delta-Aminolevulinate synthetases in the liver cytosol fraction and mitochondria of mice treated with allylisopropylacetamide and 3,5-dicarbethoxyl-1,4-dihydrocollidine. (1/369)

Hepatic delta-aminolevulinate (ALA) synthetase was induced in mice by the administration of allylisopropylacetamide (AIA) and 3,5-dicarbethoxy-1,4-dihydrocollidine (DDC). In both cases, a significant amount of ALA synthetase accumulated in the liver cytosol fraction as well as in the mitochondria. The apparent molecular weight of the cytosol ALA synthetase was estimated to be 320,000 by gel filtration, but when the cytosol ALA synthetase was subjected to sucrose density gradient centrifugation, it showed a molecular weight of 110,000. In the mitochondria, there were two different sizes of ALA synthetase with molecular weights of 150,000 and 110,000, respectively; the larger enzyme was predominant in DDC-treated mice, whereas in AIA-treated mice and normal mice the enzyme existed mostly in the smaller form. When hemin was injected into mice pretreated with DDC, the molecular size of the mitochondrial ALA synthetase changed from 150,000 to 110,000. The half-life of ALA synthetase in the liver cytosol fraction was about 30 min in both the AIA-treated and DDC-treated mice. The half-life of the mitochondrial ALA synthetase in AIA-treated mice and normal mice was about 60 min, but in DDC-treated mice the half-life was as long as 150 min. The data suggest that the cytosol ALA synthetase of mouse liver is a protein complex with properties very similar to those of the cytosol ALA synthetase of rat liver, which has been shown to be composed of the enzyme active protein and two catalytically inactive binding proteins, and that ALA synthetase may be transferred from the liver cytosol fraction to the mitochondria with a size of about 150,000 daltons, followed by its conversion to enzyme with a molecular weight of 110,000 within the mitochondria. The process of intramitochondrial enzyme degradation seems to be affected in DDC-treated animals.  (+info)

Four new mutations in the erythroid-specific 5-aminolevulinate synthase (ALAS2) gene causing X-linked sideroblastic anemia: increased pyridoxine responsiveness after removal of iron overload by phlebotomy and coinheritance of hereditary hemochromatosis. (2/369)

X-linked sideroblastic anemia (XLSA) in four unrelated male probands was caused by missense mutations in the erythroid-specific 5-aminolevulinate synthase gene (ALAS2). All were new mutations: T647C, C1283T, G1395A, and C1406T predicting amino acid substitutions Y199H, R411C, R448Q, and R452C. All probands were clinically pyridoxine-responsive. The mutation Y199H was shown to be the first de novo XLSA mutation and occurred in a gamete of the proband's maternal grandfather. There was a significantly higher frequency of coinheritance of the hereditary hemochromatosis (HH) HFE mutant allele C282Y in 18 unrelated XLSA hemizygotes than found in the normal population, indicating a role for coinheritance of HFE alleles in the expression of this disorder. One proband (Y199H) with severe and early iron loading coinherited HH as a C282Y homozygote. The clinical and hematologic histories of two XLSA probands suggest that iron overload suppresses pyridoxine responsiveness. Notably, reversal of the iron overload in the Y199H proband by phlebotomy resulted in higher hemoglobin concentrations during pyridoxine supplementation. The proband with the R452C mutation was symptom-free on occasional phlebotomy and daily pyridoxine. These studies indicate the value of combined phlebotomy and pyridoxine supplementation in the management of XLSA probands in order to prevent a downward spiral of iron toxicity and refractory anemia.  (+info)

Properties of 5-aminolaevulinate synthetase and its relationship to microsomal mixed-function oxidation in the southern armyworm (Spodoptera eridania). (3/369)

1. Activity of 5-aminolaevulinate synthetase was measured in the midgut and other tissues of the last larval instar of the southern armyworm (Spodoptera eridania Cramer, formerly Prodenia eridania Cramer). 2. Optimum conditions for measuring the activity were established with respect to all variables involved and considerable differences from those reported for mammalian enzyme preparations were found. 3. Maximum activity (20 nmol/h per mg of protein) occurs 18-24 h after the fifth moult and thereafter decreases to trace amounts as the larvae age and approach pupation. 4. Synthetase activity was rapidly induced by oral administration (in the diet) of pentamethylbenzene, phenobarbital, diethyl 1,4-dihydro-2,4,6-trimethylpyridine-3, 5-dicarboxylate, and 2-allyl-2-isopropylacetamide. 5. Puromycin inhibited the induction of synthetase by pentamethylbenzene. 6. Induction of 5-aminolaevulinate synthetase correlated well with the induction of microsomal N-demethylation of p-chloro-N-methylaniline, except for phenobarbital, which induced the microsomal oxidase relatively more than the synthetase.  (+info)

Pre-steady-state reaction of 5-aminolevulinate synthase. Evidence for a rate-determining product release. (4/369)

5-Aminolevulinate synthase (ALAS) is the first enzyme of the heme biosynthetic pathway in non-plant eukaryotes and the alpha-subclass of purple bacteria. The pyridoxal 5'-phosphate cofactor at the active site undergoes changes in absorptive properties during substrate binding and catalysis that have allowed us to study the kinetics of these reactions spectroscopically. Rapid scanning stopped-flow experiments of murine erythroid 5-aminolevulinate synthase demonstrate that reaction with glycine plus succinyl-CoA results in a pre-steady-state burst of quinonoid intermediate formation. Thus, a step following binding of substrates and initial quinonoid intermediate formation is rate-determining. The steady-state spectrum of the enzyme is similar to that formed in the presence of 5-aminolevulinate, suggesting that release of this product limits the overall rate. Reaction of either glycine or 5-aminolevulinate with ALAS is slow (kf = 0.15 s-1) and approximates kcat. The rate constant for reaction with glycine is increased at least 90-fold in the presence of succinyl-CoA and most likely represents a slow conformational change of the enzyme that is accelerated by succinyl-CoA. The slow rate of reaction of 5-aminolevulinate with ALAS is 5-aminolevulinate-independent, suggesting that it also represents a slow isomerization of the enzyme. Reaction of succinyl-CoA with the enzyme-glycine complex to form a quinonoid intermediate is a biphasic process and may be irreversible. Taken together, the data suggest that turnover is limited by release of 5-aminolevulinate or a conformational change associated with 5-aminolevulinate release.  (+info)

Phylogenetic analysis of the 5-aminolevulinate synthase gene. (5/369)

The evolution of 5-aminolevulinate synthase (ALS) was studied by acquiring sequence data and generating phylogenetic trees. Gene sequences were already available for a variety of vertebrates (which have both a housekeeping and an erythroid form of the gene), fungi, alpha-proteobacteria, and one protist and one protostome. In order to generate representative trees, ALS sequence data were acquired from various deuterostomes and protostomes. The species and tissues selected for study were beluga whale liver, hagfish blood, sea urchin gonadal tissue, cuttlefish hepatopancreas, horseshoe crab hepatopancreas, and bloodworm blood. The new sequences and those previously published were examined for the presence of heme-regulatory motifs (HRMs) and iron-responsive elements (IREs). The HRMs are present in almost all eukaryotic species, which suggests their fundamental role in the regulation of ALS. The IREs are present in all vertebrate erythroid forms of ALS, which indicates that in those animals, expression of the erythroid form of the enzyme and, hence, hemoglobin production can be influenced by the intracellular content of iron. The new sequences were aligned with previously reported ALS sequences, and phylogenetic analyses were performed. The resulting trees provided evidence regarding the timing of the gene duplication event that led to the two forms of the ALS gene in vertebrates. It appears that the housekeeping and erythroid forms of ALS probably arose before the divergence of hagfish from the deuterostome line leading to the vertebrates. The data also add to the evidence indicating that alpha-proteobacteria are the nearest contemporary relatives of mitochondria.  (+info)

Respiratory uncoupling induces delta-aminolevulinate synthase expression through a nuclear respiratory factor-1-dependent mechanism in HeLa cells. (6/369)

Nuclear respiratory factor (NRF)-1 appears to be important for the expression of several respiratory genes, but there is no direct evidence that NRF-1 transduces a physiological signal into the production of an enzyme critical for mitochondrial biogenesis. We generated HeLa cells containing plasmids allowing doxycycline-inducible expression of uncoupling protein (UCP)-1. In the absence of doxycycline, UCP-1 mRNA and protein were undetectable. In the presence of doxycycline, UCP-1 was expressed and oxygen consumption doubled. This rise in oxygen consumption was associated with an increase in NRF-1 mRNA. It was also associated with an increase in NRF-1 protein binding activity as determined by electrophoretic mobility shift assay using a functional NRF-1 binding site from the delta-aminolevulinate (ALA) synthase promoter. Respiratory uncoupling also caused a time-dependent increase in protein levels of ALA synthase, an early marker for mitochondrial biogenesis. ALA synthase induction by respiratory uncoupling was prevented by transfecting cells with an oligonucleotide antisense to the region of the NRF-1 initiation codon; a scrambled oligonucleotide with the same base composition had no effect. Respiratory uncoupling increases oxygen consumption and lowers energy reserves. In HeLa cells, uncoupling also increases ALA synthase, an enzyme critical for mitochondrial respiration, but only if translatable mRNA for NRF-1 is available. These data suggest that the transcription factor NRF-1 plays a key role in cellular adaptation to energy demands by translating physiological signals into an increased capacity for generating energy.  (+info)

Characterization of the rhodobacter sphaeroides 5-aminolaevulinic acid synthase isoenzymes, HemA and HemT, isolated from recombinant Escherichia coli. (7/369)

The hemA and hemT genes encoding 5-aminolaevulinic acid synthase (ALAS) from the photosynthetic bacterium Rhodobacter sphaeroides, were cloned to allow high expression in Escherichia coli. Both HemA and HemT appeared to be active in vivo as plasmids carrying the respective genes complemented an E. coli hemA strain (glutamyl-tRNA reductase deficient). The over-expressed isoenzymes were isolated and purified to homogeneity. Isolated HemA was soluble and catalytically active whereas HemT was largely insoluble and failed to show any activity ex vivo. Pure HemA was recovered in yields of 5-7 mg x L-1 of starting bacterial culture and pure HemT at 10 mg x L-1 x HemA has a final specific activity of 13 U x mg-1 with 1 unit defined as 1 micromol of 5-aminolaevulinic acid formed per hour at 37 degrees C. The Km values for HemA are 1.9 mM for glycine and 17 microM for succinyl-CoA, with the enzyme showing a turnover number of 430 h-1. In common with other ALASs the recombinant R. sphaeroides HemA requires pyridoxal 5'-phosphate (PLP) as a cofactor for catalysis. Removal of this cofactor resulted in inactive apo-ALAS. Similarly, reduction of the HemA-PLP complex using sodium borohydride led to > 90% inactivation of the enzyme. Ultraviolet-visible spectroscopy with HemA suggested the presence of an aldimine linkage between the enzyme and pyridoxal 5'-phosphate that was not observed when HemT was incubated with the cofactor. HemA was found to be sensitive to reagents that modify histidine, arginine and cysteine amino acid residues and the enzyme was also highly sensitive to tryptic cleavage between Arg151 and Ser152 in the presence or absence of PLP and substrates. Antibodies were raised to both HemA and HemT but the respective antisera were not only found to bind both enzymes but also to cross-react with mouse ALAS, indicating that all of the proteins have conserved epitopes.  (+info)

A photosensitising adenovirus for photodynamic therapy. (8/369)

We have developed a new approach to photodynamic therapy based on adenoviral transduction of the rate-limiting enzyme in heme synthesis. Conventional phototherapy uses porphyrin-based chemical photosensitisers, including delta-aminolaevulinic acid (ALA) which is converted to protoporphyrin IX (PpIX) by the enzymes of the heme biosynthetic pathway. The lack of a specific mechanism for targeting chemical photosensitisers and PpIX to tumour cells means that therapeutic irradiation can damage normal tissue and exposure to sunlight following treatment can cause severe burns. The rate limiting enzyme in PpIX synthesis is ALA-synthase (ALA-S). We have developed a new yeast vector system for manipulation of the adeno- virus genome and used it to construct a virus expressing a mutant form of ALA-S lacking the iron response elements which regulate ALA-S translation and the heme regulatory motifs which regulate import of ALA-S into mitochondria. The virus induces a large increase in PpIX expression and confers photosensitivity on cultured cells. Unlike conventional photodynamic therapy, a viral approach makes it possible to restrict photosensitivity by biological rather than purely physical or chemical means. As with HSV thymidine kinase, ALA-S expression is a general mechanism for sensitisation to a therapeutic agent which can easily be adapted to whatever means of gene delivery is most effective.  (+info)

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