Stereoselectivity in the 2-methylbutyrate incorporation into anteiso fatty acids in Bacillus subtilis mutants.

Two Bacillus subtilis mutants defective in branched-chain alpha-ketoacid dehydrogenase can grow when 2-methylbutyrate is provided in trypticase soy medium. Both enantiomers of the acid supported growth of the mutants but the (S)-(+)-isomer (natural) was more active than the (R)-(-)-isomer (unnatural). The mutants utilized these isomers as primer to specifically synthesize either enantiomer of anteiso fatty acids. No racemization of the isomer primers was observed during the synthesis. Thus, cells grown with (-)-isomer possessed anteiso fatty acids (over 80%) of the total fatty acids, being entirely the unnatural enantiomer. The stereospecific synthesis was found to be controlled at the step of 2-methylbutyryl-CoA synthesis. In a wild strain, only (+)-specific acyl-CoA synthetase was detected. In the mutants, either enantiomer of 2-methylbutyrate could simultaneously induce both types, (+)-specific and (-)-specific, of acyl-CoA synthetase. (+)-Specific synthetase had a higher activity and affinity towards substrate than (-)-specific synthetase. The detailed preparative procedures for (R)-(-)- and 2-[3,4-3H]methylbutyric acid are described.

Studies on the biosynthesis of beta-amino acids, the lipid moiety of iturins A, in Bacillus subtilis.

The biosynthesis of the beta-amino acid components of iturins A was studied in comparison to the biosynthesis of fatty acids. Palmitic acid was incorporated into the lipid moiety of iturins A when it was added to the culture medium of the iturin producer Bacillus subtilis. Addition of unlabeled palmitic acid enhanced the formation of straight-chain beta-amino acids and addition of valine or leucine increased the production of branched beta-amino acids. These modifications correlated with modifications in the corresponding biosynthesized fatty acids.

Inhibitory effect of FO-1561 (S-adenosyl-L-methionine sulfate tosylate) on phospholipase A2.

The mechanism of the protective effect of FO-1561 (S-adenosyl-L-methionine sulfate tosylate) on mitochondria was investigated using rat heart muscle. FO-1561 stimulated CO2 production from palmitic acid in the mitochondria, indicating an increase in energy production. FO-1561 inhibited mitochondrial swelling induced by Ca2+ added exogenously or by addition of snake venom phospholipase A2. Moreover, FO-1561 inhibited phospholipase A2 activity directly. These results suggested that FO-1561 blocked mitochondrial swelling by inhibiting endogenous phospholipase A2 activity and so preventing decrease in palmitic acid oxidation.

Effect of insulin on the oxidative desaturation of fatty acids in non-diabetic rats and in isolated liver cells

Efecto de la insulina sobre la desaturación oxidativa de los ácidos grasos en ratas no diabéticas y en células aisladas de hígado. Se estudió el efecto de la insulina en la desaturación oxidativa del ácido palmítico 1- 14C a ácido palmitoleico y linoleico 1- 14C a ácido gama -linolénico, en microsomas de hígado de rata, y del ácido eicosa-8,11,14-trienoico 1- 14C a ácido araquidónico en microsomas de hígado de rata y células HTC. No se observaron cambios en la actividad de la DELTA9 desaturasa y disminuyó la actividad de la DELTA5 desaturasa después de 12 h de la inyección de insulina en dosis de 5 U/kg peso corporal. La conversión de ácido linoleico a gama -linolénico disminuyó cuando la cantidad de insulina inyectada fue de 5 U/kg peso o más. La conversión de ácido palmítico a palmitoleico no mostró cambios importantes entre 1 y 12 h después de la inyección de 5 U/kg peso corporal de insulina. Durante todo ese lapso y en las mismas condiciones experimentales disminuyó la actividad de la DELTA5 desaturasa. La conversión de ácido linoleico a gama -linolénico se incrementó levemente después de 1 h de tratamiento insulínico y luego disminuyó significativamente hasta finalizar el experimento. El agregado de 400 mU/ml o más de insulina al medio incubación de células HTC produjo un descenso significativo en la conversión de ácido eicosatrienoico en araquidónico. El efecto de la insulina sobre la desaturación oxidativa de los ácidos grasos en microsomas de hígado de ratas normales difiere que se observa en ratas diabéticas. Se discute el papel de la insulina en relación con otras hormonas, el metabolismo glucídico y la biosíntesis de lípidos

[Production of extracellular higher fatty acids by yeasts grown on hexadecanoic acid]. / Obrazovanie vnekletochnykh vysshikh zhirnykh kislot drozhzhami pri roste na geksadekane.

Production of exocellular higher fatty acids by Candida yeasts was studied during their growth in a mineral medium with hexadecane. The qualitative and quantitative composition of exocellular higher fatty acids was investigated during cultivation of Candida lipolytica VCM Y 2378(695) under the conditions of different aeration (5 and 80% saturation of the medium with oxygen). Palmitic (C16:0), palmitooleic (C16:1), oleic (C18:1) and linoleic (C18:2) acids predominated among other higher fatty acids. The overall amount of fatty acids increased and the content of unsaturated fatty acids decreased when the yeast growth was limited with oxygen.

Hybridization studies of chicken liver fatty acid synthetase. Evidence for the participation in palmitate synthesis of cysteine and phosphopantetheine sulfhydryl groups on adjacent subunits.

Enzymatically inactive variants of chicken liver fatty acid synthetase have been prepared by specific chemical modification of the active cysteine SH group with iodoacetamide, and the phosphopantetheine SH group with chloroacetyl-CoA. Hybridization of each of these variants with the unmodified enzyme yielded (modified)-(unmodified) hybrid dimers which possessed 50% synthetase activity. A 50% active (iodoacetamide-modified)-(chloroacetyl-CoA-modified) hybrid dimer was also demonstrated by recombination of these variants with each other. These results indicate that the two functional sites on the synthetase are independently active, and that each is comprised of a cysteine SH group from one subunit and a complementary phosphopantetheine SH group from the other subunit as depicted by the head-to-tail arrangement proposed by Wakil and co-workers (Wakil, S. J., Stoops, J. K., and Joshi, V.C.

Stereochemistry of the reactions catalyzed by chicken liver fatty acid synthase.

The stereochemistry of the four partial reactions catalyzed by chicken liver fatty acid synthase that lead to the synthesis of palmitic acid has been determined. The reduction of acetoacetyl-CoA to 3-hydroxybutyryl-CoA by NADPH proceeds with the transfer of the pro-4S hydrogen of NADPH to form D-3-hydroxybutyryl-CoA. During the subsequent dehydration of D-3-hydroxybutyryl-CoA the pro-2S hydrogen and the 3-hydroxyl group are removed in a syn elimination to form crotonyl-CoA. Crotonyl-CoA is reduced to butyryl-CoA by NADPH, with the transfer of the pro-4R hydrogen of NADPH to the pro-3R position in butyryl-CoA and the transfer of a solvent hydrogen to the pro-2S position. The occurrence of the syn dehydration, when combined with the results of a previous study [ Sedgwick , B., & Cornforth , J. W. (1977) Eur. J. Biochem. 75, 465-479], implies that the condensation of the enzyme-bound malonyl moiety with the enzyme-bound saturated fatty acid to form a 3-keto intermediate proceeds with inversion at C-2 of the malonyl. The stereochemistry of the hydration was derived from an analysis of the spin-spin coupling constant of 3-hydroxy[2-2H]butyric acid benzylamides obtained from 3-hydroxy[2-2H]butyryl-CoA synthesized by fatty acid synthase. The elucidation of the stereochemistry of the reduction of crotonyl-CoA relied on the previously established stereochemistry of pork liver acyl-CoA dehydrogenase. The source of all 28 prochiral hydrogens of the palmitic acid synthesized by chicken liver fatty acid synthase was inferred from the results of this work.

[Essential fatty acids: its transformations and functions]. / Los ácidos grasos esenciales: sus transformaciones y funciones.

Essential fatty acids, in animals, pertain to two different fatty acid families: the linoleic and the linolenic. These, and the non-essential families of oleic and palmitoleic are produced by action of the enzymes proper. The lack of essential fatty acids produces typical symptoms that are accompanied by fatty acid compositions, also typical, utilized with diagnostic value. The biological effects of essential fatty acids can be specific and nonspecific. The latter manifest themselves particularly in the phospholipid composition and, therefore, in the structure and fluency of the membranes. In contrast, specific essential fatty acids act in the formation of prostaglandins, prostacyclins, tromboxans and leucotriens. Each essential fatty acid produces specific effects, depending on the prostanoids formed and the tissue in question.

Incorporation of 14C acetate into the phospholipids and fatty acids of rabbit lens in organ culture.

Lenses were cultured in Medium 199 containing 14C acetate for 3, 6, 12 and 20 hours. There was a linear increase in incorporation into the total lipids with time. The total lipid extract was subjected to two-dimensional chromatography and incorporation into the individual classes of lipids was measured. For all time periods approximately 70% of all incorporated radioactivity was seen in the sterol and phosphatidyl choline (PC) fractions. Incorporation was observed in phosphatidyl ethanolamine (PE), aldehydes of PC and PE plasmalogens, phosphatidyl inositol (PI), PC plasmalogens, PE plasmalogens, sphingomyelin (SM), phosphatidic acid (PA) and phosphatidyl serine (PS). The fatty acids of the total lipids were methylated and subjected to gas chromatography, and individual fatty acids were collected. The greatest incorporation occurred in palmitic acid (16:0) and myristic acid (14:0) with minor amounts in lauric acid (12:0). These are the major products of de novo synthesis. The remainder of the radioactivity appeared in fatty acids 18 carbons or longer, which are the products of an elongation mechanism.

Purification and properties of fatty acid synthetase from a human breast cell line.

A human mammary epithelial cell line (SKBr3) has been identified in which fatty acid synthetase constitutes up to 28%, by weight of the cytosolic proteins. The enzymes has been purified to near homogeneity from this cell line and some of its properties studied. In common with fatty acid synthetases from other animal tissues, the enzyme is a 480 000 dalton dimer of similar molecular weight subunits, it synthesizes predominantly palmitic acid and is inactive in the absence of free coenzyme A. The kinetic properties and amino acid composition of the enzyme are also similar to those of fatty acid synthetases from various tissues of other animals. Appreciable structural resemblance between human and rodent fatty acid synthetases is indicated by studies on the immunological cross-reactivities of these enzymes.

The effects of cerulenin on lipid metabolism in vitro in cellular preparations from the rat.

The effects of the fatty acid-like antibiotic, cerulenin, on fatty acid biosynthesis in preparations of rat adipocytes and mammary cells in vitro have been investigated. Synthesis of palmitic acid was most strongly inhibited, although the magnitude of the effect was dependent on the nature of the tissue, and was especially diminished in the larger adipocytes from older rats. Cerulenin had no effect on the chain-elongation of preformed fatty acids in any of the tissues studied. Some inhibition of the esterification of preformed palmitic acid was also observed, but this appeared to be due to disruption of the cells rather than direct inhibition of the acyltransferases. It is concluded that cerulenin is a valuable experimental tool in studies of lipogenesis in preparations of intact mammalian cells in vitro.

Mechanism of chain length determination in biosynthesis of milk fatty acids.

Fatty acid synthetases isolated from all mammalian tissues synthesize predominately palmitic acid. However, in vivo the mammary gland fatty acid synthetases of some species are responsible for the synthesis of medium chain fatty acids. The objective of this presentation is to outline the mechanism which regulates the product specificity of fatty acid synthetases in general and to illustrate how this control is modified in the mammary gland. Fatty acid synthetases isolated from mammalian tissues are composed of two similar, probably identical, polypeptides, each carrying as many as seven enzyme components. Thioesterase I, the component which functions to terminate growth of acyl chains on the multienzyme, is located at one terminus of each polyfunctional polypeptide and can be detached by limited proteolysis with trypsin. By studying separately the kinetics of chain elongation by the core of the trypsinized complex and of chain termination by the isolated thioesterase I component, it has been possible to establish that the specificities of the elongation and termination reactions account for the synthesis of predominantly the carbon-16 fatty acid by purified fatty acid synthetases. Mammary glands of some species contain an additonal enzyme, thioesterase II, which can modify the product specificity of fatty acid synthetase by hydrolyzing medium chain acyl moieties from thioester linkage to the 4'phosphopantetheine of the multienzyme. At all stages of development of rat mammary gland, the amount of theoesterase II present correlates well with the proportion of medium chain fatty acids synthesized by the gland. This mammary gland-specific thioesterase appears responsible for the ability of this tissue to synthesize medium chain fatty acids characteristic of milk fat.

Some characteristics of soluble fatty acid synthesis in germinating pea seeds.

Soluble fractions from germinating pea synthesize palmitic acid de novo and stearic acid by elongation. Malonyl CoA, acyl carrier protein and NADPH are required for both reactions. In contrast to some other plant systems, no requirement was found for divalent cations. On the other hand, the formation of both stearate and palmitate was inhibited by sulphydryl reagents and palmitate elongation was sensitive to arsenite. The products of the reactions were examined and found to be principally acyl-acyl carrier proteins and unesterified fatty acids. Unlike the pea microsomal fractions, the soluble enzymes are stimulated only slightly by the addition of exogenous lipids. The substrate for palmitate elongation is palmitoylacyl carrier protein, which is quantitatively elongated to stearate. Comparisons are made with membrane-localised fatty acid synthesis from the same tissue.

Effect of CO2 concentration on phosphatidylcholine and phosphatidylglycerol metabolism in surfactant and residual lung fractions.

An investigation of the effect of change of total CO(2) concentration from 7 to 43 mM at pH 7.35 in the medium perfusing isolated rat lungs on [U-(14)C]glucose incorporation into lung phospholipids has been carried out. The incorporation of [U-(14)C]glucose into phosphatidylcholine and phosphatidylglycerol of the surfactant fraction and of the remaining lung tissue (residual fraction) was observed. Increased CO(2) concentration increased [U-(14)C]glucose incorporation into phosphatidylcholine of the surfactant fraction and residual fraction by 43 and 50%, respectively, during a 2 hr perfusion. Likewise, incorporation of [U-(14)C]glucose into phosphatidylglycerol was increased 22 and 34% into the surfactant and residual fractions, respectively. The percentage of [U-(14)C]glucose incorporated into the fatty acid moieties of phosphatidylcholine of both fractions increased as a result of increased CO(2) concentration. The increase in the incorporation of [U-(14)C]glucose into the fatty acid moieties of phosphatidylcholine was confirmed by an average increase of 56 and 77% in the specific activity of palmitic acid isolated from phosphatidylcholine of the surfactant and residual fraction, respectively, as a result of increased CO(2) concentration. The results suggest that alteration in extracellular CO(2) concentration affects the de novo synthesis from glucose of phosphatidylcholine and phosphatidylglycerol of the surfactant-lipoprotein fraction of lung.

Fatty acid synthesis in testes of fat-deficient and fat-supplemented rats.

Fatty acid synthesis was studied in testes of rats fed a fat-free or fat-supplemented diet. Testes of fat-deficient rats incorporated nearly twice as much intratesticularly injected [1-14C]acetate into total fatty acids (primarily into palmitic acid) as did supplemented rats. To determine the mechanism for the increased synthesis, the activities of the following enzymes were determined in the cytoplasmic fraction of testicular homogenates: fatty acid synthetase, acetyl CoA carboxylase [EC 6.4.1.2], citrate-cleavage [EC 4.1.3.8], malic [EC 1.1.1.38], and the glucose-l-phosphate dehydrogenase [EC 1.1.1.49]: 6-phosphogluconate dehydrogenase pair [EC 1.1.1.44]. Although the activity of fatty acid synthetase did increase in livers from fat-deficient rats, no change was observed in corresponding testes. No difference between the two groups could be demonstrated in testicular activity of citrate-cleavage enzyme, malic enzyme, or the glucose-6-phosphate dehydrogenase: 6-phosphogluconate dehydrogenase pair. However, the activity of cytoplasmic acetyl CoA carboxylase in testes of rats fed the fat-deficient diet was 1.4 times higher than the activity in testes of rats fed the supplemented diet. Fat deficiency did not affect the specific activity of the testicular microsomal elongation system, assayed by incubation with 14C-malonyl CoA. The concentration of unesterified fatty acids was lower in testes of the fat-deficient compared to supplemented rats, indicating that decreased inhibition of acetyl CoA carboxylase in the fat-deficient rats testes might have been responsible for the observed increased de novo synthesis of palmitic acid.