Utilization of different fatty acyl-CoA thioesters by serine palmitoyltransferase from rat brain

Serine palmitoyltransferase (EC 2.3.1.50) catalyzes the first unique reaction of sphingolipid biosynthesis. Activities were determined with different fatty acyl-CoA substrates to describe the range of long-chain bases that could be made by rat brain microsomes. The activities were greatest with palmitoyl-CoA and palmitelaidoyl-CoA, followed by fully saturated homologs differing from these by only one carbon atom, and diminished considerably as the alkyl-chain length increased or decreased, or with the presence of a cis-double bond. These characteristics explain the predominance of long-chain bases with 18 carbon atoms in brain sphingolipids, and account for the minor variants such as the C17- and C20-long chain bases.     

Effects of liver failure on the enzymes in the branched-chain amino acid catabolic pathway

Branched-chain alpha-keto acid dehydrogenase (BCKDH) complex catalyzes the committed step of the catabolism of branched-chain amino acids (BCAA). The liver cirrhosis chemically induced in rats raised the activity of hepatic BCKDH complex and decreased plasma BCAA and branched-chain alpha-keto acid concentrations, suggesting that the BCAA requirement is increased in liver cirrhosis. Since the effects of liver cirrhosis on the BCKDH complex in human liver are different from those in rat liver, further studies are needed to clarify the differences between rats and humans. In the valine catabolic pathway, crotonase and beta-hydroxyisobutyryl-CoA hydrolase are very important to regulate the toxic concentration of mitochondrial methacrylyl-CoA, which occurs in the middle part of valine pathway and highly reacts with free thiol compounds. Both enzyme activities in human and rat livers are very high compared to that of BCKDH complex. It has been found that both enzyme activities in human livers were significantly reduced by liver cirrhosis and hepatocellular carcinoma, suggesting a decrease in the capability to dispose methacrylyl-CoA. The findings described here suggest that alterations in hepatic enzyme activities in the BCAA catabolism are associated with liver failure.     

Determination and characterization of long-chain fatty acyl-CoA thioesters from yeast and mammalian liver

The long-chain fatty acyl-CoA content of various biological materials, i.e., baker's yeast and mammalian liver, has been determined under standard and several other metabolic conditions, using optimized methods for cell disruption, separating acid-soluble and acid-insoluble CoA from each other, and assaying. After studying the optimization of the extraction of long-chain acyl-CoA compounds and the purification of the extracts, acyl-CoA fractions from several biological sources have been isolated and characterized on behalf of their fatty acid residues by gas-liquid chromatography of the methyl ester derivatives.     

The adverse metabolic effects of branched-chain amino acids are mediated by isoleucine and valine

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Odd- and branched-chain fatty acids in milk fat from Holstein dairy cows are influenced by physiological factors

Dairy products are the major source of odd- and branched-chain fatty acids (OBCFAs), a group of nutrients with emerging health benefits. The animal diet is known to influence milk fat OBCFAs of dairy cows; however, little is known about the effects of physiological factors. The objective of this study was to investigate the effects of parity and lactation stage on OBCFAs in milk fat of dairy cows. Holstein dairy cows (n = 157) were selected according to parity (first, second, third, or greater) and days in milk (DIM) (≤21 DIM, 21 < DIM ≤ 100, 100 < DIM ≤ 200, >200 DIM). All cows were fed the same total mixed ration for three weeks. Milk samples were collected during the last three days of each lactation stage for fatty acid (FA) analyses via gas chromatography. Results showed that first- and second-parity cows displayed significantly higher proportions and yields of iso-14:0, iso-15:0, iso-16:0, total iso-FA, and total branched-chain FA (P < 0.05) compared with other parities. The proportions of C17:0 and C17:1 cis-9 were also greater in first-parity cows (P < 0.05), while the yields of C17:0 and C17:1 cis-9 were similar among different parities (P > 0.05). The proportions of total OBCFAs were greater in first- and second-parity cows (P < 0.05), whereas the highest yield was observed in second-parity cows. Lactation dairy cows in ≤ 21 DIM group displayed lower proportions of iso-13:0, anteiso-13:0, C13:0, iso-14:0, C15:0, iso-16:0, total iso-FA, and total OBCFAs compared with that of the other groups (P < 0.05), and also lower yields of iso-14:0 and iso-16:0 (P < 0.05). In contrast, C17:0 and C17:1 cis-9 proportions and yields were higher in dairy cows with ≤ 21 DIM (P < 0.05). Iso-17:0 and anteiso-17:0 were not affected by lactation stage (P > 0.05). Taken together, our data showed that both parity and lactation stage have considerable effects on milk fat OBCFAs of dairy cows. In summary, first- and second-parity cows had higher milk OBCFAs compared with later parity cows, and OBCFAs with medium chain lengths were lower in dairy cows with ≤ 21 DIM, while C17:0 and C17:1 cis-9 were higher. These findings show that milk OBCFA contents are differentially modulated by physiological state. They will be useful in future studies that seek to alter OBCFA composition of Holstein dairy cow milk fats.     

Hydroxycinnamate (hca) catabolic genes from Acinetobacter sp. strain ADP1 are repressed by HcaR and are induced by hydroxycinnamoyl-coenzyme A thioesters

Hydroxycinnamates are plant products catabolized through the diphenol protocatechuate in the naturally transformable bacterium Acinetobacter sp. strain ADP1. Genes for protocatechuate catabolism are central to the dca-pca-qui-pob-hca chromosomal island, for which gene designations corresponding to catabolic function are dca (dicarboxylic acid), pca (protocatechuate), qui (quinate), pob (p-hydroxybenzoate), and hca (hydroxycinnamate). Acinetobacter hcaC had been cloned and shown to encode a hydroxycinnamate:coenzyme A (CoA) SH ligase that acts upon caffeate, p-coumarate, and ferulate, but genes for conversion of hydroxycinnamoyl-CoA to protocatechuate had not been characterized. In this investigation, DNA from pobS to an XbaI site 5.3 kb beyond hcaC was captured in the plasmid pZR8200 by a strategy that involved in vivo integration of a cloning vector near the hca region of the chromosome. pZR8200 enabled Escherichia coli to convert p-coumarate to protocatechuate in vivo. Sequence analysis of the newly cloned DNA identified five open reading frames designated hcaA, hcaB, hcaK, hcaR, and ORF1. An Acinetobacter strain with a knockout of HcaA, a homolog of hydroxycinnamoyl-CoA hydratase/lyases, was unable to grow at the expense of hydroxycinnamates, whereas a strain mutated in HcaB, homologous to aldehyde dehydrogenases, grew poorly with ferulate and caffeate but well with p-coumarate. A chromosomal fusion of lacZ to the hcaE gene was used to monitor expression of the hcaABCDE promoter. LacZ was induced over 100-fold by growth in the presence of caffeate, p-coumarate, or ferulate. The protein deduced to be encoded by hcaR shares 28% identity with the aligned E. coli repressor, MarR. A knockout of hcaR produced a constitutive phenotype, as assessed in the hcaE::lacZ-Km(r) genetic background, revealing HcaR to be a repressor as well. Expression of hcaE::lacZ in strains with knockouts in hcaA, hcaB, or hcaC revealed unambiguously that hydroxycinnamoyl-CoA thioesters relieve repression of the hcaABCDE genes by HcaR.     

Administration of branched-chain amino acids during sustained exercise--effects on performance and on plasma concentration of some amino acids.

Previous studies have shown that sustained exercise in human subjects causes an increase in the plasma concentration ratio of free tryptophan: other large neutral amino acids [including the branched-chain amino acids (BCAA)]. This should favour the transport of tryptophan into the brain and also the synthesis of 5-hydroxytryptamine, which is thought to contribute to fatigue during prolonged exercise. A mixture of the three BCAA was given to subjects during a 30-km cross-country race or a marathon (42.2 km) and the effects on mental and physical performances were measured. The mental performance, measured as the performance in the Stroop Colour and Word Test (CWT), was improved after, as compared to before the 30-km cross-country race when a BCAA supplement was given during the race, whereas the CWT scores were similar before and after in the placebo group. The running performance in the marathon was improved for the "slower" runners (3.05 h-3.30 h) when BCAA was taken during the race; however, there was no significant effect on the performance in the "faster" runners (less than 3.05 h). The results showed that both mental and physical performance was improved by an intake of BCAA during exercise. In addition, the effects of exercise on the plasma concentration of the aromatic amino acids were altered when a BCAA supplement was given during the marathon.     

Solubilization and reconstitution of sodium-dependent transport system for branched-chain amino acids from Pseudomonas aeruginosa

The sodium-dependent transport system for branched-chain amino acids of Pseudomonas aeruginosa was solubilized with n-octyl-beta-D-glucopyranoside and reconstituted into liposomes by a detergent-Sephadex G-50 gel filtration procedure. The reconstituted proteoliposomes exhibited Na+-dependent counterflow and Na+-gradient-driven transport of L-leucine, L-isoleucine, and L-valine. The leucine counterflow was specifically inhibited by only branched-chain amino acids and the uphill transport of two species of amino acids among the three was induced by counterflow of the other substrate. These results show that the transport system for branched-chain amino acids was reconstituted into liposomes from P. aeruginosa cells and strongly suggest that three branched-chain amino acids are transported by a common carrier system.     

Pharmacological activities of branched-chain amino acids: specificity of tissue and signal transduction

Branched-chain amino acid (BCAA: Leu, Ile, and Val) mixture has been used for treatment of hypoalbuminemia in patients with decompensated liver cirrhosis in Japan. It has been known that BCAA, especially leucine, activates mTOR signals and inhibition of protein degradation results in promoting protein synthesis in vitro. Furthermore, leucine activates glycogen synthase via mTOR signals in L6 cell, but not hepatocyte, and it has been shown that leucine improved glucose metabolism in normal and cirrhosis model rats. In this review, it will be proposed about the pharmacological activity of branched-chain amino acids, mainly leucine, on tissue specificity of cirrhotic disease.     

Regulation of global and specific mRNA translation by oral administration of branched-chain amino acids

The importance of branched-chain amino acids as nutrient regulators of protein synthesis in skeletal muscle was recognized more than 20 years ago. Of the branched-chain amino acids, leucine in particular was shown to play a central role in promoting muscle protein synthesis. However, it was only recently that the mechanism(s) involved in the stimulation of protein synthesis by leucine has begun to be defined. Studies performed in our laboratory during the past few years have revealed that oral administration of leucine to fasted rats enhances protein synthesis in association with increased phosphorylation of two proteins downstream of the protein kinase referred to as the mammalian target of rapamycin (mTOR). These proteins, eukaryotic initiation factor eIF4E binding protein (4E-BP)1 and ribosomal protein S6 kinase S6K1, control in part the step in translation initiation involving the binding of mRNA to the 40S ribosomal subunit. In theory the translation of all mRNAs can be regulated through such mechanisms, however, some mRNAs are more sensitive to the changes than others, resulting in modulation of gene expression through altered patterns of translation of specific mRNAs. Moreover, although a basal amount of plasma insulin is required for leucine to enhance signaling downstream of mTOR, the concentration observed in plasma of fasted rats is sufficient to observe maximal changes in phosphorylation of 4E-BP1 and S6K1.     

Deoxycholic acid degradation by a Pseudomonas species. Acidic intermediates from the initial part of the catabolic pathway.

The microbial catabolism of deoxycholic acid by a Pseudomonas species was studied, and three acidic products were isolated as their methyl esters. Evidence is presented that the compounds are methyl 3 alpha,12 alpha-dihydroxy-23,24-dinor-5 beta-cholan-22-oate, methyl 12 alpha-hydroxy-3-oxo-5 beta-cholan-24-oate and methyl 12 alpha-hydroxy-3-oxo-23,24-dinor-5 beta-cholan-22-oate.     

Chemoenzymatic synthesis of surfactants from carbohydrates, amino acids, and fatty acids

The chemoenzymatic synthesis of new surfactants is reported; they were prepared from unprotected carbohydrates, amino acids, and fatty acids. This study pointed out the factors that govern the possibility to enzymatically bind the carbohydrate to the amino acid.     

Analysis of corynomycolic acids and other fatty acids produced by Corynebacterium lepus grown on kerosene.

The saponifiable carboxylic acids of the extracellular product of Corynebacterium lepus grown on kerosene have been isolated and characterized. About 25% of these acids were a mixture of simple, saturated fatty acids ranging from C13 to C24 and including both even and odd homologues. The distribution of these acids was bimodal, with maxima at C15 and C21. The other 75% of the acids was a mixture of corynomycolic acids [R1--CH(OH)--CH(R2)--COOH] ranging from C28 to C43. The R1 alkyl fragments varied from C16 to C25, and R2 fragments varied from C6 to C14. Both even and odd corynomycolic acid homologues were observed, and the distribution had a single pronounced maximum at C32 and C33. Bacterial utilization of the carboxylic oxidation products of the kerosene substrate is suggested to account for the wide distribution in chain length of these saturated fatty acids and for the observation of both even and odd homologues.     

Free fatty acids are produced in and secreted from target cells very early in cytotoxic T lymphocyte-mediated killing

Conjugation of CTL with their cognate targets elicits a number of early changes within the target cell that are thought to play an important role in the lytic mechanism. We now report that at times earlier than 5 min after conjugation with allospecific CTL, free fatty acids (FFA) are produced in and then secreted from alloantigen-bearing target cells. Using murine CTL clones with different alloantigen specificities, stimulation of FFA production from target cells was found to be Ag specific. FFA production does not appear to be specific for any particular FFA species. Indeed, a wide spectrum of cis unsaturated as well as saturated FFA are produced. FFA production is well correlated with, and specific for, CTL-mediated target cell lysis. Other means of perturbing or lysing target cells, including freeze/thaw disruption, detergent solubilization, or increasing membrane permeabilization with ionomycin, do not stimulate FFA production. In particular, FFA production is not stimulated by treatment with pore-forming granules under conditions that cause more than 90% target cell lysis. These results suggest that FFA production plays an important role in CTL-mediated lysis because stimulation of FFA release specifically requires an event that is CTL induced, occurs very early after conjugation, and is strongly correlated with CTL-mediated lysis.     

Production of C20 polyunsaturated fatty acids (PUFAs) by pathway engineering: identification of a PUFA elongase component from Caenorhabditis elegans

Using a combination of database-mining and functional characterization, we have identified a component of the polyunsaturated fatty acid (PUFA) elongase. Co-expression of this elongating activity with fatty acid desaturases has allowed us to heterologously reconstitute the PUFA biosynthetic pathway. Both these enzymes (desaturases and elongase components) have undergone gene-duplication events which provide a paradigm for the diverged nature of PUFA biosynthetic activities.