14)C ratios of brain phosphatidylcholine and injected lysophosphatidylcholine demonstrates that formation of the former occurred predominantly via direct acylation. 6. Analysis of the water-soluble products from lysophosphatidylcholine catabolism revealed that appreciable glycerophosphoryl-[Me-(3)H]choline did not accumulate in the brain and that radioactivity was incorporated into choline, acetylcholine, phosphorylcholine and betaine. 7. The role of plasma lysophosphatidylcholine as both a precursor of brain phosphatidylcholine and a source of free choline for the brain is discussed.

Acetylcholine↗

PubMed1995

Apical release of base-labile fatty acyl groups commensurate with stimulation of glycoprotein sialosyl Lewis(a) secretion in colorectal carcinoma cells.

The rate of polarized secretion of a putative adhesion ligand, sialosyl Lewis(a) (19-9), by SW1116 colorectal carcinoma cells is stimulated at least 20-fold after pre-incubation with, and the incorporation of, retinoic acid (RA). In order to investigate the possible involvement of fatty acylation in the export of the epitope, purified ligands from carcinoma-cell membranes, membrane subfractions and media were analyzed during RA-induced secretion. Incorporation of radioactivity from (3H)palmitate into membrane subfractions and purified sialosyl Lewis(a) antigenic molecular species of M(r) > 150,000 (SiaLeams) was stimulated by RA treatment. Most of the intracellular lipid radioactivity which bound to solid-phase 19-9 antibody behaved chromatographically, either like ganglioside or like NH2 OH-labile acyl groups, but most of the (3H) bound to SiaLeams of post-incubation media behaved like base-labile fatty acyl groups, or free fatty acid. Release of base-labile lipid radioactivity after 3 hr (associated with antigen) was almost exclusively into the apical media of membrane inserts. Gas-liquid chromatography/mass spec. analyses of purified Sialeams revealed the presence of palmitate (16:0), as well as stearate (18:0) and oleate (18:1) fatty acyl groups. Our results suggest that fatty acylation of SiaLeams may be co-ordinated with alterations in glycosylation and participate in directing these molecules to the apical surface. Lipid analyses were consistent with ganglioside chaperonage of SiaLeams to the apical surface, where N-fatty-acylated gangliosides remain for the most part integrated into the bilayer, but some oxyester or thioester bonds may be cleaved to permit release of SiaLeams to the apical medium.

Acylation↗

PubMed1987

Phosphatidylinositol anchor of HeLa cell alkaline phosphatase.

Alkaline phosphatase from cancer cells, HeLa TCRC-1, was biosynthetically labeled with either 3H-fatty acids or [3H]ethanolamine as analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and fluorography of immunoprecipitated material. Phosphatidylinositol-specific phospholipase C (PI-PLC) released a substantial proportion of the 3H-fatty acid label from immunoaffinity-purified alkaline phosphatase but had no effect on the radioactivity of [3H]ethanolamine-labeled material. PI-PLC also liberated catalytically active alkaline phosphatase from viable cells, and this could be selectively blocked by monoclonal antibodies to alkaline phosphatase. However, the alkaline phosphatase released from 3H-fatty acid labeled cells by PI-PLC was not radioactive. By contrast, treatment with bromelain removed both the 3H-fatty acid and the [3H]ethanolamine label from the purified alkaline phosphatase. Subtilisin was also able to remove the [3H]ethanolamine-labeled from purified alkaline phosphatase. The 3H radioactivity in alkaline phosphatase purified from [3H]ethanolamine-labeled cells comigrated with authentic [3H]ethanolamine by anion-exchange chromatography after acid hydrolysis. The data suggest that the 3H-fatty acid and [3H]ethanolamine are covalently attached to the carboxyl-terminal segment since bromelain and subtilisin both release alkaline phosphatase from the membrane by cleavage at that end of the polypeptide chain. The data are consistent with findings for other proteins recently shown to be anchored in the membrane through a glycosylphosphatidylinositol structure and indicate that a similar structure contributes to the membrane anchoring of alkaline phosphatase.

Alkaline Phosphatase↗

PubMed1989

Acylation of proteins by myristic acid in isolated mitochondria.

Isolated and highly purified mitochondria from rat liver were incubated with [1-14C]myristate, solubilized in boiling sodium dodecyl sulfate, and analyzed by polyacrylamide gel electrophoresis and autoradiography. Six to eight protein bands were found to be radioactively labeled. If the mitochondria were heated for 5 min at 95 degrees C prior to incubation with this fatty acid, no labeling was observed. By preexposing the mitochondria to unlabeled fatty acids of varying chain lengths, the extent of labeling by [1-14C]myristate was reduced in a chain length-dependent manner, exhibiting maximal inhibition at lauric acid. Reversibility of the labeling was demonstrated by chasing the incorporated radioactivity with unlabeled fatty acids of varying chain length, resulting in a maximal displacement of the tracer again by lauric acid. Fractionation of the labeled mitochondria into mitochondrial matrix and inner mitochondrial membrane components before or after labeling showed that the modified proteins are located inside the inner mitochondrial membrane. In both cases, the pattern of labeling was different from the one observed with intact mitochondria. The labeled bands in the gel were sensitive to alkaline methanol or hydroxylamine treatment. The radioactivity recovered after this treatment co-migrated with myristic acid on thin layer chromatography plates. The chain length specificity and the rapid reversibility of the observed acylation argue for a new type of reaction, different from the acylation observed in whole cells. The possible involvement of the acylated proteins in the regulation of oxidative phosphorylation is discussed.

Acylation↗

PubMed1971

Lipogenesis from glucose-2- 14 C and acetate-1- 14 C in aorta.

Lipogenesis was measured with glucose-2-(14)C and acetate-1-(14)C in the everted aortas of normal and atherosclerotic rabbits. More glucose-2-(14)C than acetate-1-(14)C was incorporated into lipids in both the normal and the atherosclerotic aorta. Radiocarbon from glucose-2-(14)C appeared mainly in triglycerides and phospholipids with a small amount in cholesteryl esters. Incorporation increased almost threefold with atherosclerosis, most of the radioactivity being in the glycerol moiety; radioactivity was predominantly in carbon 2 of glycerol. About 70% of the acetate-1-(14)C incorporated into phospholipids and triglycerides was in the fatty acids, and the remainder was in glyceride-glycerol; 98% of the radioactivity in cholesteryl esters was in the fatty acid moiety. Incorporation into cholesteryl esters was increased most during the development of atherosclerosis. Fatty acid synthesis was similar from both acetate-1-(14)C and the 2 carbon unit derived from glucose-2-(14)C, viz., predominantly de novo synthesis of fatty acids with 14 and 16 carbon atoms, and elongation for those of 18 carbons and longer.

Acetates↗

PubMed1982

[Lipid biosynthesis in mitochondria from leaves of olive tree (author's transl)].

Phospholipids, neutral lipids and, to a lesser extent, glycolipids are the principal lipid components of mitochondria of young olive tree leaves. These lipids are rich in palmitic and oleic acid content. The in vivo incorporation of 1-14C-acetate into the mitochondrial lipids takes place especially in phosphatidylcholine, phosphatidylethanolamine and phosphatidylglycerol. The saturated, and especially monounsaturated, acids of these lipids constitute the most actively synthesized fatty acids. In vitro, neutral lipids (mono- and diacylglycerol) and phospholipids (lysophospholipids, phosphatidylglycerol and phosphatidylcholine) incorporate a major percentage of radioactivity and in them the saturated and monounsaturated fatty acids are most actively synthesized.

Lipids↗

PubMed1980

Effect of uremia on incorporation of acetate into rat plasma and tissue lipids.

Experimental uremia was induced in rats by means of bilateral nephrectomy or bilateral ureteral ligation. Incorporation of acetate-1-14C into expired 14CO2 and into plasma and tissue lipids was studied immediately after surgery and at 48 hr, when the rats were uremic. In rats studied immediately after surgery, bilateral nephrectomy, but not bilateral ureteral ligation, significantly decreased the conversion of acetate-1-14C into expired 14CO2. In uremic rats at 48 hr, acetate-1-14C metabolism to 14CO2 was not significantly altered in either group. Plasma triglyceride concentrations and 14C-acetate incorporation into triglycerides were increased in the 48-hr uremic groups, but plasma and liver triglyceride specific radioactivities were not significantly altered. Plasma free fatty acid concentrations and incorporation of acetate into free fatty acids were lower in the 48-hr uremic groups than in controls. Plasma cholesterol concentrations and specific radioactivities were increased in these uremic groups, as were liver free cholesterol specific activities. These results suggest that increased triglyceride and cholesterol synthesis from acetate may contribute to the hypertriglyceridemia and hypercholesterolemia observed in uremic rats.

Acetates↗

PubMed1986

In vivo incorporation of [2-3H]-myo-inositol into frog opsin.

The in vivo incorporation of [2-3H]-myo-inositol into frog retinal rod outer segment membranes was examined. About 25% of the recovered radioactivity was found to be protein-associated. Following acid hydrolysis of this material and extraction with hexane, all the radioactivity remained in the aqueous phase, indicating that the label was not in fatty acids. Following ion exchange column chromatography of the hydrolysate, the major radioactive compound comigrated on TLC with an internal standard of [U-14C]-myo-inositol. SDS polyacrylamide gel electrophoresis of unextracted membranes indicated that the majority of the label was associated with opsin. These results indicate that [2-3H]-myo-inositol was incorporated in vivo into opsin, presumably with retention of its chemical identity.

Animals↗

PubMed1972

Metabolism of long-chain polyunsaturated alcohols in myelinating brain.

cis-9-[1-(14)C]Octadecenol, cis,cis-9,12-[1-(14)C]octadecadienol, and cis,cis,cis-9,12,15-[1-(14)C]octadecatrienol were administered intracerebrally to 18-day-old rats. Incorporation of radioactivity into the constituent alkyl, alk-1-enyl, and acyl moieties of the ethanolamine phosphatides of brain was determined after 3, 6, 24, and 48 hr. Incorporation of radioactivity from each precursor proceeded at approximately the same rate leading to mono-, di-, and triunsaturated alkyl and alk-1-enyl glycerols. In addition, the labeled alcohols were found to be oxidized to the corresponding fatty acids which were incorporated into acyl groups; radioactivity derived from di- and triunsaturated alcohols was found mainly in acyl moieties produced through chain elongation and desaturation reactions of di- and triunsaturated fatty acids.

Animals↗

PubMed1986

Spinach Leaves Desaturate Exogenous [C]Palmitate to Hexadecatrienoate : Evidence that de Novo Glycerolipid Synthesis in Chloroplasts Can Utilize Free Fatty Acids Imported from Other Cellular Compartments.

Long-chain (14)C-fatty acids applied to the surface of expanding spinach leaves were incorporated into all major lipid classes. When applied in diethyleneglycol monomethyl ether solution, as done by previous workers, [(14)C]palmitic acid uptake was much lower than that of [(14)C] oleic acid. However, when applied in a thin film of liquid paraffin the rate of [(14)C] palmitic acid metabolism was rapid and virtually complete. Considerable radioactivity from [(14)C]palmitate incorporated into lipids following either application method gradually appeared in polyunsaturated C(16) fatty acids esterified to those molecular species of galactolipids previously thought to be made using only fatty acids synthesized and retained within the chloroplast. Evidence for the incorporation of radioactivity from exogenous [(14)C]oleate into those same molecular species of galactolipids was less compelling. The unexpected availability of fatty acids bound to extrachloroplastidal lipids for incorporation into galactolipids characteristically assembled entirely within the chloroplast emphasizes the need to reassess interrelations between the "prokaryotic" and "eukaryotic" pathways of galactolipid formation.

Journal Article↗