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Lipoic acid content of Escherichia coli and other microorganisms
Abstract
A mutant strain of Escherichia coli K-12 requiring lipoic acid. W1485lip 2 (ATCC 25645), was used to develop a turbidimetric assay for lipoic acid and a polarographic assay based on the oxidation of pyruvate by suspensions of lipoic acid-deficient organisms. The turbidmetric assay was more sensitive with a working range equivalent to 0.2-2.0 ng of DL-alpha-lipoic acid compared with 5-50 ng for the polarographic method. The mutant responded equally to racemic mixtures of alpha-lipoic acid, beta-lipoic acid and dihydrolipoic acid but gave little response to lipoamide, and other derivatives without prior hydrolysis; 8-methyllipoic acid was a competitive inhibitor of the response to lipoic acid. A high specificity of the mutant for the natural steroisomer was indicated by the fact that (+)-alpha-lipoic acid had twice the activity of the racemic mixture. Escherichia coli K12 contained less than 0.05 ng of free (+)-alpha-lipoic acid per mg dry weight but, depending on the growth substrate, the equivalent of between 13 and 47 ng of (+)-alpha-lipoic acid per mg dry weight after acid extraction. There was a strong correlation between the lipoic acid content and the sum of the specific activities for the pyruvate and alpha-ketoglutarate dehydrogenase complexes. Experiments with washed suspensions of Escherichia coli showed only small increases in lipoic acid content (18%) when incubated with pyruvate, cysteine and methionine. When supplied with exogenous lipoic acid the mutant, W1485lip2, accumulated very little more than was demanded by its metabolism. The lipoic acid contents of several organisms were measured and correlated with their metabolism.
... LA content from dietary sources is very low. Ten tons of liver contains only 30 mg of LA [25,26]. Chemical synthesis of LA requires toxic catalysts to achieve addition of two sulfur atoms, which creates an environmental concern over increased pollution [27]. ...
... LA was released from the lipoylated-lipoyl domain (holo-lipoyl domain) by hydrolysis with 3 M H 2 SO 4 in a 200-mL round-bottom flask. After all lipoyl domains had been incubated at 120˚C for 2 hours, the pH was adjusted to 7.0 with 4 N NaOH [25,36]. The organic phase was separated after the addition of an equal volume of benzene. ...
... Biological method: LA concentration was assayed with E. coli mutant strain TM179 in which the lipA and lipB mutant cannot synthesize LA by itself, as described previously [25]. Standard LA was added in the range of 0.2 to 2.0 ng per culture medium. ...
Alpha-lipoic acid (LA) is an important enzyme cofactor widely used by organisms and is also a natural antioxidant for the treatment of pathologies driven by low levels of endogenous antioxidants. In order to establish a safer and more efficient process for LA production, we developed a new biological method for LA synthesis based on the emerging knowledge of lipoic acid biosynthesis. We first cloned the lipD gene, which encodes the lipoyl domain of the E2 subunit of pyruvate dehydrogenase, allowing high levels of LipD production. Plasmids containing genes for the biosynthesis of LA were subsequently constructed utilizing various vectors and promotors to produce high levels of LA. These plasmids were transformed into the Escherichia coli strain BL21. Octanoic acid (OA) was used as the substrate for LA synthesis. One transformant, YS61, which carried lipD, lplA, and lipA, produced LA at levels over 200-fold greater than the wild-type strain, showing that LA could be produced efficiently in E. coli using genetic engineering methods.
... Lipoic acid, a widely occurring coenzyme found in prokariotic and eukariotic microorganisms [6] as well as in animals and plants [18], was first isolated by Reed and co-workers [49]. However, its existence was known since the 1930s when it was discovered that a so-called 'potato growth factor' was necessary for the growth of certain bacteria. ...
... Techniques for the determination of LA rely on colorimetric [10,27] or microbiological assays [18], after hydrolysis of the amide bond of lipoyl-N-ε-lysine. Colorimetric assays are neither sensitive nor specific because they do not distinguish between LA and DHLA. ...
... However, Yasuno and Wada [69] found that in 4-week-old Arabidopsis thaliana LA synthase is located only in mitochondria. The activity of the enzyme resulted higher in flowers and leaves than in roots, in agreement with the amounts of LA determined by the microbiological assay [18]. These authors found that LA in leaves, roots and flowers was 0.17, 0.09 and 0.29 ppm on fresh weight basis, respectively. ...
Lipoic acid (1,2-dithiolane-pentanoic acid) is a dithiol which is effective in affording protection against oxidative stress by virtue of its two sulphydryl moieties. It is present in all kinds of eukaryotic and prokaryotic cells. As lipoamide, it functions as a cofactor in the multienzyme complexes that catalyse the oxidative decarboxylation of α-keto acids such as pyruvate, α-ketoglutarate, and branched-chain α-keto acids. The complete enzyme pathway responsible for the de novo synthesis of lipoic acid has not yet been elucidated. Octanoic acid appears to be the precursor for the eight-carbon fatty acid chain, and cysteine the source of sulfur. Lipoic acid is unique, among antioxidants, because it retains powerful antioxidant properties in both its reduced (dihydrolipoic acid) and oxidised (lipoic acid) forms. Both lipoic and dihydrolipoic acids have metal-chelating ability and quench activated oxygen species either in the cytosol or in the hydrophobic domains. Dihydrolipoic acid has more antioxidant properties than lipoic acid, and it plays an important role in the recycling of other oxidised radical scavengers such as glutathione, ascorbate and tocopherol. However, dihydrolipoic acid can also exert pro-oxidant properties both by its iron-reducing ability and by its ability to generate sulfur-containing radicals that can damage proteins. There are few quantitative data on lipoic acid contents in vegetables. It has been found in asparagus, wheat and potatoes, and recently, the presence of both lipoic and dihydrolipoic acids in roots, leaves and in the stroma of wheat has been demonstrated.
... Unbound lipoic acid and lipoamide levels were also measured since these represent possible reductants for Ohr; both were below measureable levels. Prior determinations of lipoic acid in bacteria have measured the amount released by acid hydrolysis of cell pellets and reflect protein-bound lipoyl residues in amounts as high as ϳ0.2 mol per g (dry weight) (27). ...
... Lipoamide itself is sufficient for the in vitro assay, but neither free lipoamide nor lipoic acid can function as the cellular reductant, because they are expected to rapidly diffuse out of the cell (25,62) and their cellular levels are undetectable ( Table 2). Protein-bound lipoyl residues have been measured at levels as high as ϳ0.2 mol per gram (27). Both Lpd and DlaT are more widely distributed in bacteria than Ohr, so a reduction system for Ohr might be available if the ohr gene were acquired by lateral gene transfer. ...
The mshA::Tn5 mutant of Mycobacterium smegmatis does not produce mycothiol (MSH) and was found to markedly overproduce both ergothioneine and an ∼15-kDa protein determined
to be organic hydroperoxide resistance protein (Ohr). An mshA(G32D) mutant lacking MSH overproduced ergothioneine but not Ohr. Comparison of the mutant phenotypes with those of the wild-type
strain indicated the following: Ohr protects against organic hydroperoxide toxicity, whereas ergothioneine does not; an additional
MSH-dependent organic hydroperoxide peroxidase exists; and elevated isoniazid resistance in the mutant is associated with
both Ohr and the absence of MSH. Purified Ohr showed high activity with linoleic acid hydroperoxide, indicating lipid hydroperoxides
as the likely physiologic targets. The reduction of oxidized Ohr by NADH was shown to be catalyzed by lipoamide dehydrogenase
and either lipoamide or DlaT (SucB). Since free lipoamide and lipoic acid levels were shown to be undetectable in M. smegmatis, the bound lipoyl residues of DlaT are the likely source of the physiological dithiol reductant for Ohr. The pattern of occurrence
of homologs of Ohr among bacteria suggests that the ohr gene has been distributed by lateral transfer. The finding of multiple Ohr homologs with various sequence identities in some
bacterial genomes indicates that there may be multiple physiologic targets for Ohr proteins.
... No known microorganisms, including E. coli, produce any detectable amounts of free LA secreted into the growth media (Herbert and Guest, 1975). We previously described a basal LA cell factory that constitutively over-expresses a truncated AceF lipoylation domain and IPTG-inducible LipA, which we found to be toxic to cell growth if induced too highly based on a decrease of final ODs in liquid culture (Bali et al., 2020). ...
L-Lipoic acid (LA) is an important antioxidant with various industrial applications as a nutraceutical and therapeutic. Currently, LA is produced by chemical synthesis. Cell factory development is complex as LA and its direct precursors only occur naturally in protein-bound forms. Here we report a rationally engineered LA cell factory and demonstrate de novo free LA production from glucose for the first time in E. coli. The pathway represents a significant challenge as the three key enzymes, native Octanoyltransferase (LipB) and Lipoyl Synthase (LipA), and heterologous Lipoamidase (LpA), are all toxic to overexpress in E. coli. To overcome the toxicity of LipB, functional metagenomic selection was used to identify a highly active and non-toxic LipB and LipA from S. liquefaciens. Using high through put screening, we balanced translation initiation rates and dual, orthogonal induction systems for the toxic genes, LipA and LpA. The optimized strain yielded 2.5 mg free LA per gram of glucose in minimal media, expressing carefully balanced LipB and LipA, Enterococcus faecalis LpA, and a truncated, native, Dihydrolipoyllysine-residue acetyltransferase (AceF) lipoylation domain. When the optimized cell factory strain was cultivated in a fed-batch fermentation, a titer of 87 mg/L free LA in the supernatant was reached after 48 h. This titer is ∼3000-fold higher than previously reported free LA titer and ∼8-fold higher than the previous best total, protein-bound LA titer. The strategies presented here could be helpful in designing, constructing and balancing biosynthetic pathways that harbor toxic enzymes with protein-bound intermediates or products.
... PBA regulated cellular lipid homeostasis through the induction of CPT1A and INSIG2 expression via an epigenetic mechanism involving the acetylation of histone H3, histone H4, and CBP-p300 at the CPT1A and INSIG2 promoters. and plant plastids [7,8]. LA can also be absorbed from foods (leafy green vegetables and red meats) and dietary supplements. ...
The constitutive activation of the mechanistic target of rapamycin complex 1 (mTORC1) leads to the overproduction of apoB-containing triacylglycerol-rich lipoproteins in HepG2 cells. R-α-lipoic acid (LA) and 4-phenylbutyric acid (PBA) have hypolipidemic function but their mechanisms of action are not well understood. Here, we reported that LA and PBA regulate hepatocellular lipid metabolism via distinct mechanisms. The use of SQ22536, an inhibitor of adenylyl cyclase, revealed cAMP’s involvement in the upregulation of CPT1A expression by LA but not by PBA. LA decreased the secretion of proprotein convertase subtilisin/kexin type 9 (PCSK9) in the culture media of hepatic cells and increased the abundance of LDL receptor (LDLR) in cellular extracts in part through transcriptional upregulation. Although PBA induced LDLR gene expression, it did not translate into more LDLR proteins. PBA regulated cellular lipid homeostasis through the induction of CPT1A and INSIG2 expression via an epigenetic mechanism involving the acetylation of histone H3, histone H4, and CBP-p300 at the CPT1A and INSIG2 promoters.
... Escherichia coli: the lplA and lipB genes define redundant pathways for ligation of lipoyl groups to apoprotein. J. Bacteriol. 177, 1-10.3.Herbert,A. A., and Guest, J. R. (1975) Lipoic acid content of Escherichia coli and other microorganisms. Arch. Microbiol. 106, 259-266. 4. Vanden Boom, T. J., Reed, K. E., and Cronan, J. E., Jr. (1991) Lipoic acid metabolism in Escherichia coli: isolation of null mutants defective in lipoic acid biosynthesis, molecular cloning and characterization of the E. coli lip locus, ...
... Alpha lipoic acid (α-LA) is a disulphide compound found naturally in diverse group of micro-organisms and in a variety of plant and animal tissues (Herbert et al., 1975). It is mainly present in mitochondria and plays a pivotal role in energy metabolism and was first isolated from bovine liver in 1951 (Reed et al., 1951). ...
Lipoic acid plays vital role in energy metabolism. It is an excellent antioxidant acting inside the cell and at plasma membrane levels. It also acts as therapeutic agents in different diseases including diabetes, neurodegeneration, hypertension, HIV, breast cancer, heavy metal poisoning, radiation injury etc. Considering the nutritional and therapeutic perspectives, this is very important to identify α-lipoic acid-rich food. It is also required to determine whether concentration of α-lipoic acid gets changed with heat treatment. In this investigation, plant and animal foodstuffs were first blended with methanol, and then acidified, after that sonicated and finally centrifuged to extract α-lipoic acid. The concentrations of α-lipoic acid were estimated by the reverse phase high performance liquid chromatography (HPLC). Concentration of α-lipoic acid in the raw form of plant and animal foodstuffs ranged from 1.270671 ng/mg to 22.35785 ng/mg and 2.214241 ng/mg to 13.55982 ng/mg respectively. The level of α-lipoic acid in the boiled form of plant and animal foodstuffs ranged from 0.203982 ng/mg to 3.59858 ng/mg and 1.24481 ng/mg to 7.198254 ng/mg respectively. It was found that raw sample had higher concentration of α-lipoic acid than boiled sample. In our study, loss of α-lipoic acid due to boiling ranged from 10.85% to not detectable level for plant sample and 17.23% to 75.40% for animal sample indicating that α-lipoic acid in animal sample was more stable to boiling than that of plant sample. Further research should be continued to improve the stability of α-lipoic acid in different processed forms of foodstuffs.
... In some microorganisms, it can degrade branched chain amino acids. A number of saprophytic food microorganisms exhibit the potential for lipoic acid biosynthesis; these are mainly representatives of the genera Bacillus, Pseudomonas, Pedicoccus, Rhodospirillum, Anacystis, Gloecaps, Nostoc, and Saccharomyces [71]. Mitochondrial lipoyl synthase enzyme was found, in the proteome of G. geotrichum 38; this is responsible for the synthesis of lipoic acid. ...
Fried cottage cheese is a dairy product, popular in some parts of Poland. Proteomic analysis of a culture of the mold Galactomyces geotrichum 38 isolated from fried cottage cheese was performed using UHPLC/MS. From the proteins identified, we selected those involved in the biosynthesis of bioactive compounds and those useful in industry. In the G. geotrichum 38 culture, the production quantities of vitamin B2 (224 μg/L), ergosterol (54.63 mg/kg), and trehalose (0.91 g/L) were determined by HPLC. The identified proteins were also used to prepare a hypothetical fatty acid biosynthesis pathway, and the percentage of individual sphingolipids in the culture was determined. Sphingolipids are also bioactive compounds. During culturing of G. geotrichum 38, the percentage of three sphingolipids increased. The last step of the research was to prepare a model of fried cottage cheese. The mold G. geotrichum 38, used in the process of ripening fried cottage cheese, synthesized vitamin B2 and erogsterol, which influenced the nutritional value of the product.
... But the enzyme immunoassay is not sensitive, it can't distinguish the two forms of lipoic acid [16]. Hydrolysis of lipoyl-N-ε-lysine amide bond can be determined by colorimetric method, but this method is not sensitive and specific, or microbial determination, but takes more time and effort [17][18][19]. Endogenous lipoic acid in isolated lipoate-containing proteins was measured in form of lipoyllysine by enzymatic hydrolysis, using HPLC equipped with an ultraviolet (UV) detector (330 nm). This method has the advantage of increasing the recovery of endogenous lipoic acid. ...
Lipoic acid is a kind of small molecular compound with strong oxidizing properties. It has been widely used in medicine and has achieved good results since its discovery. However, it is less used in plants, and the biosynthetic pathway is not clear. The content in the plant is mainly measured by high-performance liquid chromatography(HPLC). At present, it is mainly used as an additive to the culture medium for plant tissue culture and Agrobacterium-mediated plant genetic transformation, in order to reduce the browning rate of explants, improve Agrobacterium-mediated genetic transformation efficiency.
... α-Lipoic acid (α-LA) is a naturally occurring enzyme cofactor synthesized from octanoic acid in most prokaryotic and eukaryotic microorganisms as well as plant and animal mitochondria and plant plastids [90,91]. α-LA can also be absorbed from foods (leafy green vegetables and red meats) and dietary supplements. ...
Fibroblast growth factor 21 (FGF21) gene expression is altered by a wide array of physiological, metabolic, and environmental factors. Among dietary factors, high dextrose, low protein, methionine restriction, short-chain fatty acids (butyric acid and lipoic acid), and all-trans-retinoic acid were repeatedly shown to induce FGF21 expression and circulating levels. These effects are usually more pronounced in liver or isolated hepatocytes than in adipose tissue or isolated fat cells. Although peroxisome proliferator-activated receptor α (PPARα) is a key mediator of hepatic FGF21 expression and function, including the regulation of gluconeogenesis, ketogenesis, torpor, and growth inhibition, there is increasing evidence of PPARα-independent transactivation of the FGF21 gene by dietary molecules. FGF21 expression is believed to follow the circadian rhythm and be placed under the control of first order clock-controlled transcription factors, retinoic acid receptor-related orphan receptors (RORs) and nuclear receptors subfamily 1 group D (REV-ERBs), with FGF21 rhythm being anti-phase to REV-ERBs. Key metabolic hormones such as glucagon, insulin, and thyroid hormone have presumed or clearly demonstrated roles in regulating FGF21 transcription and secretion. The control of the FGF21 gene by glucagon and insulin appears more complex than first anticipated. Some discrepancies are noted and will need continued studies. The complexity in assessing the significance of FGF21 gene expression resides in the difficulty to ascertain (i) when transcription results in local or systemic increase of FGF21 protein; (ii) if FGF21 is among the first or second order genes upregulated by physiological, metabolic, and environmental stimuli, or merely an epiphenomenon; and (iii) whether FGF21 may have some adverse effects alongside beneficial outcomes.
... Based on these observations, two E. coli pathways for protein lipoylation were proposed ( Fig. 5) (70,74,77), as follows. When free lipoic acid is available in the medium, E. coli incorporates and attaches extracellular lipoate (78)(79)(80) via the LplA-dependent scavenging pathway that proceeds through lipoyl-AMP (Fig. 5). When lipoate is absent from the medium, lipoyl groups must be made by de novo synthesis. ...
Although the structure of lipoic acid and its role in bacterial metabolism were clear over 50 years ago, it is only in the past decade that the pathways of biosynthesis of this universally conserved cofactor have become understood. Unlike most cofactors, lipoic acid must be covalently bound to its cognate enzyme proteins (the 2-oxoacid dehydrogenases and the glycine cleavage system) in order to function in central metabolism. Indeed, the cofactor is assembled on its cognate proteins rather than being assembled and subsequently attached as in the typical pathway, like that of biotin attachment. The first lipoate biosynthetic pathway determined was that of
Escherichia coli
, which utilizes two enzymes to form the active lipoylated protein from a fatty acid biosynthetic intermediate. Recently, a more complex pathway requiring four proteins was discovered in
Bacillus subtilis
, which is probably an evolutionary relic. This pathway requires the H protein of the glycine cleavage system of single-carbon metabolism to form active (lipoyl) 2-oxoacid dehydrogenases. The bacterial pathways inform the lipoate pathways of eukaryotic organisms. Plants use the
E. coli
pathway, whereas mammals and fungi probably use the
B. subtilis
pathway. The lipoate metabolism enzymes (except those of sulfur insertion) are members of PFAM family PF03099 (the cofactor transferase family). Although these enzymes share some sequence similarity, they catalyze three markedly distinct enzyme reactions, making the usual assignment of function based on alignments prone to frequent mistaken annotations. This state of affairs has possibly clouded the interpretation of one of the disorders of human lipoate metabolism.
... Lipoic acid (LA, 1,2-dithiolane-3-pentanoic acid, Figure 1(a)), along with its reduced form (dihydrolipoic acid, DHLA, Figure 1(b)), is an important cofactor of mitochondrial enzymes and a natural antioxidant. It is present in both eukaryotic and prokaryotic microorganisms [1] and in all plant and animal cells [2]. In oxidative decarboxylation of pyruvate, -ketoglutarate, branched-chain -keto acids and glycine it acts as a catalyst [3,4]. ...
The present study offers results of analysis concerning the course of reaction between reduced α-lipoic acid (LA) and 2-chloro-1-methylquinolinium tetrafluoroborate (CMQT). In water environments, the reaction between CMQT and hydrophilic thiols proceeds very rapidly and the resultant products are stable. For the described analysis, optimum reaction conditions, such as concentration of the reducing agent, environment pH, and concentration of the reagent were carefully selected. The spectrophotometric assay was carried out measuring absorbance at
λ
=
348
nm (i.e., the spectral band of the obtained reaction product). Furthermore, the calibration curve of lipoic acid was registered. It was concluded that the Lambert-Beer law was observed within the range 1–10 μmol L−1. Later, the reaction between LA and CMQT was used as precolumn derivatization in a chromatographic determination of the lipoic acid in the range 2.5–50 μmol L−1. Practical applicability of the designed methods was evaluated by determining lipoic acid in Revitanerv pharmaceutical preparation which contains 300 mg LA in a single capsule. The error of the determination did not exceed 0.5% in relation to the declared value.
... Based on these observations, a two pathway E. coli lipoylation system was proposed (198,201,218) (Fig. 10). When presented with free lipoic acid in the medium E. coli incorporates extracellular lipoate (217,220) via the LplA-dependent scavenging pathway which utilizes ATP to activate lipoic acid in the form of lipoyl-AMP. When lipoate is absent from the medium lipoyl groups must be made by de novo synthesis. ...
Biotin and Lipoic Acid: Synthesis, Attachment, and Regulation, Page 1 of 2
Abstract
Two vitamins, biotin and lipoic acid, are essential in all three domains of life. Both coenzymes function only when covalently attached to key metabolic enzymes. There they act as “swinging arms” that shuttle intermediates between two active sites (= covalent substrate channeling) of key metabolic enzymes. Although biotin was discovered over 100 years ago and lipoic acid 60 years ago, it was not known how either coenzyme is made until recently. In Escherichia coli the synthetic pathways for both coenzymes have now been worked out for the first time. The late steps of biotin synthesis, those involved in assembling the fused rings, were well described biochemically years ago, although recent progress has been made on the BioB reaction, the last step of the pathway in which the biotin sulfur moiety is inserted. In contrast, the early steps of biotin synthesis, assembly of the fatty acid-like “arm” of biotin were unknown. It has now been demonstrated that the arm is made by using disguised substrates to gain entry into the fatty acid synthesis pathway followed by removal of the disguise when the proper chain length is attained. The BioC methyltransferase is responsible for introducing the disguise, and the BioH esterase is responsible for its removal. In contrast to biotin, which is attached to its cognate proteins as a finished molecule, lipoic acid is assembled on its cognate proteins. An octanoyl moiety is transferred from the octanoyl acyl carrier protein of fatty acid synthesis to a specific lysine residue of a cognate protein by the LipB octanoyltransferase followed by sulfur insertion at carbons C-6 and C-8 by the LipA lipoyl synthetase. Assembly on the cognate proteins regulates the amount of lipoic acid synthesized, and, thus, there is no transcriptional control of the synthetic genes. In contrast, transcriptional control of the biotin synthetic genes is wielded by a remarkably sophisticated, yet simple, system, exerted through BirA, a dual-function protein that both represses biotin operon transcription and ligates biotin to its cognate proteins.
... 16 LDH is essential for anaerobic glycolysis and hence necessary for muscular work in the absence of oxygen. 17 In chickens with clinical leucosis increased concentrations of LDH were reported 18 .LDH was chosen for this study because a good deal of information is available about its enzymatic characteristic and genetic control. ...
Aim of the study is to investigate the effect of ethanol by the action of lipoic acid on certain liver
parameters like lactate dehydrogenase, succinate dehydrogenase, and glucose-6-phosphatase etc.
Scientific outcome that lipoic acid possess hepatoprotective as well as antioxidant properties. During the
study we found that all parameters are up to the mark and scientific outcomes ensures the quality of the
work. Hence this study needs attention further for its practice in real pharma as well as scientific
industries to deliver a quality outcome for a society.
... LA has been used therapeutically in diabetic neuropathy and retinopathy (1, 6, 19, 33, 36, 38, 46, 49 -52), but its TG-lowering properties have only recently been recognized, first in laboratory animals (7,22,43,47) and then in humans (27,48). LA is a naturally occurring, essential, dithiol-containing cofactor synthesized enzymatically from octanoate in most prokaryotic and eukaryotic micro-organisms as well as plant and animal mitochondria (15). The presence of an asymmetric carbon yields two enantiomers, R-LA and S-LA, during organic syn-thesis. ...
We report on the characterization of lipogenic tissue transcriptional networks that support physiological responses of obese rats to a lipid-lowering bioactive food compound, R-α-lipoic acid (LA). Nine-week old male Zucker Diabetic Fatty (fa/fa) rats were fed a chow diet supplemented with 3 g LA per kg diet or pair fed for two weeks. At the end of the trial, high-quality RNA was extracted from the liver and epididymal fat and subjected to transcriptome analysis using RNA-Seq technology. Results showed a substantially higher number of differentially expressed genes (DEG, False Discovery Rate adjusted p ≤ 0.05 and absolute log2(fold change) ≥ 1) in liver (110 genes) vs. epididymal fat (10 genes). Most epididymal fat DEG were also differentially expressed in liver and shared directionality of change. Gene Ontology (GO) analysis of these transcripts revealed significant enrichment of GO categories related to immune response, stress response, lipid metabolism, and carboxylic acid metabolic processes. Of interest interferon-related genes involved in defense against microorganism and innate immune response were induced by LA. Lipid metabolism-related transcript changes observed in LA-fed animals included downregulation of lipogenic genes (Pnpla3, Pnpla5, Elovl6, Acly, Gpam, and Aacs) and concomitant upregulation of short-, medium- and long-chain fatty acid metabolic processes (Acot1, Acot2, Acsf2, and Crat). Transcriptional changes were accompanied by the lowering of abdominal adiposity and blood triacylglycerol levels. We conclude that LA dietary supplementation induces prominent gene expression changes in liver in support of significant improvement of whole-body lipid status.
... Keywords Antioxidant Á Characiformes Á Dehydroascorbic acid Á Pacu Á Scurvy-prone Á Tocopherol Introduction a-Lipoic acid (LA) is a naturally-occurring thiol compound found ubiquitously in microorganisms, plants, and animals (Reed et al. 1966;Herbert and Guest 1975). LA mainly exists in a bound form to the e-amino group of the lysine side chain present in pyruvate dehydrogenase. ...
Effects of dietary α-lipoic acid (LA) and ascorbic acid (AA) on the growth, tissue vitaminC and tocopherol (vitaminE) levels, and malondialdehyde levels were examined in the tropical fish pacu, Piaractus mesopotamicus. Pacu juveniles were fed one of four casein-gelatin-based diets for 8 weeks: with 0.05% AA and 0.1% LA (+AA+LA), with AA and without LA (+AA–LA), without AA and with LA (−AA+LA), and without AA and LA (−AA−LA). When the fish received quantities of feed equal to 1.9–2.5% of its body weight, growth was not influenced, regardless of the presence of AA or LA throughout most of the experimental period. Growth was, however, slightly but significantly lower at week8 in the AA-deficient/LA-supplemented group. An AA-deficient diet caused a highly significant reduction in both total AA and dehydroascorbic acid content in the liver and gill tissues. This reduction of tissue AA concentrations was reversed in a significant manner by LA (antioxidant-sparing effect). The 8-week-long vitaminC deprivation was sufficient to initiate the reduction in tissue ascorbic acid; however, total ascorbate in the liver of fish in the (−)AA/(+)LA group was 127.7±54.3nmol g−1 tissue, whereas it was 28.6±26.3nmol g−1 in the (−)AA/(−)LA group, a 4.4-fold difference. This mitigating effect of the addition of the endogenous antioxidant LA to the diet indicates that LA exerts a vitaminC-sparing effect in teleost fish that by far exceeds the phenomena demonstrated in non-scurvy-prone mammals. There was no difference among the different diet groups for vitaminE and malondialdehyde levels in the liver. These results suggest that LA is a potent substance for the prevention of AA deficiency in cultured fishes. The optimal dietary level of LA needs to be determined in the light of the slight reduction in body weight gain after 8weeks of feeding in the absence of AA.
... Alpha-lipoic acid (LA) and its reduced form, dihydrolipoic acid (DHLA), are naturally occurring thiol compounds found ubiquitously in microorganisms, plants and animals (Reed 1974;Herbert and Guest 1975) and are potent antioxidants with the capacity to scavenge reactive oxygen species (ROS) and recycle endogenous antioxidants (Packer et al. 1995;Li et al. 2004). In cells, very little lipoate exists in the free acid form; almost all is tethered to the e-amino group of conserved lysine residues on lipoylaccepting domains of proteins (Biewenga et al. 1997a;Booker 2004). ...
Cytochrome b561 (Cyt-b561) proteins constitute a family of trans-membrane proteins that are present in a wide variety of organisms. Two of their characteristic properties are the reducibility by ascorbate (ASC) and the presence of two distinct b-type hemes localized on two opposite sides of the membrane. Here we show that the tonoplast-localized and the putative tumor suppressor Cyt-b561 proteins can be reduced by other reductants than ASC and dithionite. A detailed spectral analysis of the ASC-dependent and dihydrolipoic acid (DHLA)-dependent reduction of these two Cyt-b561 proteins is also presented. Our results are discussed in relation to the known antioxidant capability of DHLA as well as its role in the regeneration of other antioxidant compounds of cells. These results allow us to speculate on new biological functions for the trans-membrane Cyt-b561 proteins.
... LipA encodes the lipoate synthase which catalyzes the last step in lipoate biosynthesis and incorporation. Lipoate is an important co-factor of LpdA that is contained in the the pyruvate dehydrogenase complex, oxoglutarate dehydrogenase and the glycine cleavage complex [18]. ...
Although Escherichia coli is one of the best studied model organisms, a comprehensive understanding of its gene regulation is not yet achieved. There exist many approaches to reconstruct regulatory interaction networks from gene expression experiments. Mutual information based approaches are most useful for large-scale network inference.
We used a three-step approach in which we combined gene regulatory network inference based on directed information (DTI) and sequence analysis. DTI values were calculated on a set of gene expression profiles from 19 time course experiments extracted from the Many Microbes Microarray Database. Focusing on influences between pairs of genes in which one partner encodes a transcription factor (TF) we derived a network which contains 878 TF - gene interactions of which 166 are known according to RegulonDB. Afterward, we selected a subset of 109 interactions that could be confirmed by the presence of a phylogenetically conserved binding site of the respective regulator. By this second step, the fraction of known interactions increased from 19% to 60%. In the last step, we checked the 44 of the 109 interactions not yet included in RegulonDB for functional relationships between the regulator and the target and, thus, obtained ten TF - target gene interactions. Five of them concern the regulator LexA and have already been reported in the literature. The remaining five influences describe regulations by Fis (with two novel targets), PhdR, PhoP, and KdgR. For the validation of our approach, one of them, the regulation of lipoate synthase (LipA) by the pyruvate-sensing pyruvate dehydrogenate repressor (PdhR), was experimentally checked and confirmed.
We predicted a set of five novel TF - target gene interactions in E. coli. One of them, the regulation of lipA by the transcriptional regulator PdhR was validated experimentally. Furthermore, we developed DTInfer, a new R-package for the inference of gene-regulatory networks from microarrays using directed information.
... Escherichia coli: the lplA and lipB genes define redundant pathways for ligation of lipoyl groups to apoprotein. J. Bacteriol. 177, 1-10.3.Herbert,A. A., and Guest, J. R. (1975) Lipoic acid content of Escherichia coli and other microorganisms. Arch. Microbiol. 106, 259-266. 4. Vanden Boom, T. J., Reed, K. E., and Cronan, J. E., Jr. (1991) Lipoic acid metabolism in Escherichia coli: isolation of null mutants defective in lipoic acid biosynthesis, molecular cloning and characterization of the E. coli lip locus, ...
Thesis (Ph.D.)--Pennsylvania State University, 2005. Microfilm (positive).
... Fate of cleaved lipoic acid Prior evidence suggests that E. coli does not maintain large intracellular stores of free lipoate. Herbert and Guest measured PDH and KDH activities in E. coli extracts and found that these enzymatic activities correlated well with the total cellular content of lipoic acid, even when an excess of lipoic acid was added to the growth medium [18]. These results suggest that either E. coli limits the uptake of lipoic acid, or that the bacterium fails to retain excess lipoic acid that is not needed to activate the two ketoacid dehydrogenases. ...
In the 1950s, Reed and coworkers discovered an enzyme activity in Streptococcus faecalis (Enterococcus faecalis) extracts that inactivated the Escherichia. coli and E. faecalis pyruvate dehydrogenase complexes through cleavage of the lipoamide bond. The enzyme that caused this lipoamidase activity remained unidentified until Jiang and Cronan discovered the gene encoding lipoamidase (Lpa) through the screening of an expression library. Subsequent cloning and characterization of the recombinant enzyme revealed that lipoamidase is an 80 kDa protein composed of an amidase domain containing a classic Ser-Ser-Lys catalytic triad and a carboxy-terminal domain of unknown function. Here, we show that the amidase domain can be used as an in vivo probe which specifically inactivates lipoylated enzymes.
We evaluated whether Lpa could function as an inducible probe of alpha-ketoacid dehydrogenase inactivation using E. coli as a model system. Lpa expression resulted in cleavage of lipoic acid from the three lipoylated proteins expressed in E. coli, but did not result in cleavage of biotin from the sole biotinylated protein, the biotin carboxyl carrier protein. When expressed in lipoylation deficient E. coli, Lpa is not toxic, indicating that Lpa does not interfere with any other critical metabolic pathways. When truncated to the amidase domain, Lpa retained lipoamidase activity without acquiring biotinidase activity, indicating that the carboxy-terminal domain is not essential for substrate recognition or function. Substitution of any of the three catalytic triad amino acids with alanine produced inactive Lpa proteins.
The enzyme lipoamidase is active against a broad range of lipoylated proteins in vivo, but does not affect the growth of lipoylation deficient E. coli. Lpa can be truncated to 60% of its original size with only a partial loss of activity, resulting in a smaller probe that can be used to study the effects of alpha-ketoacid dehydrogenase inactivation in vivo.
... Introduction α -Lipoic acid (6,8-thioctic acid, LA) is an eight-carbon fatty acid containing a thiolane ring with a disulfide bond joining carbons 6 and 8 (Ou et al. 1995). LA is a naturally occurring cofactor that has been reported to be present in a diverse group of microorganisms and a variety of plant and animal tissues (Herbert and Guest 1975). The cofactor is essential for the function of several key enzymes involved in oxidative metabolism, including pyruvate dehydrogenase, 2-oxoglutarate dehydrogenase, branched-chain 2oxoacid dehydrogenase, and the glycine cleavage system (Reed and Cronan 1993). ...
Alpha-lipoic acid (LA), a naturally occurring cofactor reported to be present in a diverse group of microorganisms, plants, and animal tissues, has been widely and successfully used as a therapy for a variety of diseases, including diabetes and heart disease. However, to date, recombinant DNA technology has not been applied for higher LA production due mainly to difficulties in the functional expression of key enzymes involved in LA production. Here, we report a study for higher LA production with the aid of chaperone plasmids, DnaKJE and trigger factor (Tf). The lipA and lplA genes encoding lipoate synthase and lipoate protein ligase in Pseudomonas fluorescens, respectively, were cloned and transformed into Escherichia coli K12. When they were overexpressed in E. coli, both LipA and LplA were expressed as inclusion bodies leading to no increase in LA production. However, when chaperone plasmids DnaKJE and Tf were coexpressed with lipA and lplA, the resulting recombinant E. coli strains showed higher LA production than the wild-type E. coli by 32-111%, respectively.
Prostate cancer (PCa) progression leads to bone modulation in approximately 70% of affected men. A nutraceutical, namely, α-lipoic acid (α-LA), is known for its potent anti-cancer properties towards various cancers and has been implicated in treating and promoting bone health. Our study aimed to explore the molecular mechanism behind the role of α-LA as therapeutics in preventing PCa and its associated bone modulation. Notably, α-LA treatment significantly reduced the cell viability, migration, and invasion of PCa cell lines in a dose-dependent manner. In addition, α-LA supplementation dramatically increased reactive oxygen species (ROS) levels and HIF-1α expression, which started the downstream molecular cascade and activated JNK/caspase-3 signaling pathway. Flow cytometry data revealed the arrest of the cell cycle in the S-phase, which has led to apoptosis of PCa cells. Furthermore, the results of ALP (Alkaline phosphatase) and TRAP (tartrate-resistant acid phosphatase) staining signifies that α-LA supplementation diminished the PCa-mediated differentiation of osteoblasts and osteoclasts, respectively, in the MC3T3-E1 and bone marrow macrophages (BMMs) cells. In summary, α-LA supplementation enhanced cellular apoptosis via increased ROS levels, HIF-1α expression, and JNK/caspase-3 signaling pathway in advanced human PCa cell lines. Also, the treatment of α-LA improved bone health by reducing PCa-mediated bone cell modulation.
Although the role of mechanistic target of rapamycin complex 1 (mTORC1) in lipid metabolism has been the subject of previous research, its function in chylomicron production is not known. In this study, we created three stable human colorectal adenocarcinoma Caco-2 cell lines exhibiting normal, low or high mTORC1 kinase activity, and used these cells to investigate the consequences of manipulating mTORC1 activity on enterocyte differentiation and chylomicron-like particle production. Constitutively active mTORC1 induced Caco-2 cell proliferation and differentiation (as judged by alkaline phosphatase activity) but weakened transepithelial electrical resistance (TEER). Repressed mTORC1 activity due to the knockdown of RPTOR significantly decreased the expression of lipogenic genes FASN, DGAT1 and DGAT2, lipoprotein assembly genes APOB and MTTP, reduced protein expression of APOB, MTTP and FASN, downregulated the gene expression of very long-chain fatty acyl-CoA ligase (FATP2), acyl-CoA binding protein (DBI), and prechylomicron transport vesicle-associated proteins VAMP7 (vesicle-associated membrane protein 7) and SAR1B (secretion associated Ras related GTPase 1B) resulting in the repression of apoB-containing triacylglycerol-rich lipoprotein secretion. Exposure of Caco-2 cells harboring a constitutively active mTORC1 to short-chain fatty acid derivatives, R-α-lipoic acid and 4-phenylbutyric acid, downregulated chylomicron-like particle secretion by interfering with the lipidation and assembly of the particles, and concomitantly repressed mTORC1 activity with no change to Raptor abundance or PRAS40 (Thr246) phosphorylation. R-α-lipoic acid and 4-phenylbutyric acid may be useful to mitigate intestinal lipoprotein overproduction and associated postprandial inflammation.
Lipoic acid is an essential sulfur-containing cofactor used by several multienzyme complexes involved in energy metabolism and the breakdown of certain amino acids. It is composed of n-octanoic acid with sulfur atoms appended at C6 and C8. Lipoic acid is biosynthesized de novo in its cofactor form, in which it is covalently bound in an amide linkage to a target lysyl residue on a lipoyl carrier protein (LCP). The n-octanoyl moiety of the cofactor is derived from type 2 fatty acid biosynthesis and is transferred to an LCP to afford an octanoyllysyl amino acid. Next, lipoyl synthase (LipA in bacteria) catalyzes the attachment of the two sulfur atoms to afford the intact cofactor. LipA is a radical S-adenosylmethionine (SAM) enzyme that contains two [4Fe-4S] clusters. One [4Fe-4S] cluster is used to facilitate a reductive cleavage of SAM to render the highly oxidizing 5′-deoxyadenosyl 5′-radical needed to abstract C6 and C8 hydrogen atoms to allow for sulfur attachment. By contrast, the second cluster is the sulfur source, necessitating its destruction during turnover. In Escherichia coli, this auxiliary cluster can be restored after each turnover by NfuA or IscU, which are two iron-sulfur cluster carrier proteins that are implicated in iron-sulfur cluster biogenesis. In this chapter, we describe methods for purifying and characterizing LipA and NfuA from Mycobacterium tuberculosis, a human pathogen for which endogenously synthesized lipoic acid is essential. These studies provide the foundation for assessing lipoic acid biosynthesis as a potential target for the design of novel antituberculosis agents.
A facile and eco-friendly approach for the synthesis of water-soluble WS2 quantum dots (QDs) was developed via ultrasonication and hydrothermal process from bulk WS2. In this strategy, the dispersity of bulk WS2 in aqueous phase was improved with the aid of the surfactant (CTAB), which could shorten the exfoliation time and improve the exfoliation efficiency to form layered WS2 nanosheets. Through hydrothermal treatment, the nanosheets were further scissored into QDs with high quality. The QDs show excellent features with narrow size distribution, good water solubility and stable fluorescence. We find that the fluorescence of WS2 QDs can be quenched by Fe3+ through photo-induced electron transfer, and a wide detection linear range for Fe3+ is acquired. It indicates that WS2 QD can be used as a “turn-off” probe for Fe3+. In the presence of lipoic acid (LA), the fluorescence was recovered due to the stronger interaction between LA and Fe3+ than WS2 QDs. And a “turn-on” sensor for LA was developed with a linear range from 1 to 10 μM and a detection limit of 0.59 μM. The strategy might be suitable for the facile synthesis of other water-soluble transition metal dichalcogenide QDs. It is expected that the water-soluble QDs have great potential applications in biological system.
Two vitamins, biotin and lipoic acid, are essential in all three domains of life. Both coenzymes function only when covalently attached to key metabolic enzymes. There they act as "swinging arms" that shuttle intermediates between two active sites (= covalent substrate channeling) of key metabolic enzymes. Although biotin was discovered over 100 years ago and lipoic acid was discovered 60 years ago, it was not known how either coenzyme is made until recently. In Escherichia coli the synthetic pathways for both coenzymes have now been worked out for the first time. The late steps of biotin synthesis, those involved in assembling the fused rings, were well described biochemically years ago, although recent progress has been made on the BioB reaction, the last step of the pathway, in which the biotin sulfur moiety is inserted. In contrast, the early steps of biotin synthesis, assembly of the fatty acid-like "arm" of biotin, were unknown. It has now been demonstrated that the arm is made by using disguised substrates to gain entry into the fatty acid synthesis pathway followed by removal of the disguise when the proper chain length is attained. The BioC methyltransferase is responsible for introducing the disguise and the BioH esterase for its removal. In contrast to biotin, which is attached to its cognate proteins as a finished molecule, lipoic acid is assembled on its cognate proteins. An octanoyl moiety is transferred from the octanoyl-ACP of fatty acid synthesis to a specific lysine residue of a cognate protein by the LipB octanoyl transferase, followed by sulfur insertion at carbons C6 and C8 by the LipA lipoyl synthetase. Assembly on the cognate proteins regulates the amount of lipoic acid synthesized, and thus there is no transcriptional control of the synthetic genes. In contrast, transcriptional control of the biotin synthetic genes is wielded by a remarkably sophisticated, yet simple, system exerted through BirA, a dual-function protein that both represses biotin operon transcription and ligates biotin to its cognate protein.
Lipoic acid is an essential cofactor for a variety of mitochondrial enzymes. We have characterised a gene from Saccharomyces cerevisiae which appears to encode a protein involved in the attachment of lipoic acid groups to the pyruvate dehydrogenase and glycine decarboxylase complexes. The predicted protein product of this gene has significant identity to the lipoyl ligase B of both Escherichia toll and Kluyveromyces lactis. A strain harbouring a null allele of this S. cerevisiae gene is respiratory deficient due to inactive pyruvate dehydrogenase, and is unable to utilise glycine as a sole nitrogen source.
The activation of hepatic kinase mechanistic target of rapamycin complex 1 (mTORC1) is implicated in the development of obesity related metabolic disorders. This study investigated the metabolic sequelae of mTORC1 hyperactivation in human hepatoma cells and the lipid-regulating mechanisms of two short-chain fatty acids; 4-phenylbutyric acid (PBA) and (R)-α-lipoic acid (LA). We created three stable cell lines that exhibit low, normal or high mTORC1 activity. mTORC1 hyperactivation induced the expression of lipogenic (DGAT1 and DGAT2) and lipoprotein assembly (MTP and APOB) genes thereby raising cellular triacylglyceride (TG) and exacerbating secretion of apoB-containing TG-rich lipoproteins. LYS6K2, a specific inhibitor of the p70 S6 kinase branch of mTORC1 signaling, reversed these effects. PBA and LA decreased secreted TG through distinct mechanisms. PBA repressed apoB expression (both mRNA and protein) and lowered secreted TG without mitigation of mTORC1 hyperactivity or activation of AMPK. LA decreased cellular and secreted TG by attenuating mTORC1 signaling in an AMPK independent manner. LA did not regulate apoB expression but led to the secretion of apoB-containing TG-poor lipoproteins by repressing the expression of lipogenic genes, FASN, DGAT1, and DGAT2. Our studies provide new mechanistic insight into the hypolipidemic activity of PBA and LA in the context of mTORC1 hyperactivation, and suggest that the short-chain fatty acids may aid in the prevention and treatment of hypertriglyceridemia.
IntroductionWater-Soluble VitaminsFat-Soluble VitaminsConcluding Remarks
Elizabeth S. Billgren was raised in Bemus Point, NY where she graduated from Maple Grove Jr./Sr. High School in 2001 with the intention of pursuing a degree in theater. She began studies at Jamestown Community College (Jamestown, NY) where she became interested in science and graduated in 2003 with an AA in liberal arts and humanities and an AS in mathematics and science. She went on to earn a B.S. from The State University of New York at Fredonia in biology in 2005. She was first introduced to laboratory research by Dr. Wayne Yunghans and received a grant from the Biology Research Endowment Fund to complete a research project in cell biology during the summer of 2004. She then attended The Pennsylvania State University (University Park, PA) and earned an M.S. in biochemistry, microbiology, and molecular biology in the laboratory of Dr. Squire Booker in 2008. Elizabeth is currently employed by United Chemical Technologies, Inc. (Lewistown, PA) as an analytical chemist.
Considering the current obesity epidemic in the United States (>100 million adults are overweight or obese), the prevalence of hypertriglyceridemia is likely to grow beyond present statistics of ∼30% of the population. Conventional therapies for managing hypertriglyceridemia include lifestyle modifications such as diet and exercise, pharmacological approaches, and nutritional supplements. It is critically important to identify new strategies that would be safe and effective in lowering hypertriglyceridemia. α-Lipoic acid (LA) is a naturally occurring enzyme cofactor found in the human body in small quantities. A growing body of evidence indicates a role of LA in ameliorating metabolic dysfunction and lipid anomalies primarily in animals. Limited human studies suggest LA is most efficacious in situations where blood triglycerides are markedly elevated. LA is commercially available as dietary supplements and is clinically shown to be safe and effective against diabetic polyneuropathies. LA is described as a potent biological antioxidant, a detoxification agent, and a diabetes medicine. Given its strong safety record, LA may be a useful nutraceutical, either alone or in combination with other lipid-lowering strategies, when treating severe hypertriglyceridemia and diabetic dyslipidemia. This review examines the current evidence regarding the use of LA as a means of normalizing blood triglycerides. Also presented are the leading mechanisms of action of LA on triglyceride metabolism.
Osteoporosis is a chronic disease associated with decreased bone density that afflicts millions of people worldwide. Current pharmacological treatments are limited, costly, and linked to several negative side effects. These factors are driving current interest in the clinical use of naturally occurring bioactive compounds to mitigate bone loss. Alpha-lipoic acid, a potent antioxidant and essential member of mitochondrial dehydrogenases, has shown considerable promise as an antiosteoclastogenic agent due to its potent reactive oxygen species-scavenging capabilities along with a proven clinical safety record. Collectively, current data indicate that alpha-lipoic acid protects from bone loss via a 2-pronged mechanism involving inhibition of osteoclastogenic reactive oxygen species generation and upregulation of redox gene expression.
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An acetonitrile–salt stacking method was established for the assay of lipoic acid (LA) in biological samples. Samples were deproteinized with acetonitrile at a final concentration of 60 % (v/v) and then injected hydrodynamically at 3.45 × 103 Pa for 180.0 s. The optimum background electrolyte was found to be 90.0 mmol L−1 pH 9.1 borate buffer. LA could be detected within 35 min at +7.0 kV with satisfactory repeatability (relative standard deviations, RSDs, of migration times and peak areas were both below 10 % for intraday and interday; n = 6/9) and a relatively low limit of detection of ca. 0.5 μmol L−1.
The present study deals with the development of a method for the quantitative determination of lipoic acid in a dietary supplement preparation. A rapid capillary electrophoretic method is developed using UV detection at 208 nm. Although lipoic acid is only weakly UV-absorbing, at this wavelength it could be detected with sufficient sensitivity with an LOD and LOQ of 0.8 and 2.5 μg/ml, respectively. Analysis time was less than 9 min. The compound was extracted from tablets with a recovery of 98.3% and a precision of 2.8% RSD.
We simulated the docking of α-lipoic acid (α-LA) in β-cyclodextrin (β-CD) using two models. We considered in this study complexes formed by 1:1 host–guest stoichiometry in vacuo and in aqueous phase, using PM6, DFT and ONIOM2 hybrid calculations. The results obtained with PM6 method clearly indicate that the complexes formed are energetically favored with or without solvent, model 2 (α-LA entering the cavity of β-CD from its wide side by COOH group) is found more favored than model 1 (α-LA entering into the cavity of β-CD from its wide side by cyclic group), the preference being greater in the case of ONIOM2 calculations. In addition, NBO analysis gives that mutual interactions between the donor and acceptor orbitals of α-lipoic acid and β-CD plays an important role to the stabilization of such a complex. Finally, 1H nuclear magnetic resonance (NMR) chemical shifts of free and complexed α-LA were calculated by the Gauge-Including Atomic Orbital (GIAO) method and compared with available experimental data. The results of GIAO calculations were analyzed and discussed.
Synthesis of α-lipoic acid has been achieved by a simple sequence of reactions. The synthesis highlights the use of α-chloroesters in a Reformatsky reaction. The intermediate keto acid is an intermediate from which both isomers of lipoic acid can be prepared.
A simple and reliable TLC method for analysis of -lipoic acid ( LA) with post-chromatographic derivatisation with palladium(II) chloride immersion reagent has been developed and evaluated. Separation of LA was performed on 20 cm x 10 cm RPTLC plates with 2-propanol-methanol-acetone-water-acetic acid 6: 4: 2: 8: 0.2 (v/v) as mobile phase. Yellow complexes formed in situ were scanned at 375 nm. The migration distance of LA was 43.0 mm. The relationship between peak area and amount of LA applied was evaluated by use of linear (1.0-3.0 mu g per band) and second-degree polynomial ( 0.5-5.0 mu g per band) regression functions. The correlation coefficient (r = 0.999), the limit of quantification (0.39 mu g per band), recovery (98.5-105.2%), and precision (1.8-2.9%) obtained by use of the procedure were satisfactory. The method was used for analysis of LA in several drug formulations and selected dietary supplement preparations. The LA content was 99.5-101.0% in the drug formulations, 98.8-99.5% in three of five dietary supplements tested, and 48.0-185.0% in two other dietary supplements.
Direct electrochemistry of alpha‐lipoic acid (ALA) was performed at a glassy carbon electrode using cyclic, differential pulse and square wave voltammetry over a wide range of pH. The oxidation of ALA is an irreversible process, pH independent, and involves the charge transfer of one electron. The diffusion coefficient of ALA was calculated from the results obtained at pH 6.9 in 0.1 M phosphate buffer and was shown to be D0=1.1×10 cm s. The limits of detection (LOD) and quantification (LOQ) calculated from the results obtained at this pH are 1.8 and 6.1 µM, respectively.The lipoic acid content in two dietary supplements samples, a syrup containing ALA and capsules of ALA, has been determined directly at the glassy carbon electrode by differential pulse voltammetry using the standard addition method.
Turkey is one of the richest countries in terms of flora in Europea and
Middle East. The aim of this study, to arouse the interest to Gagea Salisb
species which is part of our flora. Therefore, Gagea species was investigated
around Muğla province in 2000-2006. It has been found 8 species, one of them is endemic (G. bithynica Pascher) and the other one is new record for
C2 square(Muğla(G. bohemica (Zauschn.) Schultes & Schultes). Since this
region usually hilly and rocky, Gagea species didn't exposed to
anthropogenic factors. But G. fibrosa (Desf.) Schultes & Schultes fil
(Kotekli village) and G. juliae Pascher (Yaraş village and Kavaklidere
Region) species situations are different from others. Area of these species
are getting smaller because of increase of the instruction activities in these
fields, all localities are high and hilly places and grass down. This situation
may cause to danger for the survival of these species during the time. In the
future, increasing of the investigation to understand these species
phytochemical structure and to prove phytochemical structure of them may
cause to interest of pharmacologist to use Gagea Salisb species to prepare
the medicine.
IntroductionWater-Soluble VitaminsFat-Soluble VitaminsConcluding Remarks
Relative α-lipoic acid content of diabetic livers was considerably less than that of normal livers as determined by gas chromatography.
It was not possible to detect any dihydrolipoic acid in the livers. Biochemical abnormalities such as hyperglycaemia, ketonemia,
reduction in liver glycogen and impaired incorporation of [2-14C] -acetate into fatty acids in alloxan diabetic rats were brought to near normal levels by the oral or intraperitoneal administration
of dihydrolipoic acid. The effect of α-lipoic acid was comparable to that of dihydrolipoic acid in reducing the blood sugar
levels of diabetic rabbits during a glucose tolerance test.
The results suggest that the mode of action of lipoic acid was through stimulation of pyruvate dehydrogenase.
Intraperitoneal administration of lipoic acid (10 mg/100 g) does not effect changes in serum insulin levels in normal and
alloxan diabetic rats, while normalising increased serum pyruvate, and impaired liver pyruvic dehydrogenase characteristic
of the diabetic state. Dihydrolipoic acid has been shown to participate in activation of fatty acids with equal facility as
coenzyme A. Fatty acyl dihydrolipoic acid however is sparsely thiolyzed to yield acetyl dihydrolipoic acid. Also acetyl dihydrolipoic
acid does not activate pyruvate carboxylase unlike acetyl coenzyme A. The reduced thiolysis of Β-keto fatty acyl dihydrolipoic
acid esters and the lack of activation of pyruvic carboxylase by acetyl dihydrolipoic acid could account for the antiketotic
and antigluconeogenic effects of lipoic acid
Zero-current potential measurements (gold electrode) are suitable for continuously following the oxidation-reduction reactions of exogenic lipoic acid during Escherichia coli bacterial growth. This paper relates to a mathematical modeling of the experimental time-courses of potential.First, an empirical mathematical relation was obtained in vitro (i.e. in a sterile culture broth) between the zero-current potential and the concentrations of electroactive species that coexist and prevail in vivo (i.e. during the cultures).Secondly, a system of simple kinetic equations was proposed to express the metabolic, physical or chemical processes responsible for the in vivo time evolutions of the concentrations of electroactive species, from which the time evolution of the electrode potential during the cultures was obtained. Most of these equations have been standardized by direct measurements. Numerical values could be applied to the remaining parameters of the model by comparing the computer-simulated time-courses of potential with experimental potential-time signals.The model properly fitted the experimental reality. It substantiated a theoretical correlation between the time evolutions of potential and the reductive activity of cultures by means of growth parameters relative to the population of organisms and transport or consumption parameters relative to the bacterial cell.
Lipoic acid is a sulfur-containing cofactor required for the function of several multienzyme complexes involved in the oxidative decarboxylation of alpha-keto acids and glycine. Mechanistic details of lipoic acid metabolism are unclear in eukaryotes, despite two well defined pathways for synthesis and covalent attachment of lipoic acid in prokaryotes. We report here the involvement of four genes in the synthesis and attachment of lipoic acid in Saccharomyces cerevisiae. LIP2 and LIP5 are required for lipoylation of all three mitochondrial target proteins: Lat1 and Kgd2, the respective E2 subunits of pyruvate dehydrogenase and alpha-ketoglutarate dehydrogenase, and Gcv3, the H protein of the glycine cleavage enzyme. LIP3, which encodes a lipoate-protein ligase homolog, is necessary for lipoylation of Lat1 and Kgd2, and the enzymatic activity of Lip3 is essential for this function. Finally, GCV3, encoding the H protein target of lipoylation, is itself absolutely required for lipoylation of Lat1 and Kgd2. We show that lipoylated Gcv3, and not glycine cleavage activity per se, is responsible for this function. Demonstration that a target of lipoylation is required for lipoylation is a novel result. Through analysis of the role of these genes in protein lipoylation, we conclude that only one pathway for de novo synthesis and attachment of lipoic acid exists in yeast. We propose a model for protein lipoylation in which Lip2, Lip3, Lip5, and Gcv3 function in a complex, which may be regulated by the availability of acetyl-CoA, and which in turn may regulate mitochondrial gene expression.
1. The synthesis of dibutylchloromethyltin chloride, a new covalent inhibitor of the mitochondrial ATP synthase [oligomycin-sensitive ATPase (adenosine triphosphatase)] complex is described, together with a method for preparing dibutylchloro[(3)H]methyltin chloride. 2. Studies with the yeast mitochondrial oligomycin-sensitive ATPase complex show that dibutylchloromethyltin chloride inhibits both the membrane-bound enzyme and also the purified Triton X-100-dispersed preparation. 3. F(1)-ATPase is not inhibited even at 500nmol of dibutylchloromethyltin chloride/mg of protein, and the general inhibitory properties are similar to those of triethyltin, oligomycin and dicyclohexylcarbodi-imide, known energy-transfer inhibitors of oxidative phosphorylation. 4. Binding studies with yeast submitochondrial particles show that dibutylchloromethyltin chloride antagonizes the binding of triethyl[(113)Sn]tin, indicating that there is an interaction between the two inhibitor-binding sites. 5. Unlike triethyltin, inhibition by dibutylchloromethyltin chloride is due to a covalent interaction which titrates a component of the inner mitochondrial membrane present at a concentration of 8-9nmol/mg of protein. 6. All of the labelled component can be extracted with chloroform/methanol (2:1, v/v), and sodium dodecyl sulphate/polyacrylamide-gel electrophoresis of the chloroform/methanol extract indicates that the labelled component has an apparent mol.wt. of 6000-8000. However, t.l.c. reveals the presence of only one labelled component which is lipophilic and non-protein and is distinct from the free inhibitor, mitochondrial phospholipids and the dicyclohexylcarbodi-imide-binding protein (subunit 9). 7. Inhibition of mitochondrial ATPase and oxidative phosphorylation is correlated with specific interaction with a non-protein lipophilic component of the mitochondrial inner membrane which is proposed to be a co-factor or intermediate of oxidative phosphorylation.
Lipoic acid can be detected and assayed manometrically, at levels of 1 to 10 m% by measuring oxygen utilization of a pyruvate dehydrogenase apoenzyme system in Streptococcus ]aecalis, strain 10C1. The apoenzyme is prepared by growing the cells in a synthetic medium deficient in lipoic acid. This assay can be performed with cell suspensions 2 or more conveniently with vacuum-dried cells 3 in which the necessary enzymes are stable for a period ranging up to five years. A curve relating the rates of oxygen consumption to the amounts of pure lipoic acid serves as standard. Dihydrolipoic acid, reduced lipoic acid, 6,8-dithioloctanoic acid (DMO), can be prepared chemically from lipoic acid—the disulfide—by chemical reduction. Acetyl dihydrolipoic acid, (+)-6-acetyl-6,8-dithioloctanoic acid (6-Ac-DMO), can be prepared chemically from (–)-dihydrolipoic acid by enzymatic acetylation.
The principal physiological function of lipoic acid appears to be its action as coenzyme in the oxidative decarboxylation of α-oxo acids. In order to find out whether lipoic acid is essential to higher organisms, structurally modified lipoic acids were prepared as potential antagonists. Only 4-oxalipoamide promotes the growth of S. faecalis 8043; the other substances tested are inactive or else inhibit growth. In experiments to determine the substrate specificity of lipoamide oxidoreductase and its inhibition by structurally modified lipoic acids, no strong inhibitors were discovered. –; Ring strain, spectra, and ring cleavage of compounds containing the 1, 2-dithiolane ring are discussed in relation to the biological function and the reactivity of lipoic acid.
(+)-α-Lipoic acid has been synthesized. DL-3-Acetylthio-7-carbethoxyheptanoic acid was converted into its acid chloride which was reduced with sodium borohydride to yield a mixture of reduction products. Alkaline hydrolysis of this mixture produced 8-hydroxy-6-thioloctanoic acid. Replacement of the hydroxyl with sulfhydryl followed by oxidation gave DL-α-lipoic acid. Resolution of DL-3-acetylthio-7-carbethoxyheptanoic acid supplied the starting materials for the synthesis of the (+)- and (-)-α-lipoic acids.
Several observations are recorded concerning the behavior of α-lipoic acid (I) in solution or in a fluid state. The preparation of the benzhydrylammonium salts of (+)-, (-)- and DL-α-lipoic acid is described, and the synthesis of DL-α-lipoamide (IV) and DL-α-lipol (V) from the intermediate acid chloride, DL-α-lipoyl chloride (III), is reported.
This chapter describes the turbidimetric and polarographic assays for lipoic acid using mutants of Escherichia coli. The mutants of Escherichia coli that are unable to synthesize lipoic acid are characterized in the chapter. Their lipoic acid requirement is approximately 0.5ng/ml for half-maximal aerobic growth, but in glucose minimal medium, this requirement can be spared or replaced by acetate plus succinate or lysine plus methionine. However, with succinate as carbon and energy source, the requirement for lipoic acid is absolute, and acetate, glucose, lysine, and methionine are without effect. The organisms respond to α- and to β-lipoic acid but not to lipoamide, lipoylglycinamide, or conjugated derivatives present in biological material unless they are first hydrolyzed. In addition, the suspensions of organisms grown in the absence of lipoic acid cannot oxidize pyruvate or α-ketoglutarate unless this cofactor is supplied.
Several microorganisms were examined for the content of lipoic acid by using a strain of Streptococcus faecalis deficient in this coenzyme. In comparison to this, the specific activity levels were determined for the pyruvate: ferredoxin oxidoreductase and the pyruvate dehydrogenase complex, which both catalyse the cleavage of pyruvate and coenzyme A to acetyl coenzyme A, CO2 and two reducing equivalents. Anabaena cylindrica, Chlorobium, Clostridium pasteurianum and kluyveri, where only the pyruvate: ferredoxin oxidoreductase can be demonstrated, were found to contain minute levels of lipoic acid. Thus lipoic acid does not appear to be a cofactor of the decarboxylation catalysed by the pyruvate: ferredoxin oxidoreductase. On the other hand, the amount of lipoic acid is at least ten times higher in Ankistrodesmus, Chlamydomonas, Anacystis, Micrococcus, Azotobacter and Escherichia coli which have the dehydrogenase complex.
SUMMARY Streptococcus faecalis IOCI and 6783 grew anaerobically on glucose in media lacking lipoic acid and acetate, and assays with Streptococcus cremoris KH indicated that extracts of S. faecalis IOCI contained lipoic acid activity equivalent to 200 ng of a-D,L-lipoic acid/mg protein. The Yslllcose value for a continuous culture of strain IOCI grown anaerobically in lipoic acid-deficient medium was 37.2, about the same as that for IOCI in lipoic acid-sufficient medium (35.5). Strains IOCI and 6783 meta- bolized low concentrations of glucose (I to 8 pmol/ml) non-homolactically and produced per mol of glucose about 1.0 mol lactate, 1-2 mol acetate, and 0.4 mol ethanol. Appreciable amounts of pyruvate, formate, glycerol, acetoin, diacetyl, and 2,3-butylene glycol were not detected. The Yglucoee values for strain IOCI were lower (25, 17 and 21, respectively) if IO-~ M-arsenite or 0.5 % acetate was added or the pH value in the fermentor was decreased to 5.3. With acetate added or the pH value reduced, the glucose was metabolized mostly to lactate. Results indicate that S. Jizecalis IOCI used the pyruvate-dehydrogenase-enzyme complex rather than the phosphoroclastic mechanism in metabolizing anaerobically to acetate approxi- mately half of the pyruvate produced from low concentrations of glucose.
Extracts from the nitrogen fixing blue-green algaAnabaena cylindrica catalyse a pyruvate decarboxylation, which is dependent on ferredoxin and stimulated by coenzyme A, ATP and a SH-protecting compound. This pyruvate clastic reaction is completely reversible: The net synthesis of pyruvate requires CO2, acetyl-coenzyme A and reduced ferredoxin. Preparations fromAnabaena cylindrica also catalyse the exchange reaction between CO2 and the carboxyl group of pyruvate. Thus the enzyme fromAnabaena cylindrica has essentially all the characteristics known for the pyruvate: ferredoxin oxidoreductase from anaerobic bacteria.
The activity of the pyruvate: ferredoxin oxidoreductase inAnabaena grown with ammonia is lower than one-fifth of that in cells grown with molecular nitrogen or nitrate as the nitrogen source. From this, it will be concluded that a physiological role of the reaction is to generate reduced ferredoxin for the assimilation of nitrogen to ammonia. The pyruvate synthesis is probably not physiological inA. cylindrica.
In addition, extracts fromA. cylindrica also catalyse a ferredoxin dependent decarboxylation of α-ketoglutarate. It is not yet clear, whether this ketoglutarate cleavage has a function inA. cylindrica.
A selection procedure was developed for the isolation of mutants of Escherichia coli which require lipoic acid, acetate+succinate, or lysine + methionine for aerobic growth with glucose. The properties of the mutants requiring lipoic acid (lip
-) were compared with those of suc
- mutants which lack α-ketoglutarate dehydrogenase and require succinate or lysine + methionine. Genetic analysis by conjugation with Hfr and F’ donors indicated that the genetic loci of some 36 independently isolated lip
- mutants are confined to a small segment of the E. coli chromosome between the purE and suc sites. By using phage P1 no cotransduction of lip with suc, glt A or purE could be demonstrated, but suc, gltA and gal were cotransducible and the relative order of these sites was determined.
1The synthesis of the pyruvate dehydrogenase complex in Escherichia coli K12 is inducible, and pyruvate is the metabolite causing induction.In a mutant lacking the activities of phosphoenol pyruvate synthase and dihydrolipoamide transacetylase (a component of the pyruvate dehydrogenase complex) pyruvate is no longer significantly metabolized under certain growth conditions. In such a mutant pyruvate as well as α-ketobutyrate induces pyruvate dehydrogenase synthesis. Decreasing inducing activity was found with increasing chain length (or branching) of homologous α-ketoacids.Apo-pyruvate dehydrogenase is produced when thiamine requiring strains are grown in the absence of thiamine, i. e., the pyruvate dehydrogenase complex formed is inactive. Such strains, possessing wild type pyruvate dehydrogenase complex, accumulate pyruvate when growing without thiamine. Only with thiamine starvation could the synthesis of wild type pyruvate dehydrogenase complex be fully induced. Also, in merodiploid strains homogenotic for the wild type ace locus a gene dosage effect was found only upon thiamine deprivation. Inducibility is thus separable from enzymatic activity; enzymatically active pyruvate dehydrogenase complex removes the effector inducing its synthesis thus preventing full induction during normal growth conditions.No evidence was found for an indirect action by pyruvate, i. e., pyruvate does not induce by inhibition or activation of some other reaction (in the citric acid cycle or glycolysis), thereby causing an increased concentration of another metabolite which is the true inducer.2It is possible that pyruvate serves not only as inducing metabolite for pyruvate dehydrogenase complex synthesis but also as repressing metabolite for isocitric lyase synthesis (or for a glyoxylate cycle operon).3Both ace mutants and wild type possess pyruvate oxidase, an acetate producing enzyme system independent of the pyruvate dehydrogenase complex. It appears that the activity of the oxidase is too low to provide enough acetate which can replace that normally produced via pyruvate dehydrogenase complex. A physiological role for the oxidase has not been found.
Differential rates of incorporation of sugars, organic acids, and amino acids during autotrophic growth of several blue-green algae and thiobacilli have been determined. In obligate autotrophs (both blue-green algae and thiobacilli), exogenously furnished organic compounds make a very small contribution to cellular carbon; acetate, the most readily incorporated compound of those studied, contributes about 10% of newly synthesized cellular carbon. In Thiobacillus intermedius, a facultative chemoautotroph, acetate contributes over 40% of newly synthesized cellular carbon, and succinate and glutamate almost 90%. In the obligate autotrophs, carbon from pyruvate, acetate, and glutamate is incorporated into restricted groups of cellular amino acids, and the patterns of incorporation in all five organisms are essentially identical. These patterns suggest that the tricarboxylic acid cycle is blocked at the level of alpha-ketoglutarate oxidation. Enzymatic analyses confirmed the absence of alpha-ketoglutarate dehydrogenase in the obligate autotrophs, and also revealed that they lacked reduced nicotinamide adenine dinucleotide oxidase, and had extremely low levels of malic and succinic dehydrogenase. These enzymatic deficiencies were not manifested by the two facultative chemoautotrophs examined. On the basis of the data obtained, an interpretation of obligate autotrophy in both physiological and evolutionary terms has been developed.
1. Diaminopimelate epimerase from a soluble extract of Bacillus megaterium N.C.I.B. 7581 was purified about 25-fold by fractionation with ammonium sulphate and chromatography on calcium phosphate gel-cellulose. The product was impure but was unstable on further purification. 2. Quantitative assay methods for the enzyme were devised in which meso- or ll-diaminopimelic acid may be the substrate. 3. Between 25 degrees and 45 degrees at pH7.0 enzyme action leads to an equilibrium mixture containing 65% meso-isomer and 35% ll-isomer. 4. The initial rate of epimerization was 2-3 times as fast with ll-diaminopimelic acid as substrate as with the meso-isomer; a number of other amino acids were not racemized by the enzyme. The Michaelis constants at 37 degrees were 6.7mm (ll-isomer) and 100mm (meso-isomer); with both substrates enzyme activity was maximal at pH7-8. The relative rates of epimerization of ll-diaminopimelic acid at 25 degrees , 37 degrees and 45 degrees were 0.77:1.00:1.15. 5. A thiol compound (of which 2,3-dimercaptopropan-1-ol was the most effective) was needed as an activator of the purified enzyme. 6. Carbonylbinding reagents and several other compounds did not inhibit diaminopimelate epimerase. 7. Pyridoxal phosphate did not stimulate enzymic activity even in preparations that had been almost completely freed of derivatives of vitamin B(6) (as shown by microbiological assay).
1. The breakdown of pyruvate was examined in whole cells and cell-free extracts of the blue-green alga Anabaena variabilis. Decarboxylation of specifically labelled pyruvate indicated a similar metabolic route to that of acetate, although no pyruvate oxidase was present. Pyruvate: ferredoxin oxidoreductase was detected in cell-free extracts and after DEAE-cellulose treatment, addition of ferredoxin was necessary for pyruvate decarboxylation; acetyl-CoA was the first product of the reaction.2. The formation of acetyl-CoA from pyruvate required ATP; kinetic evidence as well as experiments with [γ-32P]ATP indicated that this serves as an activator and not as a substrate in the reaction. The importance of this reaction in the control of biosynthesis and nitrogen fixation is discussed.
By repeated subcultivation of the parent strain Pediococcus cerevisiae ATCC8081 in the presence of methicillin, a substrain P. cerevisiae 8081 CRD was developed which grew only when the partly defined medium was supplemented with methicillin or certain other penicillins. The methicillin-dependent strain was not highly resistant to methicillin and grew only in the presence of a limited range of concentrations of it (about 10-300 µg./ml.). Even with an optimal growth concentration of methicillin (50-100 µg./ml.), the dependent organisms grew less well than did the parent strain without methicillin and showed a longer lag period before growth became visible.
Although other derivatives of 6-aminopenicillanic acid (but not of 7-aminocephalosporanic acid) supported moderate growth of Pediococcus cerevisiae 8081 CRD, none was as effective as methicillin, nor was there marked cross-resistance to any of these other derivatives. The more potent the penicillin as inhibitor of growth of the parent strain, the smaller was the optimal concentration needed to support growth of the dependent sub-strain. Several other antibiotics were ineffective as growth factors.
When methicillin was hydrolysed with acid, alkali or pencillinase, activity as a growth factor was lost. During growth of the parent strain and of the methicillin-dependent strain at pH 6.5 material was produced in the medium which was able to destroy the antibiotic potency of methicillin or other penicillins. The substance was not an enzyme, and the presence of methicillin was not necessary to induce its formation by the parent strain.
The methicillin-dependent strain did not grow, with or without methicillin, when sodium acetate was omitted from the medium. No substance of known chemical structure was found which could replace acetate for growth. Pediococcus cerevisiae 8081 CRD grew rapidly in the absence of both acetate and methicillin when the medium was supplemented with yeast extract However, when acetate was present as well as yeast extract, methicillin again became necessary for growth of the dependent organisms.
Wittenberger, Charles L. (National Institute of Dental Research, U.S. Public Health Service, Bethesda, Md.), and Ann S. Haaf. Lactate-degrading system in Butyribacterium rettgeri subject to glucose repression. J. Bacteriol.
88
896–903. 1964.—The ability of Butyribacterium rettgeri to utilize lactate as the main energy source for growth requires the formation of a lactate-degrading system. The precise nature of this system is unknown, but preliminary evidence suggests that cellular acquisition of lactate-decomposing activity involves the formation of a nonpyridine nucleotide-linked lactic dehydrogenase. This enzyme, which can couple lactate oxidation to the reduction of ferricyanide [K3Fe(CN)6-lactic de-hydrogenase (LDH)], is absent from glucose-grown cells; this observation appears to account for the inability of such cells to decompose lactate even though they may form lactate from glucose. The formation of K3Fe(CN)6-LDH in growing cultures requires the addition of lipoic acid to the medium, and is repressed by glucose, pyruvate, or fructose. When any of the latter substrates are included in the growth medium with lactate, nicotinamide adenine dinucleotide-linked LDH activity is present in cells at markedly higher levels than it is in cells grown on lactate alone.
SUMMARY The synthesis of an inducible amidase by Pseudomonas mginosu 86021~ was studied in cultures growing exponentially in succinate medium. Induction by both the substrate inducer acetamide, and the non-substrate inducer N-acetylacetamide, was repressed by cyanoacetamide. Induction by 10-2M-N-acetylacetamide was significantly repressed by lO-%-cyano- acetamide, but repression of induction by 10-3~-acetamide required a tenfold excess of cyanoacetamide. Amidase synthesis in a medium in which acetamide was the sole carbon+nitrogen source was also repressed by cyanoacetamide, which under these conditions inhibited the growth of non-induced bacteria. Several tricarboxylic acid cycle intermediates, and related compounds, repressed amidase synthesis in exponentially growing organisms. Catabolite repression by propionate in succinate medium was decreased by increasing the concentration of acetamide. These findings are discussed in relation to general theories of regulation of microbial enzyme synthesis.
Since 1922 when Wu proposed the use of the Folin phenol reagent for
the measurement of proteins (l), a number of modified analytical pro-
cedures ut.ilizing this reagent have been reported for the determination
of proteins in serum (2-G), in antigen-antibody precipitates (7-9), and
in insulin (10).
Although the reagent would seem to be recommended by its great sen-
sitivity and the simplicity of procedure possible with its use, it has not
found great favor for general biochemical purposes.
In the belief that this reagent, nevertheless, has considerable merit for
certain application, but that its peculiarities and limitations need to be
understood for its fullest exploitation, it has been studied with regard t.o
effects of variations in pH, time of reaction, and concentration of react-
ants, permissible levels of reagents commonly used in handling proteins,
and interfering subst.ances. Procedures are described for measuring pro-
tein in solution or after precipitation wit,h acids or other agents, and for
the determination of as little as 0.2 y of protein.
1. The type of metabolism adopted by Pseudomonas oxalaticus during growth on a variety of carbon sources was studied. 2. The only substrate upon which autotrophic growth was observed is formate. 3. In mixtures of formate and those substrates upon which the organism can grow faster than on formate, e.g. succinate, lactate or citrate, heterotrophic metabolism results. 4. In mixtures of formate and those substrates upon which the organism can grow at a similar rate to that on formate, e.g. glycollate or glyoxylate, the predominant mode of metabolism adopted is heterotrophic utilization of the C(2) substrate coupled with oxidation of formate as ancillary energy source. 5. P. oxalaticus grows on oxalate 30% slower than on formate. In mixtures of formate and oxalate, the predominant mode of metabolism adopted is autotrophic utilization of formate coupled with oxidation of oxalate as ancillary energy source. 6. In mixtures of formate and those substrates upon which the organism grows at a much lower rate than on formate, e.g. glycerol and malonate, the predominant mode of metabolism adopted is autotrophic utilization of formate. 7. It is concluded that synthesis of the enzymes involved in autotrophic metabolism is controlled by a combination of induction and metabolite repression.
1. The activities of the enzymes of the citric acid cycle, the glyoxylate by-pass and some other enzymes acting on the substrates of these cycles have been measured at the pH of the yeast cell during the aerobic growth of yeast on different carbon sources and in different growth media. 2. Sugars induced an anaerobic type of metabolism as measured by ethanol production. Glucose was much more effective in inducing the anaerobic pathways than was galactose. The production of ethanol by cells grown on pyruvate was very small. 3. Glucose was also a more effective repressor than was galactose of the citric acid-cycle enzymes but both were equally effective in repressing almost completely the enzymes of the glyoxylate by-pass. 4. Disappearance of the sugars from the growth medium resulted in an increase in the activities of the enzymes of the citric acid cycle and in the appearance of substantial activities of the enzymes of the glyoxylate cycle. By contrast, the activities of purely biosynthetic enzymes (glutamate-oxaloacetate transaminase, NADP(+)-linked glutamate dehydrogenase) and of pyruvate decarboxylase were decreased. 5. The 2-oxoglutarate-oxidase system was found to be the least active enzyme of the citric acid cycle. 6. The regulatory control at the levels of pyruvate and acetaldehyde and the control of the citric acid cycle are discussed.
Studies on the biosynthesis of lipoic acid
- L J Reed
- T Okaichi
- I Nakanishi
Synthesis of metabolic intermediates In: The biology of blue-green algae
- A J Smith
Synthesis of metabolic intermediates
- A J Smith
- A. J. Smith
Ein Regulationsmechanismus beim Abbau der Brenztraubensäure durch Escherichia coli
- U Henning
- U. Henning
Discussion. Chemical and photochemical reactions of thioctic acid and related disulfides
- E L R Stokstad
- E. L. R. Stokstad