An assay for δ-aminolevulinic acid synthetase based on a specific, semiautomatic determination of picomole quantitites of δ-[14C]aminolevulinate

Bishop, David F.; Wood, William F.

doi:10.1016/0003-2697(77)90669-8

Cited by 11 publications

(4 citation statements)

References 37 publications

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“…Cells were harvested from 5-ml samples, suspended in 1 ml of 50 mM Hepes, pH 7.5/2 mM dithiothreitol/0.2 mM phenylmethylsulfonyl fluoride/2 mM EDTA/aprotinin (3 ,ug/ml), and frozen. The thawed lysate was centrifuged at 10,000 x g for 30 min, and the supernatant was assayed for ALAS activity as described (22) and with detection by Ehrlich's reagent after reaction with ethyl acetoacetate (23). One unit of activity is that amount of enzyme required to catalyze the production of 1 nmol of 8-aminolevulinate per hour under the conditions of the assay.…”

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confidence: 99%

Enzymatic defect in "X-linked" sideroblastic anemia: molecular evidence for erythroid delta-aminolevulinate synthase deficiency.

Cotter

Baumann

Bishop

1992

Proc. Natl. Acad. Sci. U.S.A.

140

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Recently, the human gene encoding erythroid-specific -aminolevulinate synthase was localized to the chromosomal region Xp2l-Xq2l, identifying this gene as the logical candidate for the enzymatic defect causing "X -linked" sideroblastic anemia. To investigate this hypothesis, the 11 exonic coding regions of the -aminolevulinate synthase gene were amplified and sequenced from a 30-year-old Chinese male with a pyridoxine-responsive form of X-linked sideroblastic anemia. A single T --A transition was found in codon 471 in a highly conserved region of exon 9, resulting in an ile -+ Asn substitution. This mutation interrupted contiguous hydrophobic residues and was predicted to transform a region of (3-sheet structure to a random-coil structure. Prokaryotic expression of the normal and mutant cDNAs revealed that the mutant construct expressed low levels of enzymatic activity that required higher concentrations of pyridoxal 5'-phosphate to achieve maximal activation than did the normal enzyme. The amino acid substitution occurred in the exon containing the putative pyridoxal 5'-phosphate binding site and may account for the reduced ability of the cofactor to catalyze the formation of 6aminolevulinic acid.The sideroblastic anemias are a clinically heterogeneous group of disorders resulting from inherited or acquired causes (1, 2). The inherited types are X chromosome-linked or autosomal, and the acquired forms are secondary to chemical induction or to inflammatory disease or are idiopathic. Patients in these classifications can be further subdivided into those who are responsive to pyridoxine and those who are refractory. Sideroblastic anemia is classified as a disorder of heme synthesis since the activities of certain heme biosynthetic enzymes were reported to be deficient (3), whereas globin synthesis was normal. Since many patients had reduced 8-aminolevulinate synthase [ALAS; succinylCoA:glycine C-succinyltransferase (decarboxylating); EC 2.3.1.37] enzymatic activity in bone marrow (4, 5) and because pyridoxal 5'-phosphate (PLP), the pyridoxine metabolite, is a required cofactor, ALAS has been suggested as a candidate gene for mutations leading to some types of sideroblastic anemia (6).The human genes encoding the housekeeping and erythroid forms of ALAS were isolated and characterized (7-9). Although the two genes predicted proteins with 59% amino acid identity, they were mapped to different chromosomes by somatic cell hybrid and in situ hybridization methods. The housekeeping gene mapped to chromosome 3p2l, and the erythroid gene was localized to the X chromosomal region Xp2l-Xq2l (9-13). The X-chromosomal assignment suggested that the erythroid form of ALAS (designated ALAS2) might be the enzymatic defect in "X-linked" sideroblastic anemia (XLSA).To investigate this hypothesis, the erythroid ALAS2 coding regions were amplified and sequenced from a male affected with pyridoxine-responsive XLSA. In this communication, an exonic point mutation in the ALAS2 gene that predicted the substitution of asparagine f...

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confidence: 99%

Enzymatic defect in "X-linked" sideroblastic anemia: molecular evidence for erythroid delta-aminolevulinate synthase deficiency.

Cotter

Baumann

Bishop

1992

Proc. Natl. Acad. Sci. U.S.A.

140

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“…ALA eluted from the latter was then converted to the pyrrole and isolated by another round of anion-exchange chromatography. Bishop and Wood (1977) used a semi-automated amino acid analyzer and counted the radioactivity in the fraction corresponding to authentic ALA. Brooker et al (1982) performed cation-exchange chromatography as described by Ebert et al (1970), but converted ALA in the final eluate to the pyrrole, extracted it into ethyl acetate, evaporated the extract to dryness, and counted the radioactivity in the entire dried residue. Minaga et al (1978) reported that the major co-eluting contaminant was a breakdown product of succinyl-CoA, succinyl-cysteamine thioester, formed via the action of an amidase or peptidase.…”

Section: 27mentioning

confidence: 99%

Measurement of ALA Synthase Activity

1999

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“…To remove possible low levels of impurities in the radioactive succinate which may contaminate the iso lated 5-ALA [1,4], the original material can be diluted to 0.1 ml of 50 mmol/1 sodium acetate buffer, pH 3.9, applied to a Dowex AG50W-X8 ion exchange column (pre-equilibrated with the same buffer) and washed through with water. The [2,3-l4C]-succinate is ad justed to pH 7.4 with 0.1 mol/1 NaOH [5] and then added to the 5-mmol/l succinate solution to the re quired specific activity.…”

Section: Methodsmentioning

confidence: 99%

“…However, with this assay procedure, a number of problems have arisen, particularly in the isolation of [I4C]-8-ALA free from other contaminants. Several groups [1,2,26,28] have reported that 6-ALA isolated after the enzyme assay contains varying amounts of unknown metabolites making quantitation of 5-ALA synthase activity difficult. Bishop and Wood [1] showed that one of these con taminants comigrated with 5-ALA on cation exchange resins and was only resolved by partition chromatography in an analytical amino acid analyzer.…”

Section: Introductionmentioning

confidence: 99%