Elongation of acyl-CoAs by microsomes from etiolated leek seedlings

Long chain fatty acid synthesis was studied using etiolated leek seedling microsomes. In the presence of ATP, [2-¹⁴C]malonyl-CoA was incorporated into fatty acids of C₁₆C₂₆. The omission of ATP, even in the presence of acetyl-CoA, led to a complete loss of activity, which was restored by addition of exogeneous acyl-CoAs. Comparison of acyl-CoA (C₁₂C₂₄) elongation showed that stearoyl-CoA, in the presence of [2-¹⁴C]malonyl-CoA, was the more efficient precursor leading to the formation of fatty acids having a chain length of C₂₀C₂₆. [1-¹⁴C]C₁₆CoA and [1-¹⁴C]C₁₈CoA were elongated in the presence of malonyl-CoA, without degradation of the acyl chain. The time-course and the malonyl-CoA concentration curves showed that [1-¹⁴C]C₁₈CoA was a better primer than [1-¹⁴C]C₁₆CoA. Acyl-CoA elongation was also studied over the concentration range 4.5–45 μM [1-¹⁴C]C₁₈CoA. Comparison of the radioactivity incorporated into the fatty acids formed using [2-¹⁴C]malonyl-CoA in the presence of C₁₈CoA, on the one hand, and [1-¹⁴C]C₁₈CoA in the presence of malonyl-CoA, on the other, demonstrated clearly that the acyl chain of the acyl-CoA was elongated by malonyl-CoA.     

Biosynthesis of saturated very long chain fatty acids by purified membrane fractions from leek epidermal cells

Elongation of fatty acids by microsomal fractions obtained from leek epidermal cells was measured by the incorporation of [1-¹⁴C]stearate, [1-¹⁴C]stearyl CoA, and [1-¹⁴C]stearyl ACP in the presence of malonyl CoA and NADPH. Stearoyl CoA appears to be the primer of the elongase (s) rather than stearoyl ACP. There are at least two elongases, the first elongating C₁₈ to C₂₀, the second synthesizing C₂₂ to C₃₀ fatty acids from C₂₀. The main site of the elongase (s) is a subcellular fraction enriched in endoplasmic reticulum. The plasma membrane-enriched fraction, which contains large amounts of saturated very long chain fatty acids, synthesizes only minor amounts of them.     

Glucose metabolism during osmotic stress in isolated axons of Eriocheir sinensis

Isolated axons of $\text{Eriocheir sinensis}$ show high ratios of ¹⁴CO₂ production from glucose-1-¹⁴C to ¹⁴CO₂ production from glucose-6-¹⁴C (ratio C₁/C₆). During osmotic stresses, there is a modification in ¹⁴CO production from glucose-6-¹⁴C as well as in the ratio C₁/C₆ while ¹⁴CO₂ production from glucose-1-¹⁴C does not change significantly. These results are interpreted in terms of activity of oxidative and non-oxidative pathways of glucose metabolism.     

The stereochemistry of biosynthesis of decaprenoxanthin in a cell-free system

Intact cells of Flavobacterium dehydrogenans grown on glucose or acetate did not incorporate mevalonic acid-[¹⁴C]. After treatment with lysozyme the protoplasts were lysed by sonication in a dilute medium containing mevalonic acid-[¹⁴C] and the cell-free system produced incorporated label into uncyclized C₄₀, monocyclic C₄₅ and bicyclic C₅₀ carotenoids of which decaprenoxanthin was the most abundant.With mevalonate-[2-¹⁴C,4R-4-³H₁] the ¹⁴C:³H ratios of the carotenoids showed that the hydrogen atoms at C-2 and C-6 of the ring and that at C-3 of the 1-hydroxy, 2-methyl but-2-ene-4-yl residues of decaprenoxanthin were derived from the 4-pro-R hydrogen atom of mevalonic acid.Mevalonate-[2-¹⁴C,2R-2-³H₁] and mevalonate-[2-¹⁴C,2S-2-³H₁] gave ratios which showed that the C-4 hydrogen atoms of decaprenoxanthin were derived from the 2-pro-S hydrogen atom of mevalonic acid.     

Removal of the acetate-moiety of 2,4-dichlorophenoxyacetic acid in Ribes sativum

Ten minutes after uptake of 2,4-dichlorophenoxyacetic acid-1-¹⁴C(2,4-D-1-¹⁴C) by excised Ribes sativum leaves, 37·8 % of the radioactivity in water-soluble metabolites was in glyoxylic acid. When 2,4-D- 2-¹⁴C was supplied under the same conditions, 23·0 % of the radioactivity of the water-soluble rnetabolites was in glyoxylic acid. Radioactive glycine and glyoxylic acid, isolated from Ribes sativum 6 hr after uptake of 2,4-D-1-¹⁴C, contained essentially all of the ¹⁴C in the carboxyl-carbon atoms. When 2,4-D-2-¹⁴C was the precursor, the glycine isolated contained 64·8 % of its radioactivity in C₂, while 60·0 % of the radioactivity in glyoxylic acid was in C₂. The side-chain label of 2,4-D-2-¹⁴C-4-³⁶Cl was more efficiently incorporated into ethanol-insoluble plant residue than the ring-label. The metabolism of glyoxylic acid-1-¹⁴C and 2,4-D-1-¹⁴C in excised Ribes sativum leaves were compared. The data suggest a cleavage of the acetate-moiety of 2,4-D resulting in a C₂ compound, perhaps glyoxylate.     

Participation of C6C1 unit in the biosynthesis of ephedrine in Ephedra

Feeding of benzoic acid-[7-¹⁴C], benzaldehyde-[7-¹⁴C] and cinnamic acid-[3-¹⁴C] to Ephedra distachya resulted in efficient incorporations of ¹⁴C into the α-carbon atom of the side chain of l-ephedrine. Thus ephedrine was shown to be biosynthesized by the condensation of a C₆C₁ portion which is derived from phenylalanine via cinnamate and an unidentified C₂-N fragment.     

Effect of 4-Pentenoic Acid on Intermediate Metabolism of Tetrahymena*

SYNOPSIS. The growth of Tetrahymena pyriformis strain HSM was strongly inhibited by 4-pentenoic acid. Supplementing the medium with acetate reversed the growth inhibition, but pyruvate was ineffective. Glycogen content was much lower in cells grown with 4-pentenoic acid than in controls; this effect was not reversed by acetate or by pyruvate. There was little effect of 4-pentenoic acid on the incorporation of label from [1-¹⁴C]acetate, [2-¹⁴C]glycerol, [1-³⁴]ribose, [U-¹⁴C]fructose, or [1-¹⁴C]glucose into CO₂, but incorporation of label into glycogen was inhibited, the strongest inhibition being on acetate and the weakest (～ 20%) on ribose, fructose, and glucose. A 3-compartment model for quantitation of labeled acetyl CoA fluxes was shown to be applicable to Tetrahymena grown in the presence of 4-pentenoic acid, and experiments were performed to establish the flux of [1-¹⁴C]acetyl CoA into glycogen, lipids, CO₂, glutamate, and alanine. It was evident from the results of these experiments that 4-pentenoic acid did not appreciably inhibit β-oxidation or lipogenesis, but markedly decreased the glyconeogenic flux of labeled acetyl-CoA from the peroxismal and outer mitochondrial compartments. At least 2 mechanisms have been proposed for the action of 4-pentenoic acid: (a) reduction of the levels of acetyl CoA or free CoA and (b) direct inhibition of enzymes by 4-pentenoyl CoA or its metabolites. Although 4-pentenoic acid has little effect on acetyl-CoA metabolism in the inner mitochondrial compartment, the present data suggest that the flux through the outer mitochondrial compartment of acetyl-CoA derived from pyruvate is inhibited largely by the first, and that the glyconeogenic flux of acetyl-CoA is inhibited largely by the 2nd mechanism.     

Effect of tolbutamide on the metabolism of Tetrahymena

Tolbutamide partially inhibited the growth but increased the glycogen content of Tetrahymena pyriformis in logarithmically growing cultures. Tolbutamide slightly increased ¹⁴CO² production from [1-¹⁴C] and [6-¹⁴HC] glucose and [2-¹⁴C] pyruvate, but had little effect on the oxidation of [1-¹⁴C] acetate when any of these substrates were added to the proteose-peptone medium in which the cells had been grown. Measurement of ¹⁴CO₂ production from [1-¹⁴C] and [2-^I4C]-glyoxylate showed that this substrate was primarily oxidized via the glyoxylate cycle, with little if any oxidation occurring via the peroxisomal glyoxylate oxidase. Addition of tolbutamide inhibited the glyoxylate cycle as indicated by a marked reduction in label appearing in CO₂ and in glycogen from labeled acetate. In control cells, addition of acetate strongly inhibited the oxidation of [2-¹⁴C]-pyruvate whereas addition of pyruvate had little effect on the oxidation of [1-¹⁴C]-acetate. Acetate was more effective than pyruvate in preventing the growth inhibitory and glycogen-increasing effects of tolbutamide. The data suggest that one effect of tolbutamide may be to interfere with the transfer of isocitrate and acetyl CoA across mitochondrial membranes.     

Labelling of lipids by D-[1-14C]glucose, D-[6-14C]glucose and D-[3-3H]glucose in pancreatic islets from normal and GK rats

In pancreatic islets prepared from either normal or GK rats and incubated at either low (2.8 mM) or high (16.7 mM) D-glucose concentration, the labelling of both lipids and their glycerol moiety is higher in the presence of D-[1-¹⁴C]glucose than D-[6-¹⁴C]glucose. The rise in D-glucose concentration augments the labelling of lipids, the paired ¹⁴C/³H ratio found in islets exposed to both D-[1-¹⁴C]glucose or D-[6-¹⁴C]glucose and D-[3-³H]glucose being even slightly higher at 16.7 mM D-glucose than that found, under otherwise identical conditions, at 2.8 mM D-glucose. Such a paired ratio exceeds unity in islets exposed to D-[1-¹⁴C]glucose. The labelling of islet lipids by D-[6-¹⁴C]glucose is about 30 times lower than the generation of acidic metabolites from the same tracer. These findings indicate (i) that the labelling of islet lipids accounts for only a minor fraction of D-glucose catabolism in pancreatic islets, (ii) a greater escape to L-glycerol-3-phosphate of glycerone-3-phosphate generated from the C₁-C₂-C₃ moiety of D-glucose than D-glyceraldehyde-3-phosphate produced from the C₄-C₅-C₆ moiety of the hexose, (iii) that only a limited amount of [3-³H]glycerone 3-phosphate generated from D-[3-³H]glucose is detritiated at the triose phosphate isomerase level before being converted to L-glycerol-3-phosphate, and (iv) that a rise in D-glucose concentration results in an increased labelling of islet lipids, this phenomenon being somewhat more pronounced in the case of D-[1-¹⁴C]glucose or D-[6-¹⁴C]glucose rather than D-[3-³H]glucose.     

Biosynthesis of securinine,the main alkaloid of Securinega suffruticosa

Biosynthesis of securinine was studied by incorporation experiments in Securinega suffruticosa. Among presumed precursors tested, lysine, cadaverine, and tyrosine showed the highest incorporation into securinine. Degradation experiments revealed that cadaverine-[1,5-¹⁴C] labelled specifically the piperidine ring of securinine and the radioactivity from dl-tyrosine-[2-¹⁴C] was introduced into the C-11 lactone carbonyl. Experiments with L-tyrosine-[U-¹⁴C] and L-tyrosine-[3′,5′-³H; U-¹⁴C] prove that the remaining C₆Sz.sbnd;C₂ moiety is derived from the aromatic ring and the C-2 and C-3 or tyrosine.     

Fatty acid esters of testosterone in rat brain: identification, distribution, and some properties of enzymes which synthesize and hydrolyze the esters

[4-¹⁴C]Testosterone was converted to an unknown compound with a much higher R_f on thin layer chromatogram than the substrate when it was incubated with a rat brain microsomal preparation. Evidence from its mass, infrared, and ultraviolet spectra indicated that the enzymic product is a mixture of fatty acid esters of testosterone. Saponification of the product yielded testosterone and a mixture of C_12:0, C_14:0, C_16:0, C_18:0, and C_18:1 fatty acids. The enzymic product was identical to testosterone laurate and testosterone stearate which were synthesized chemically. The enzyme system had a pH optimum at 4.9 with acetate buffer. The apparent K_m was 8.3 × 10^?5m for testosterone and 5.0 × 10^?5m for palmityl CoA. An enzyme which hydrolyzes testosterone[1-¹⁴C]oleate was also detected in rat brain. Most of this activity was in the nuclear and mitochondrial fractions. This enzyme had an optimum pH at 6.5 with phosphate buffer and its apparent K_m was 2.1 × 10^?4m. A low level of synthetic activity was found in fetal brain tissue which increased and reached a maximum at 3 weeks of age. The synthetic activity rapidly decreased with further increase in age. Hydrolytic activity was nearly undetectable in fetal rat brain, increased gradually until the animal reaches 3 weeks old, and remained at this level. Both synthetic and hydrolytic enzyme activities were higher in the brain than in other tissues examined.     

Biochemistry of Suberization: Incorporation of [1-C]Oleic Acid and [1-C]Acetate into the Aliphatic Components of Suberin in Potato Tuber Disks (Solanum tuberosum)

Biosynthesis of the aliphatic components of suberin was studied in suberizing potato (Solanum tuberosum) slices with [1-¹⁴C]oleic acid and [1-¹⁴C]acetate as precursors. In 4-day aged tissue, [1-¹⁴C]oleic acid was incorporated into an insoluble residue, which, upon hydrogenolysis (LiA1H₄), released the label into chloroform-soluble products. Radio thin layer and gas chromatographic analyses of these products showed that ¹⁴C was contained exclusively in octadecenol and octadecene-1, 18-diol. OsO₄ treatment and periodate cleavage of the resulting tetraol showed that the labeled diol was octadec-9-ene-1, 18-diol, the product expected from the two major components of suberin, namely 18-hydroxyoleic acid and the corresponding dicarboxylic acid. Aged potato slices also incorporated [1-¹⁴C]acetate into an insoluble material. Hydrogenolysis followed by radio chromatographic analyses of the products showed that ¹⁴C was contained in alkanols and alkane-α,ω-diols. In the former fraction, a substantial proportion of the label was contained in aliphatic chains longer than C₂₀, which are known to be common constituents of suberin. In the labeled diol fraction, the major component was octadec-9-ene-1,18-diol, with smaller quantities of saturated C₁₆, C₁₈, C₂₀, C₂₂, and C₂₄-α,ω-diols. Soluble lipids derived from [1-¹⁴C]acetate in the aged tissue also contained labeled very long acids from C₂₀ to C₂₈, as well as C₂₂ and C₂₄ alcohols, but no labeled ω-hydroxy acids or dicarboxylic acids were detected. Label was also found in n-alkanes isolated from the soluble lipids, and the distribution of label among them was consistent with the composition of n-alkanes found in the wound periderm of this tissue; C₂₁ and C₂₃ were the major components with lesser amounts of C₁₉ and C₂₅. The amount of ¹⁴C incorporated into these bifunctional monomers in 0-, 2-, 4-, 6-, and 8-day aged tissue were 0, 1.5, 2.5, 0.8, and 0.3% of the applied [1-¹⁴C]oleic acid, respectively. Incorporation of [1-¹⁴C]acetate into the insoluble residue was low up to the 3rd day of aging, rapid during the next 4 days of aging, and subsequently the rate decreased. These changes in the rates of incorporation of exogenous oleic acid and acetate reflected the development of diffusion resistance of the tissue surface to water vapor. As the tissue aged, increasing amounts of the [1-¹⁴C]acetate were incorporated into longer aliphatic chains of the residue and the soluble lipids, but no changes in the distribution of radioactivity among the α-ω-diols were obvious. The above results demonstrated that aging potato slices constitute a convenient system with which to study the biochemistry of suberization.     

Autotrophic CO2 fixation in Chlorobium limicola. Evidence for the operation of a reductive tricarboxylic acid cycle in growing cells

Chlorobium limicola was grown on a mineral salts medium with CO₂ as the main carbon source supplemented with specifically labeled ¹⁴C propionate and the incorporation of ¹⁴C into alanine ( intracellular pyruvate), aspartate ( oxaloacetate), and glutamate ( -ketoglutarate) was studied in long term labeling experiments. During growth in presence of propionate 30% of the cell carbon were derived from propionate and 70% from CO₂. Propionate was not oxidized to CO₂.All three amino acids were found to be labeled. The labeling patterns indicate that propionate was assimilated via propionyl CoA, methylmalonyl CoA and succinyl CoA. When 1-¹⁴C propionate was the labeled precursor no radioactivity was found in the carboxyl group(s) of alanine, aspartate and glutamate, excluding the incorporation of propionate into the amino acids via succinate oxidation to fumarate. With 1-¹⁴C propionate preferentially aspartate (C-3) and glutamate (C-2) became labeled, with 2-¹⁴C propionate alanine (C-3) and glutamate (C-4). These findings indicate that propionate was incorporated into the amino acids via succinyl CoA, -ketoglutarate, isocitrate, and citrate, followed by a si-type cleavage of citrate to oxaloacetate and acetyl CoA (or acetate). Similar experiments with U-¹⁴C acetate confirm these conclusions. Thus, all reactions of the proposed reductive tricarboxylic acid cycle could be demonstrated in autotrophically growing cells.     

Biosynthesis of methyl branched hydrocarbons of the German cockroach Blattella germanica (L.) (orthoptera,blattellidae)

The precursors and directionality of synthesis of the methyl branched cuticular hydrocarbons and the female contact sex pheromone, 3,11-dimethyl-2-nonacosanone, of the German cockroach, Blattella germanica, were investigated by radiotracer and carbon-13 NMR techniques. The amino acids [G-³H]valine, [4,5-³H]isoleucine and [3,4-¹⁴C₂]methionine labeled the hydrocarbon fraction in a manner indicating that the carbon skeletons of all three amino acids serve as the methyl branch group donor. The incorporation of [1,4-¹⁴C₂]- and [2,3-¹⁴C₂]succinates into the hydrocarbon and acylglycerol/polar lipid fractions indicated that succinate also served as a precursor to methylmalonyl-CoA. Carbon-13 NMR analyses showed that [1-¹³C]propionate labeled the carbon adjacent to the tertiary carbon, and, for the 3,x-dimethylalkanes, that carbon-4 and not carbon-2 was enriched. [1-¹³C]Acetate labeled carbon-2 of these hydrocarbons. This indicates that the methyl branching groups of the 3,x-dimethylalkanes were inserted early in the chain elongation process. [3,4,5-¹³C₃]Valine labeled the methyl, tertiary and carbon adjacent to the tertiary carbon of the methyl branched alkanes. Thus, the methyl branched hydrocarbon was formed by the insertion of methylmalonyl units derived from propionate, isoleucine, valine, methionine and succinate early in chain elongation.     

In vitro synthesis of mycolic acids by the fluffy layer fraction of Bacterionema matruchotii

Biosynthetic activity for mycolic acid occurred in the fluffy layer fraction but not in the 5000g supernatant of Bacterionema matruchotii. With [1-¹⁴C]palmitic acid as precursor for the in vitro system, the predominant product was identified as C_32:0 mycolic acid by radio-gas-liquid chromatographie (radio-GLC) and gas chromatographic/mass spectroscopic analyses; if [1-¹⁴C]stearic acid was used, two major radioactive peaks appeared on GLC: one corresponding to the peak of (C_34:0 + C_34:1) mycolic acids and the other to (C_36:0 + C_36:1) mycolic acids. By pyrolysis/radio-GLC analysis, C_32:0 mycolic acid synthesized by [1-¹⁴C]palmitic acid was pyrolyzed at 300 °C to form palmitaldehyde (the mero moiety) and methyl palmitate (the branch moiety). The pH optimum for the incorporation of [1-¹⁴C]palmitate into bacterionema mycolic acids was 6.4 and the reaction required a divalent cation. The in vitro system utilized myristic, palmitic, stearic and oleic acids (probably via their activated forms) well as precursors, among which myristic and palmitic acids were more effective than the rest. Avidin showed no effect on the biosynthesis of mycolic acid from ¹⁴C-palmitate whereas cerulenin, a specific inhibitor of β-ketoacyl synthetase in de novo fatty acid synthesis, inhibited the reaction at a relatively higher concentration. Thin-layer chromatographic analysis of lipids extracted from the reacting mixture without alkaline hydrolysis showed that both exogenous [1-¹⁴] fatty acid and synthesized mycolic acids were bound to an unknown compound by an alkali-labile linkage and this association seemed to occur prior to the condensation of two molecules of fatty acid.     

The Metabolism of Carbon-14 Specifically Labelled Glucose in Leaves Showing Tobacco Mosaic Virus Induced Local Necrotic Lesions

Glucose metabolism of healthy and tobacco mosaic virus-infected leaf-discs of Nicotiana tabocum L. var. Xanthi showing local-necrotic lesions was investigated using glucose-¹⁴C. Local lesion formation following inoculation with tobacco mosaic virus resulted in enhanced glucose metabolism reflected by an increased rate of release of ¹⁴CO₂ from glucose-U-¹⁴C and greater incorporation of ¹⁴C into all cell fractions. When specifically labelled glucose was fed to healthy and tobacco mosaic virus infected leaves, the C₆/C₁ ratio (rate of release of ¹⁴CO₂ from glucose-6-¹⁴C/rate of release of ¹⁴CO₂ from glucose-l-¹⁴C) was similar for healthy and virus-infected leaves. The C₆/C₁ ratios recorded from 0.30 to 0.50 indicate that both the glycolytic and pentose phosphate pathways participate in glucose catobolism in healthy and virus-infected leaves. Although the C₆/C₁ ratio was the same as that of the healthy leaf the rate of release of ¹⁴CO₂ from glucose-6-¹⁴C and glucose-1-¹⁴C was greatly increased in the virus-infected leaf. The increased glucose catabolism occurs by both glycolytic and pentose phosphate pathways in the virus-infected leaf.     

Anaerobic C1 Metabolism of the O-Methyl-14C-Labeled Substituent of Vanillate

The O-methyl substituents of aromatic compounds constitute a C₁ growth substrate for a number of taxonomically diverse anaerobic acetogens. In this study, strain TH-001, an O-demethylating obligate anaerobe, was chosen to represent this physiological group, and the carbon flow when cells were grown on O-methyl substituents as a C₁ substrate was determined by ¹⁴C radiotracer techniques. O-[methyl-¹⁴C]vanillate (4-hydroxy-3-methoxy-benzoate) was used as the labeled C₁ substrate. The data showed that for every O-methyl carbon converted to [¹⁴C]acetate, two were oxidized to ¹⁴CO₂. Quantitation of the carbon recovered in the two products, acetate and CO₂, indicated that acetate was formed in part by the fixation of unlabeled CO₂. The specific activity of ¹⁴C in acetate was 70% of that in the O-methyl substrate, suggesting that only one carbon of acetate was derived from the O-methyl group. Thus, it is postulated that the carboxyl carbon of the product acetate is derived from CO₂ and the methyl carbon is derived from the O-methyl substituent of vanillate. The metabolism of O-[methyl-¹⁴C]vanillate by strain TH-001 can be described as follows: 3¹⁴CH₃OC₇H₅O₃ + CO₂ + 4H₂O → ¹⁴CH₃COOH + 2¹⁴CO₂ + 10H⁺ + 10e^- + 3HOC₇H₅O₃.     

Phosphoglucoisomerase-catalyzed interconversion of hexose phosphates: isotopic discrimination between hydrogen and deuterium

Summary The discrimination between the isotopes of hydrogen in the reaction catalyzed by yeast phosphoglucoisomerase is examined by NMR, as well as by spectrofluorometric or radioisotopic methods. The monodirectional conversion of D-glucose 6-phosphate to D-fructose 6-phosphate displays a lower maximal velocity with D-[2-²H]glucose 6-phosphate than unlabelled D-glucose 6-phosphate, with little difference in the affinity of the enzyme for these two substrates. About 72% of the deuterium located on the C₂ of D-[1-¹³C,2-²H]glucose 6-phosphate is transferred intramolecularly to the C₁ of D-[1-¹³C,1-²H]fructose 6-phosphate. The velocity of the monodirectional conversion of D-[U-¹⁴C]glucose 6-phosphate (or D-[2-³H]glucose 6-phosphate) to D-fructose 6-phosphate is virtually identical in H₂O and D₂O, respectively, but is four times lower with the tritiated than ¹⁴C-labelled ester. In the monodirectional reaction, the intramolecular transfer from the C₂ of D-[2-³H]glucose 6-phosphate is higher in the presence of D₂O than H₂O. Whereas prolonged exposure of D-[1-¹³C]glucose 6-phosphate to D₂O, in the presence of phosphoglucoisomerase, leads to the formation of both D-[1-¹³C,2-²H]glucose 6-phosphate and D-[1-¹³C,1-²H]fructose 6-phosphate, no sizeable incorporation of deuterium from D₂O on the C₁ of D-[1-¹³C]fructose 1,6-bisphosphate is observed when the monodirectional conversion of D-[1-¹³C]glucose 6-phosphate occurs in the concomitant presence of phosphoglucoisomerase and phosphofructokinase. The latter finding contrasts with the incorporation of hydrogen from ¹H₂O or tritium from ³H₂O in the monodirectional conversion of D-[2-³H]glucose 6-phosphate and unlabelled D-glucose 6-phosphate, respectively, to their corresponding ketohexose esters.     

Studies on the uptake of fatty acids by Escherichia coli

Oleate uptake by Escherichia coli showed saturation kinetics with a K_m of 34 μm and an activation energy of 6.25 kcal/mole indicating that the rate limiting step in oleate uptake involves an enzyme-catalyzed step. The rate of oleate uptake was decreased by the respiratory poisons, arsenate and 4-pentenoate, which apparently is activated to pentenoyl CoA, thus reducing the intracellular concentration of free intracellular CoA. These data indicated that oleate uptake is dependent on cellular ATP and CoA. During short pulses with [1-¹⁴C]oleate, most of the radioactivity which was taken up was released as ¹⁴C0₂; cells accumulated radioactivity in phospholipids and compounds with the chromatographic mobility of Krebs cycle intermediates. Neither free fatty acid nor oleyl CoA were detectable in the cells. The results support the hypothesis that long-chain fatty acids are translocated by the long-chain fatty acyl CoA synthetase and that uptake is the rate limiting step in the utilization of exogenous fatty acid.     

Acyl CoA:Cholesterol acyl transferase activity in human placental microsomes: Inhibition by progesterone

The characteristics of acyl CoA:cholesterol acyltransferase (ACAT; EC 2.3.1.26) in microsomes prepared from human term placenta were studied and the rate of incorporation of [1-¹⁴C] oleoyl CoA into cholesteryl esters was measured. The apparent K_m of the enzyme for [1-¹⁴C] oleoyl CoA was 38 ± 9 μm and the V for the reaction was 15 ± 6 pmol × mg^? protein × min^?1. The Hill coefficient for the reaction was 1.2, indicative of some degree of positive cooperativity. Cholesterol, added to the incubation mixture, did not influence ACAT activity, indicating that endogenous microsomal cholesterol served as an effective substrate for the placental ACAT enzyme. However, [1,2-³H]cholesterol in the presence of oleoyl CoA was incorporated into cholesteryl esters by placental microsomes. When progesterone was present in the incubation mixture at a concentration of 20 μm, ACAT activity was inhibited 50%. Pregnenolone, 5α-dihydroprogesterone, 17α-hydroxyprogesterone, deoxycorticosterone, dehydroisoandrosterone, androstenedione, testosterone, and estradiol-17β also inhibited ACAT activity, whereas corticosterone, cortisol, and estriol had little effect. These results are supportive of the view that ACAT activity in human placenta may be regulated by endogenously synthesized steroid hormones.