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PubMed1982

The effects of succinylacetone (4,6-dioxoheptanoic acid) on delta-aminolevulinate synthase activity and the content of heme in monolayers of chick embryo liver cells.

Succinylacetone was shown to inhibit aminolevulinate dehydratase (5-aminolevulinate hydro-lyase (adding 5-aminolevulinate and cyclizing), EC 4.2.1.24) to reduce cellular heme and porphyrins and to induce delta-aminolevulinate synthase (succinyl-CoA:glycine C-succinyltransferase (decarboxylating), EC 2.3.1.37) in monolayers of chick embryo liver cells. Marked synergistic effects on delta-aminolevulinate synthase activity were obtained by combining succinylacetone with levulinate and porphyrogenic drugs. The time course of delta-aminolevulinate synthase activity showed a delayed synergistic response.

5-Aminolevulinate Synthetase↗
PubMed1982

delta-Aminolevulinic acid formation. Purification and properties of alanine:4,5-dioxovalerate, aminotransferase and isolation of 4,5-dioxovalerate from Clostridium tetanomorphum.

L-Alanine:4,5-dioxovalerate aminotransferase, the enzyme that catalyzes the transamination between alanine and 4,5-dioxovalerate to yield delta-aminolevulinate and pyruvate, has been purified from extracts Clostridium tetanomorphum by acetone precipitation and successive tetanomorphum by acetone precipitation and successive chromatography on Sephadex G-150, hydroxyapatite, Octyl-Sepharose, and SP-Sephadex C-50. The enzyme is pure by the criterion of disc gel electrophoresis with varying polyacrylamide concentrations. It is dimeric, and has an apparent molecular weight of 111,000. Each molecule contains 2 molecules of pyridoxal 5-phosphate. The apparent Km values for 4,5-dioxovalerate and L-alanine are 0.26 and 1.96 mM, respectively. In addition to alanine, glutamate also is an effective amino group donor. The enzyme is inhibited by various keto acids as well as by inhibitors of pyridoxal phosphate-containing enzymes. It was possible to show that 4,5-dioxovalerate is formed by cultures of C. tetanomorphum when grown in the presence of 0.2 M levulinate, an inhibitor of 5-aminolevulinate dehydratase.

Amino Acids↗
PubMed2002

The malate dehydrogenase of Ralstonia eutropha and functionality of the C(3)/C(4) metabolism in a Tn5-induced mdh mutant.

The Tn5-induced mutant VG12 of Ralstonia eutropha HF39, which was isolated in this study, revealed an interesting phenotype: it grew on fructose and pyruvate as well as autotrophically like the wild-type, whereas growth on tricarboxylic acid intermediates and glyoxylic acid was reduced, and no growth occurred if acetate, propionate or levulinate were provided as carbon source. Tn5 was mapped in a gene encoding an NAD(H)-dependent malate dehydrogenase (MDH), and MDH activity was strongly diminished in VG12. Furthermore, the mdh gene was cloned, sequenced and heterologously expressed in Escherichia coli, conferring significantly higher specific MDH activity to the recombinant strain. The phenotype of VG12 sheds light on the C(3)/C(4) metabolism of R. eutropha, which mediates between the Entner-Doudoroff pathway and the tricarboxylic acid cycle (TCC), demonstrating that enzymes catalyzing the conversion of C(3) and C(4) metabolites can circumvent the metabolic disruption of the TCC in VG12 and that the phosphoenolpyruvate carboxykinase serves a dual and important function.

Carbon↗
PubMed1975

Preparation and Baeyer-Villiger Reaction of certain 2-carbalkoxycyclopropyl methyl ketones.

Treatment of mixed anhydrides derived from cis- and trans-2-carbobenzyloxycyclopropanecarboxylic acids and ethyl chloroformate with ethoxymagnesio di-tert-butyl malonate and subsequent treatment of the resulting adducts with p-toluenesulfonic acid afforded cis- and trans-benzyl-2-acetylcyclopropanecarboxylates in good to excellent yields, with retention of the original stereochemistry of the systems. These methyl ketones and an open chain congener, benzyl levulinate, were inert toward m-chloroperbenzoic acid. The cis-isomer and benzyl levulinate underwent normal Baeyer-Villiger reactions mediated by trifluoroperacetic acid, forming moderate yields of the acetate ester insertion products.

Cyclopropanes↗
PubMed1977

Calcium levulinate medication. A pitfall in the diagnosis of organic acidurias.

Five children, who received calcium levulinate (calcium 4-oxopentanoate) intravenously in pharmacological doses excreted in the following 24-h period both 4-oxopentanoic acid (3.5--11.0 mg/24 h) and 4-hydroxypentanoic acid (4.5--10.4 mg/24 h). Attention is drawn to the fact that in gas chromatographic-mass spectrometric systems these compounds closely resemble the two acids found in children with beta-ketothiolase deficiency.

Acetyl-CoA C-Acyltransferase↗
PubMed1992

A general strategy for random insertion and substitution mutagenesis: substoichiometric coupling of trinucleotide phosphoramidites.

Results from a number of recent studies suggest that amino acid insertion mutations may provide an important alternative to substitution mutations for modifying protein structures and functional activities. To facilitate the use of single-amino acid insertions, we have developed a general strategy for inducing random, in-phase codon insertions across a defined segment of a cloned gene. In brief, a mixture of blocked and protected trinucleotide phosphoramidites is coupled at substoichiometric levels after every third monomer coupling on a conventional solid-state synthesizer. From the heterogeneous mixture of oligonucleotide sequences thus generated, those oligonucleotides that have acquired a single additional codon are purified by urea/PAGE. By using equimolar amounts of GCT and GGT trinucleotides in the oligonucleotide synthesis plus standard oligonucleotide-directed mutagenesis techniques, we have induced as many as 13 different single alanine and glycine insertion mutations into the gene for staphylococcal nuclease in one experiment. On replacement of the 5'-dimethoxytrityl blocking group on the trinucleotide phosphoramidite with an acid-stable blocking group, such as levulinate or fluoren-9-ylmethoxycarbonyl (Fmoc), this same strategy of substoichiometric couplings at codon boundaries should permit the synthesis of complex pools of oligonucleotides for the introduction, with constant efficiency, of every type of amino acid substitution at each codon across a gene segment.

Base Sequence↗
PubMed1988

Porphyrinostatic effect of isonicotinic acid hydrazide on porphyric chick embryo and yeast.

1. The common feature of "inducible" forms of hepatic porphyria is an increased activity of delta-amino-levulinate/ALA/synthase. 2. As a potential therapeutic procedure, isonicotinic acid hydrazide/INH/as inhibitor of this enzyme, was applied and found to show a porphyrinostatic effect, depending on its concentration, both in yeast and porphyric chick embryo models. 3. Moreover, pyridoxalphosphate/PDX/eliminated the porphyrinostatic effect of INH, depending again on its concentration. 4. Histochemical tests did not demonstrate any significant functional or morphological changes in the chick embryo hepatocytes following INH administration.

5-Aminolevulinate Synthetase↗
PubMed1988

Biosynthesis of delta-aminolevulinate in greening barley leaves. IX. Structure of the substrate, mode of gabaculine inhibition, and the catalytic mechanism of glutamate 1-semialdehyde aminotransferase.

Glutamic acid 1-semialdehyde hydrochloride was synthesized and purified. Its prior structural characterization was extended and confirmed by 1H NMR spectroscopy and chemical analyses. In aqueous solution at pH 1 to 2 glutamic acid 1-semialdehyde exists in a stable hydrated form, but at pH 8.0 it has a half-life of 3 to 4 min. Spontaneous degradation of the material at pH 8.0 generated some undefined condensation products, but coincidentally a significant amount isomerized to 5-aminolevulinate. At pH 6.8 to 7.0, glutamate 1-semialdehyde is sufficiently stable to permit routine and reproducible assay for glutamate 1-semialdehyde aminotransferase activity. Only about 20% of the enzyme extracted from chloroplasts was sensitive to inactivation by gabaculine with no pretreatment. However, when the enzyme was exposed to 5-aminolevulinate, levulinate or 4,5-dioxovalerate in the absence of glutamate 1-semialdehyde, it was completely inactivated by gabaculine; 4,6-dioxoheptanoate had no effect on the enzyme. These results lead to the hypothesis that the aminotransferase exists in the chloroplast in a complex with pyridoxamine phosphate, which must be converted to the pyridoxal form before it can form a stable adduct with gabaculine. We propose that the enzyme catalyzes the conversion of glutamate 1-semialdehyde to 5-aminolevulinate via 4,5-diaminovalerate.

Aminolevulinic Acid↗
PubMed1976

The biosynthesis of delta-aminolevulinic acid from the intact carbon skeleton of glutamic acid in greening barley.

The formation of delta-aminolevulinic acid in mammals birds, yeast and some bacteria is known to take place by the ALA-synthetase assisted coupling of glycine with succinylCoA. Plants, however, form the bulk of their delta-aminolevulinate in another way. We present evidence here that the intact, 5-carbon chain of glutamate becomes that of delta-aminolevulinate. Greening barley was fed specifically labelled glutamate and levulinate to create a pool of labelled delta-aminolevulinate. Levulinate, a competitive inhibitor of ALA-dehydratase, prevents the metabolism of delta-aminolevulinate. The carbon chain of the labelled delta-aminolevulinate was broken into formaldehyde and succinate by periodate to determine the position of the label. It was found that the C1, carboxyl carbon of glutamate becomes the amino-bearing (C5) carbon of delta-aminolevulinate and forms the formaldehyde on cleavage. delta-Aminolevulinate formed from C3,4-labelled glutamate bears its label in the succinate cleaved fragment. We conclude that during the light induced development of the plastid in barley the carbon chain of delta-aminolevulinate is formed from the intact chain of glutamate. ALA-synthetase catalyzed the formation of delta-aminolevulinate from glycine and succinylCoA cannot play a quantitively important role in the formation of chlorophyll.

Aminolevulinic Acid↗
PubMed1975

Agarose linked adenosine and guanosine-5'-monophosphate; a new general method for the coupling of ribonucleotides to polymers through their cis-diols.

Condensation of the cis-diol of adenosine (1a) or guanosine (1b) with ethyl levulinate led to the acetal-esters 2a or 2b. Phosphorylation with phosphorous oxide trichloride converted them into their 5'-monophosphates. On alkaline hydrolysis the acetal-esters 2a,b as well as their 5'-monophosphates gave the carboxylic acid derivatives 3a,b and 4a,b, respectively. Condensation of these acid derivatives with aminohexyl-agarose (5) using water soluble N-ethyl-N'-(3'-dimethyl-aminopropyl)-carbodiimide hydrochloride as coupling reagent led to the new polymers 6a/6b and 7a/7b. This method of preparing resin linked adenosine and guanosine derivatives should be generally applicable to any ribonucleoside or ribonucleotide with a cis-diol. Because of the widespread occurence of these molecules, particularly as cofactors of enzymes, the new polymers might be useful tools for the purification of certain classes of enzymes by affinity chromatography.

Adenosine↗
PubMed1996

Inhibition of hemoglobin expression by heterologous production of nitric oxide synthase in the K562 erythroleukemic cell line.

Recent studies have indicated that nitric oxide may affect iron metabolism through disruption of the iron-sulfur complex of iron regulatory protein-1, a translational regulator. In the present study, we report that heterologous expression of murine macrophage nitric oxide synthase (NOS-2) in the human erythroleukemic K562 cell line results in constitutive production of nitric oxide associated with inhibition of hemoglobin expression. K562 cells were transfected with an episomally-maintained, hygromycin-selectable expression vector bearing the coding region of NOS-2. Constitutive NOS expression was detected by Western blotting of cell lysates and by the accumulation of nitrite in the culture media. Although NOS-transfected cells grew more slowly than control cells, they were able to maintain constitutive expression of NOS and production of nitric oxide for more than 1 month following transfection. The hemoglobin content of NOS-transfected K562 cells was less than one-fifth that of control cells, but increased markedly if NOS inhibitor was included in the culture media. The nitric oxide-mediated inhibition of hemoglobin expression was reversed by supplementing the culture media with 20 mumol/L hemin or 0.5 mmol/L 5-amino-levulinate, indicating that nitric oxide did not directly inhibit hemoglobin synthesis, but likely acted on a step in heme synthesis. mRNA levels for globin and erythroid aminolevulinic acid synthase (eALAS) were the same in both NOS-transfected and control cells. Our observations indicate that hemoglobin expression is inhibited by nitric oxide in NOS-transfected K562 cells by posttranscriptional repression of eALAS, the first enzyme of the heme biosynthetic pathway. The most likely mechanism is a nitric oxide-mediated translational repression of eALAS, as was recently demonstrated for ferritin synthesis. These observations further illustrate the potential for endogenously produced nitric oxide to regulate cellular posttranscriptional events. In particular, our observations may be relevant to the role of nitric oxide in anemia and lowered blood hemoglobin concentrations that are associated with chronic infections, such as tuberculosis or parasitic disease.

5-Aminolevulinate Synthetase↗
PubMed1977

Cytochrome c turnover in rat skeletal muscles.

Exercise induces an increase in cytochrome c concentration in skeletal muscle. This adaptation provides an approach to studying the turnover of cytochrome c that avoids the problem of reutilization encountered with isotopic tracers. The half-life of cytochrome c was estimated from the time course of the increase in its concentration to a new, higher, steady state level in response to exercise training, and from the decrease in cytochrome c after cessation of exercise. The half-time of the increase in cytochrome c concentration was approximately 6 days, while the half-time of the decrease was 7 to 8 days in the fast red and slow red types of muscle. The finding that the half-times of the increase and of the decrease in cytochrome c concentration are similar provides evidence that the exercise-induced increase in cytochrome c is due to an increase in its rate of synthesis. These half-times are much shorter than those obtained with isotopic tracers. It had been thought that the heme precursor delta-aminolevulinate is not reutilized. However, the half-time of the decrease in radioactivity of cytochrome c labeled with delta-aminol[14C]levulinate was 45 days, and increased to 60 days in response to exercise, in fast red muscle. The half-time of the decrease in radioactivity of cytochrome c labeled with [(3H)]leucine in gastrocnemius muscle was shorter than with delta-amino[14C]levulinate (18 days compared to 38 days). These results indicate that when delta-amino(14C)levulinate is used to label heme, reutilization is a serious problem in skeletal muscle.

Aminolevulinic Acid↗
PubMed1990

Oral loading of homogentisic acid in controls and in obligate heterozygotes for hereditary tyrosinemia type I.

Homogentisic acid (HGA) (50 mg/kg) was given orally to 22 obligate heterozygotes for hereditary tyrosinemia type 1 (HT) and to 11 controls. After 1 h the mean +/- standard error (SE) plasma level of HGA was 30.42 +/- 1.41 micrograms/ml in carriers and 19.29 +/- 1.62 in controls. Mean +/- SE fasting delta-amino-levulinate dehydratase (delta-ALD) was 40.05 +/- 1.79 m microM/min/g Hb in carriers, much lower than the 60.81 +/- 5.11 found in controls. After 3 h this difference in levels of delta-ALD remained, with mean +/- SE values of 25.70 +/- 2.89 m microM/min/g Hb in carriers, compared with 48.83 +/- 5.37 in controls. Three-hour mean +/- SE excretion of fumarylacetone "equivalent" [FAc] in urine in carriers, 51.597 +/- 5.580 micrograms/mg/creatinine, was significantly higher than the 27.941 +/- 5.916 in controls. Three-hour excretion of succinylacetone "equivalent" [SAc] was also significantly higher in the urine of carriers. FAc in 3-h urine was identified by thin-layer chromatography and confirmed by gas chromatography/mass spectrometry. Multivariate stepwise discriminant analysis showed that the inclusion order of significant variables was as follows: HGA levels at 1 hr, fasting level of delta-ALD, residual level of HGA at 3 h, and 3-h excretion of [FAc]. Non-significant variables were HGA tolerance, levels of delta-ALD at 3 h, sex, and 3-h excretion of [SAc].(ABSTRACT TRUNCATED AT 250 WORDS)

Amino Acid Metabolism, Inborn Errors↗
PubMed1995

Biosynthetic preparation of isotopically labeled heme.

An efficient method for the preparation of isotopically enriched heme has been developed. This method utilizes a commercially available bacterial host and plasmid, into which a synthetic gene encoding for rat liver outer mitochondrial membrane cytochrome b5, a heme-binding protein, has been inserted. The method described in this report utilizes the efficient synthesis of the cytochrome b5 polypeptide together with the enhanced biosynthesis of heme brought about by addition of the first committed precursor in heme biosynthesis, delta-aminolevulinic acid. Apocytochrome b5 sequesters heme as the macrocycle is being synthesized in order to form holocytochrome b5, thus avoiding toxic concentrations of free macrocycle in the cell. Relatively high concentrations of free heme in the cell have been shown to stimulate excretion of heme precursors such as coproporphyrinogen and uroporphyrinogen (W. F. Harris III, R. S. Burkhalter, W. Lin and R. Timkovich, (1993) Bioorg. Chem. 21, 209-220), therefore causing isotopic dilution of the labeled material. The heme obtained using this methodology was determined to be > 85% enriched. Because the heme in cytochrome b5 is not covalently attached to the polypeptide, it can be extracted and used in other applications. Use of glutamate, a precursor of delta-amino-levulinate biosynthesis in Escherichia coli, did not result in high levels of isotopic incorporation into heme, thus pointing out to the importance of using a labeled precursor that is committed to heme biosynthesis in order to obtain high levels of isotopic labeling.

Aminolevulinic Acid↗
PubMed1988

Structure, turnover, and heme-mediated suppression of the level of mRNA encoding rat liver delta-aminolevulinate synthase.

lambda gt11 cDNA libraries were constructed with poly(A)+ RNA preparations from both porphyric chicken and rat livers. A cDNA which encodes chicken hepatic delta-aminolevulinate synthase was cloned by screening with an anti-chicken liver delta-aminolevulinate synthase antibody. Using this cDNA as a probe, cDNAs encoding the entire protein coding sequence of rat hepatic delta-aminolevulinate synthase were then cloned. The complete nucleotide sequences of the cDNAs have been determined. The result predicts that the rat hepatic pre-delta-aminolevulinate synthase comprises 642 amino acids. We measured the half-life of the hepatic delta-aminolevulinate synthase mRNA by RNA blot hybridization analysis using allylisopropylacetamide-induced porphyric rats as an experimental model and the rat cDNA as a hybridization probe. The half-life of the mRNA determined by the injection of alpha-amanitin is as short as 20 min. This value is significantly shorter than the estimated half-lives of most other mRNAs in the differentiated tissues of animals. The effect of hemin administration on the level of hepatic delta-amino-levulinate synthase mRNA was also examined. The half-disappearance time of the mRNA after the hemin administration was essentially the same as that determined by alpha-amanitin or actinomycin D, and no additive effect was observed between alpha-amanitin and hemin on the half-life determination. The results provide convincing evidence that heme inhibits the transcription of delta-aminolevulinate synthase mRNA.

5-Aminolevulinate Synthetase↗
PubMed1977

Effect of cobalt on the synthesis of liver microsomal cytochromes.

1. The amount of cytochrome b5 was not changed significantly by a single injection of cobalt (60 mg/kg body weight) or by daily injection of cobalt (30 mg/kg body weight) for 4 days or 8 days. On the other hand, the amount of cytochrome P-450 was depressed strongly by both cobalt treatments. 2. The incorporations of [3H]leucine as well as 5-amino[3H]levulinate into cytochrome b5 in cobalt-treated animals were almost the same as those in the controls 5 h after injections of the radioisotopes, whereas the radioactivity of heme labelled with 5-amino[3H]levulinate in microsomal residues after trypsin digestion, which would consist mainly of cytochrome P-450, was higher in the controls than in cobalt-treated animals after 5 h.

Aminolevulinic Acid↗