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H A Dailey

Publications and source records attributed to H A Dailey.

At least 37 records · Page 2Linked to original sources
PubMed1998

Erythroid 5-aminolevulinate synthase is required for erythroid differentiation in mouse embryonic stem cells.

We have examined the induction of the enzymes of the heme biosynthetic pathway during erythroid differentiation of mouse embryonic stem (ES) cells. Following transfer to appropriate medium all of the pathway enzymes are induced within three days. Unlike differentiating mouse erythroleukemia cells (Lake-Bullock, H. and Dailey, H.A. Mol Cell Biol 13:7122-7132, 1993), all of the enzymes appear to be induced simultaneously and not sequentially in differentiating ES cells. The role of erythroid 5-aminolevulinate synthase (ALAS-2) in this differentiation process was examined by disruption of the ALAS-2 gene. The targeting vector used for disruption replaced all of exons 4 to 6 with a selectable neomycin resistance gene. The resulting genetically modified (ALAS-2 knockout) cells, as well as normal ES cells were used to study induction of heme biosynthesis. Following 10 days of culture in methylcellulose media significant morphological differences between the embryoid bodies (EBs) of the two cell lines were observed. ES cells exhibited morphology of typical EBs with a dark field (blood island) in the center, while ALAS-2 knockout ES cells developed very poorly both in size and shape. At 8 days of differentiation, only 3% of all EBs contained visible erythropoietic cells (i.e., stained positively for hemoglobin) in the ALAS-2 knockout cell line, compared with 50% in ES cells. Most of the genes in the heme synthetic pathway were expressed to a stable level within 3 to 6 days after induction in normal ES cells, while the ALAS-2 knockout cell line failed to significantly increase the level of expression of these genes. Fetal beta-globin mRNA was not detectable in the differentiating ALAS-2 knockout cells, whereas mRNA for this gene was detected in normal ES cells within 3 days of differentiation. These results suggest that ALAS-2 is necessary for ES cell erythroid differentiation and that there is an interrelationship between heme and globin synthesis in differentiating ES cells.

5-Aminolevulinate Synthetase↗
PubMed1998

Molecular characterization of homozygous variegate porphyria.

Variegate porphyria (VP) is a low penetrance, autosomal dominant disorder that results from partial deficiency of protoporphyrinogen oxidase (PPOX) activity caused by mutation in the PPOX gene. The rare homozygous variant of VP is characterized by severe PPOX deficiency, onset of photosensitization by porphyrins in early childhood, skeletal abnormalities of the hand and, less constantly, short stature, mental retardation and convulsions. We have identified PPOX mutations on both alleles of five of the 11 unrelated patients with homozygous VP reported to date. Two patients were homoallelic for missense mutations (D349A and A433P), while three were heteroallelic. Functional analysis by prokaryotic expression showed that the D349A and A433P and one missense mutation in each of the three heteroallelic patients (G358R in two patients and A219KANA) preserved some PPOX activity (9.5-25% of wild-type). Mutations on the other allele of the heteroallelic patients abolished or markedly decreased activity. There was no relation between genotype assessed by functional analysis and the presence or severity of non-cutaneous manifestations. The mutations were absent from 104 unrelated patients with autosomal dominant VP. Our findings define the molecular pathology of homozygous VP and suggest that mild PPOX mutations occur in the general population but have very low or no clinical penetrance in heterozygotes.

Adolescent↗
PubMed1997

Expression and purification of mammalian 5-aminolevulinate synthase.

We have described a procedure for production and purification of recombinant, mature-length mouse ALAS-2. The fact that E. coli utilizes the C5 path for ALA production means that there is no problem with contamination of the recombinant ALAS-2 by host cell enzyme, such as one may have with a yeast expression system. While the detailed procedure produces enzyme in good yield with relatively common protein purification techniques, future expression systems may be developed to take advantage of the rapid purification achieved by the use of a 6-histidine (His6) aminoterminal tag and metal chelate chromatography. Such approaches in this laboratory with protoporphyrinogen oxidase, coproporphyrinogen oxidase, and uroporphyrinogen decarboxylase have resulted in the production and purification of enzymes whose kinetic and physical parameters are essentially identical to those of proteins lacking the His6 tag.

5-Aminolevulinate Synthetase↗
PubMed1997

Characteristics of human protoporphyrinogen oxidase in controls and variegate porphyrias.

Protoporphyrinogen oxidase (E.C.1.3.3.4) (PPO) catalyzes the penultimate step in the heme biosynthetic pathway. Deficiency in activity of this enzyme results in the human genetic disease variegate porphyria. Herein we detail the cloning, expression, purification and characterization of the normal and variegate porphyria forms of human PPO. The cDNA sequence for human ppo is approximately 1.8 kb in length and codes for a protein of 477 amino acids. This protein, which does not contain a typical cleavable mitochondrial targeting sequence, is approximately 51 kDa and contains a putative dinucleotide binding motif near the amino terminus. The active enzyme is a homodimer and contains an FAD. Attachment of a six his amino terminal tag allows for the rapid and efficient purification of approximately 10 mg of enzyme from one liter of E. coli culture. Three variegate porphyria mutant PPO enzymes were expressed and characterized. These mutations, R59W, R168C and A433P, result in decreased enzyme activity by causing a decrease in kcat without a significant change in Km for the substrate protoporphyrinogen IX. Purified R59W lacks the FAD cofactor which may be explained by the fact that this mutation resides within the dinucleotide binding motif of PPO.

Amino Acid Sequence↗
PubMed1997

Variegate porphyria in South Africa, 1688-1996--new developments in an old disease.

Variegate porphyria, an autosomal dominant inherited trait resulting in decreased activity of protoporphyrinogen oxidase, the penultimate haem biosynthetic enzyme, is characterised clinically by photosensitive skin disease and a propensity to acute neurovisceral crises. The disease has an exceptionally high frequency in South Africa, owing to a founder effect. The specific mutation in the protoporphyrinogen oxidase gene sequence which represents this founder gene has been identified. Genetic diagnosis is therefore now possible in families in whom the gene defect is known. However, the exact nature and degree of activity of the porphyria can only be determined by detailed quantitative biochemical analysis of excreted porphyrins. The relative contributions of the acute attack and the skin disease to the total disease burden of patients with variegate porphyria is not static, and in South Africa there have been significant changes over the past 25 years, with fewer patients presenting with acute attacks, leaving a greater proportion to present with skin disease or to remain asymptomatic with the diagnosis being made in the laboratory. The most common precipitating cause of the acute attack of VP is administration of porphyrinogenic drugs. Specific suppression of haem synthesis with intravenous haem arginate is the most useful treatment of a moderate or severe acute attack. Although cutaneous lesions are limited to the sun-exposed areas, management of the skin disease of VP remains inadequate.

Animals↗
PubMed1996

Human coproporphyrinogen oxidase is not a metalloprotein.

Coproporphyrinogen oxidase (CPO) (EC 1.3.3.3), the antepenultimate enzyme in the heme biosynthetic pathway, catalyzes the conversion of coproporphyrinogen III to protoporphyrinogen IX. Previously, based upon metal analysis and site-directed mutagenesis of purified recombinant enzyme, it has been suggested that CPO contains and requires copper for activity (Kohno, H., Furukawa, T., Tokunaga, R., Taketani, S., and Yoshinaga, T. (1996) Biochim. Biophys. Acta 1292, 156-162). To examine this putative metal site in human CPO, the cDNA encoding human CPO was engineered into an expression vector with a His6 tag at its amino terminus, and the protein was expressed in Escherichia coli and purified to apparent homogeneity using nickel-nitroliotriacetic acid resin. Activity of the purified protein was monitored by a coupled fluorometric assay that employed purified protoporphyrinogen oxidase to convert protoporphyrinogen to protoporphyrin, thereby allowing the direct fluorescent determination of protoporphyrin IX produced. CPO has an apparent Km of 0.6 microM and an apparent Kcat of 16 min-1 with coproporphyrinogen III as substrate. Metal analysis of the enzyme was carried out via ultraviolet and visible spectroscopy, inductively coupled plasma atomic emission spectroscopy metal analysis, and electron paramagnetic resonance spectroscopy. The data presented demonstrate that human CPO contains no metal center, that it is not stimulated in vitro by iron or copper, and that addition of these metals to cultures expressing the protein has no effect.

Amino Acid Sequence↗
PubMed1996

Site-directed mutagenesis and spectroscopic characterization of human ferrochelatase: identification of residues coordinating the [2Fe-2S] cluster.

The five cysteines closest to the carboxyl terminus of human ferrochelatase have been individually mutated to serine, histidine, or aspartate residues in an attempt to identify the protein ligands to the [2Fe-2S] cluster. Mutations of cysteines at positions 403, 406, and 411 (C403D, C403H, C406D, C406H, C406S, C411H, and C411S mutants) all resulted in inactive enzyme that failed to assemble the [2Fe-2S] cluster as judged by whole-cell EPR studies. In contrast, mutation of the cysteines at positions 360 and 395 to serines (C360S and C395S mutants) did not affect the enzymatic activity, and the resulting enzyme assembled a [2Fe-2S] cluster that was spectroscopically indistinguishable from the wild-type enzyme. The results indicate that three of the conserved cysteines in the 30-residue C-terminal extension of mammalian ferrochelatase are involved in ligating the [2Fe-2S] cluster. Resonance Raman and variable-temperature magnetic circular dichroism studies of heme-free preparations of human ferrochelatase are reported, and the spectra are best interpreted in terms of one non-cysteinyl, oxygenic ligand for the [2Fe-2S] cluster. Such anomalous coordination could account for the cluster lability compared to similar clusters with complete cysteinyl ligation and hence may be intrinsic to the proposed regulatory role for this cluster in mammalian ferrochelatases.

Amino Acid Sequence↗
PubMed1996

Protoporphyrinogen oxidase of Myxococcus xanthus. Expression, purification, and characterization of the cloned enzyme.

Protoporphyrinogen oxidase (EC 1.3.3.4) catalyzes the six electron oxidation of protoporphyrinogen IX to protoporphyrin IX. The enzyme from the bacterium Myxococcus xanthus has been cloned, expressed, purified, and characterized. The protein has been expressed in Escherichia coli using a Tac promoter-driven expression plasmid and purified to apparent homogeneity in a rapid procedure that yields approximately 10 mg of purified protein per liter of culture. Based upon the deduced amino acid sequence the molecular weight of a single subunit is 49,387. Gel permeation chromatography in the presence of 0.2% n-octyl-beta-D-glucopyranoside yields a molecular weight of approximately 100,000 while SDS gel electrophoresis shows a single band at 50,000. The native enzyme is, thus, a homodimer. The purified protein contains a non-covalently bound FAD but no detectable redox active metal. The M. xanthus enzyme utilizes protoporphyrinogen IX, but not coproporphyrinogen III, as substrate and produces 3 mol of H2O2/mol of protoporphyrin. The apparent Km and kcat for protoporphyrinogen in assays under atmospheric concentrations of oxygen are 1.6 microM and 5.2 min-1, respectively. The diphenyl ether herbicide acifluorfen at 1 microM strongly inhibits the enzyme's activity.

Amino Acid Sequence↗
PubMed1996

Function of the [2FE-2S] cluster in mammalian ferrochelatase: a possible role as a nitric oxide sensor.

Ferrochelatase (E.C. 4.99.1.1) is the terminal enzyme of the heme biosynthetic pathway, catalyzing the insertion of ferrous iron into protoporphyrin. In mammals the enzyme contains a labile [2Fe-2S] center. Although this cluster is absent in all prokaryotic, plant, and yeast ferrochelatases, its destruction or elimination from the mammalian enzyme results in loss of enzyme activity. In the current study we present data which clearly demonstrate that mammalian ferrochelatase is strongly inhibited by nitric oxide and that this effect is mediated via destruction of the [2Fe-2S] cluster. Carbon monoxide has no inhibitory effect, and yeast ferrochelatase, which lacks the [2Fe-2S] cluster, is not affected by NO (or CO). EPR and UV-visible absorption of purified recombinant human ferrochelatase provides evidence that NO is targeting the [2Fe-2S] center. UV-visible absorption spectroscopy of both human and murine recombinant ferrochelatase incubated with NO or the NO donor, S-nitroso-N-acetylpenicillamine (SNAP), indicate a rapid loss of the visible absorption spectrum of the [2Fe-2S] cluster. EPR studies of the resulting samples reveal the characteristic axial S = 1/2 resonance, g perpendicular = 2.033, and g parallel = 2.014 of a cysteinyl-coordinated monomeric iron-dinitrosyl cluster degradation product. Parallel spectroscopic studies of spinach ferredoxin, which also contains a [2Fe-2S] cluster, gave no indication of NO-induced cluster degradation under the same experimental conditions. Exposure of DMSO-induced murine erythroleukemia cells exposed to SNAP results in an initial decrease in heme production, suggesting that in vivo the cluster is rapidly destroyed. The potential physiological relevance of these data to the anemias that are found in individuals with chronic infections is discussed.

Animals↗
PubMed1996

Human protoporphyrinogen oxidase: expression, purification, and characterization of the cloned enzyme.

Protoporphyrinogen oxidase (E.C.1.3.3.4) catalyzes the oxygen-dependent oxidation of protoporphyrinogen IX to protoporphyrin IX. The enzyme from human placenta has been cloned, sequenced, expressed in Escherichia coli, purified to homogeneity, and characterized. Northern blot analysis of eight different human tissues show evidence for only a single transcript in all tissue types and the size of this transcript is approximately 1.8 kb. The human cDNA has been inserted into an expression vector for E. coli and the protein produced at high levels in these cells. The protein is found in both membrane and cytoplasmic fractions. The enzyme was purified to homogeneity in the presence of detergents using a metal chelate affinity column. The purified protein is a homodimer composed of subunits of molecular weight of 51,000. The enzyme contains one noncovalently bound FAD per dimer, has a monomer extinction coefficient of 48,000 at 270 nm and contains no detectable redox active metals. The apparent K(m) and Kcat for protoporphyrinogen IX are 1.7 microM and 10.5 min-1, respectively. The enzyme does not use coproporphyrinogen III as a substrate and is inhibited by micromolar concentrations of the herbicide acifluorfen. Protein database searches reveal significant homology between protoporphyrinogen oxidase and monoamine oxidase.

Amino Acid Sequence↗
PubMed1996

A R59W mutation in human protoporphyrinogen oxidase results in decreased enzyme activity and is prevalent in South Africans with variegate porphyria.

Variegate porphyria (VP), a low-penetrant autosomal dominant inherited disorder of haem metabolism, is characterised by photosensitivity (Fig. 1) and a propensity to develop acute neuropsychiatric attacks with abdominal pain, vomiting, constipation, tachycardia, hypertension, psychiatric symptoms and, in the worst cases, quadriplegia. Acute attacks, often precipitated by inappropriate drug therapy, are potentially fatal. While earlier workers thought the distal haem biosynthetic enzyme ferrochelatase may be involved in the genesis of VP, it was shown in the early 1980's, and is now accepted, that VP is associated with decreased protoporphyrinogen oxidase activity (PPO) (E.C.1.3.3.4). VP prevalence is much higher in South Africa than elsewhere; probably due to a founder effect with patients descending from a 17th century Dutch immigrant. PPO cDNAs from Bacillus subtilis, Myxococcus xanthus, human placenta and mouse liver have been cloned, sequenced and expressed. Human and mouse cDNAs consist of open reading frames 1431 nucleotides long, encoding a 477 amino acid protein. The human PPO gene contains thirteen exons, spanning approximately 4.5 kb. We have identified a C to T transition in codon 59 (in exon 3) resulting in an arginine to tryptophan substitution (R59W). A protein expressed from an in vitro-mutagenized PPO construct exhibits substantially less activity than the wild type. The R59W mutation was present in 43 of 45 patients with VP from 26 of 27 South African families investigated, but not in 34 unaffected relatives or 9 unrelated British patients with PPO deficiency. Since at least one of these families is descended from the founder of South African VP, this defect may represent the founder gene defect associated causally with VP in South Africa.

Amino Acid Sequence↗
PubMed1995

Cloning, sequence, and expression of mouse protoporphyrinogen oxidase.

Protoporphyrinogen oxidase (EC 1.3.3.4) is the penultimate enzyme in the heme biosynthetic pathway, catalyzing the six-electron oxidation of protoporphyrinogen to protoporphyrin. A dominantly inherited genetic deficiency in this enzyme results in the disease variegate porphyria. We now report the cloning, sequence, and expression of mouse protoporphyrinogen oxidase. The cDNA for mouse protoporphyrinogen oxidase was obtained by complementation of Escherichia coli SASX38, a protoporphyrinogen oxidase-deficient strain, with a mouse erythroleukemia (MEL) cell expression library. The sequence of this cDNA along with 5' untranslated sequence obtained by 5' rapid amplification of cDNA ends of MEL cell mRNA is 1814 bp in length and contains an open reading frame of 1431 bp. This encodes a protein of 477 amino acid residues with a calculated molecular weight of 50,870. The protein as expressed in E. coli is sensitive to inhibition by the diphenyl ether herbicide acifluorfen. Northern blot analyses of RNA from uninduced and induced MEL cells as well as mouse hepatoma cells all show two major mRNA species of 1.8 and 3.6 kb.

Amino Acid Sequence↗
PubMed1995

Generation of resistance to the diphenyl ether herbicide acifluorfen by MEL cells.

The diphenyl ether herbicide acifluorfen has been shown to act by inhibition of the terminal enzyme of the protoporphyrin biosynthetic pathway, protoporphyrinogen oxidase (E.C. 1.3.3.4) (PPO), in plant and animal cells. In the present study we show that long term maintenance of murine erythroleukemia (MEL) cells in acifluorfen, which is normally toxic to these cells at 5 microM concentration, results in cells that grow at a near normal rate in 100 microM acifluorfen. Acifluorfen resistant cells do not have increased levels of PPO activity, nor does the PPO made by these cells have increased resistance to acifluorfenin, but these cells accumulate porphyrin and have elevated levels of heme. Data is presented that suggests the resistance of these MEL cells to acifluorfen may be attributable to induction of a cytochrome P450(s).

Animals↗
PubMed1995

Effect of cellular location on the function of ferrochelatase.

Ferrochelatase, the terminal enzyme of the heme biosynthetic pathway, is a nuclear encoded protein that is synthesized in the cytoplasm in a precursor form and then is translocated to the matrix side of the inner mitochondrial membrane. Since the product of the enzymatic reaction, protoheme IX, is utilized almost exclusively in the cytoplasmic compartment or on the cytoplasmic side of the inner mitochondrial membrane, it was of interest to determine if the intracellular location of ferrochelatase-deficient strain of the yeast Saccharomyces cerevisiae vectors that coded for full-length ferrochelatase and a truncated form of the enzyme that lacked the mitochondrial targeting sequence were expressed. Both of these transformed cells produce approximately equal total amounts of ferrochelatase, as determined by enzyme assays and Western blot analysis, but only with the full-length construct was ferrochelatase properly localized. In cells containing the truncated construct, ferrochelatase activity was found in all membrane fractions but was not located on the matrix side of the inner mitochondrial membrane. Cells containing either construct produced heme, although the amount of heme synthesized by cells with the truncated construct was significantly less. Interestingly in cells with improperly localized ferrochelatase the amount of b-type cytochrome decreased by 80% as opposed to c- and a-type cytochromes where the decreases were only 60 and 40%, respectively.

Base Sequence↗
PubMed1995

Regulation of heme biosynthesis in Escherichia coli.

Escherichia coli is an organism that synthesizes 5-aminolevulinate (ALA), the first committed compound of the heme biosynthetic pathway, from glutamate (C-5 pathway) as opposed to glycine and succinyl CoA (C-4 pathway). While regulation of the C-4 pathway is generally acknowledged to occur at the level of formation of ALA, the mode of regulation of the C-5 pathway is currently unclear. Here we have examined one aspect of regulation of heme synthesis in E. coli: the role of the end product, heme, as a feed-back regulator of ALA production. By using plasmid-encoded ALA synthase and/or cytochrome b5 expressed in a wild type E. coli strain, it was possible to determine the role that the proposed regulatory heme pool plays in the regulation of ALA and heme production. Expression of rat-soluble cytochrome b5 results in an increase of cellular heme, indicating that the cell responds to this foreign "heme sink" by producing more heme even though the cytochrome does not participate directly in normal cellular regulation. Accumulation of pathway intermediates does not occur under these conditions. Expression of plasmid-encoded mouse ALA synthase results in increased cellular heme production as well as the accumulation of pathway intermediates either in the presence or absence of plasmid encoded cytochrome b5. These data support a regulatory scheme where the heme biosynthetic pathway in this C-5 organism is regulated at the level of ALA production in part by cellular heme content.

5-Aminolevulinate Synthetase↗
PubMed1995

A new mutation of the ALAS2 gene in a large family with X-linked sideroblastic anemia.

X-linked sideroblastic anemia is a genetic disorder characterized by a hypochromic microcytic anemia of variable intensity with the presence of ring sideroblasts in the bone marrow of the patients. Two different mutations have been reported in the ALAS2 gene in patients with this disease. We have studied a large kindred with a pyridoxine-sensitive form of X-linked sideroblastic anemia. Sequencing amplified cDNA of the proband revealed a guanine-to-adenine change at nucleotide 871 of the coding sequence (exon 7 of the gene). This results in a glycine to serine substitution that is responsible for a marked decrease in the enzymatic activity of the mutated protein. A polymerase chain reaction assay demonstrated the presence of the same mutation in three affected males and two female carriers in the kindred. The carrier status was excluded in eight females at risk. Early detection of the mutant allele in family members may thus be important for the prevention of anemia in males and of iron overload both in affected males and carrier females.

Adolescent↗