Gene/Protein Disease Symptom Drug Enzyme Compound
Pivot Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound   Target Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound

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Query: EC:2.5.1.61 (porphobilinogen deaminase)
637 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

Porphobilinogen deaminase catalyzes the condensation of four porphobilinogen monopyrrole units into hydroxymethylbilane, a linear tetrapyrrole necessary for the formation of chlorophyll and heme in higher plant cells. We report the purification to homogeneity of a chloroplast-localized form of the enzyme from pea (Pisum sativum L.) by a novel purification scheme involving dye-ligand affinity chromatography. The purified chloroplast porphobilinogen deaminase consists of a single polypeptide with a relative molecular mass of 36-45 kDa as determined by size-exclusion chromatography and sodium dodecyl sulfate polyacrylamide gel electrophoresis. The isoelectric point of the protein is acidic. The activity of the enzyme shows different levels of sensitivity to divalent cations and is most sensitive to FE2+. The amino terminus of pea enzyme has been obtained by microsequencing and determined to bear little similarity to the amino acid sequences of porphobilinogen deaminases purified from other organisms. Polyclonal antisera elicited against the purified protein has been used to examine the abundance and cellular distribution of the enzyme.
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PMID:Isolation, characterization and partial amino acid sequence of a chloroplast-localized porphobilinogen deaminase from pea (Pisum sativum L.). 198 93

Uroporphyrinogen III synthase (hydroxymethylbilane hydro-lyase (cyclizing); EC 4.2.1.75), the fourth enzyme in the heme biosynthetic pathway, was purified to homogeneity from human erythrocytes. For enzyme purification and characterization, a sensitive coupled enzyme assay was used which generated the substrate, hydroxymethylbilane; the oxidized product, uroporphyrin III, was quantitated by high pressure liquid chromatography. Uroporphyrinogen III synthase was initially separated from delta-aminolevulinate dehydratase and hydroxymethylbilane synthase by a preparative anion exchange chromatographic step. Subsequent chromatography on hydroxyapatite, phenyl-Sepharose, and Sephadex G-100 purified the enzyme about 70,000-fold with an 8% yield. Homogeneous enzyme was obtained following a final C4-reversed phase high pressure liquid chromatographic step which removed a single major and several minor protein contaminants from the enzyme. The purified enzyme had a specific activity of over 300,000 units/mg, an isoelectric point of 5.5, and was thermolabile (t1/2 at 60 degrees C approximately 1 min). Molecular weight studies by gel filtration (Mr approximately equal to 30,000) and analytical sodium dodecyl sulfate-polyacrylamide gel electrophoresis (Mr approximately equal to 29,500) were consistent with the enzyme being a monomer. Using hydroxymethylbilane as substrate, the purified enzyme formed uroporphyrinogen III in the absence of hydroxymethylbilane synthase or other cofactors. The pH optimum was 7.4 and the Km for hydroxymethylbilane was 5-20 microM. The enzyme was activated by Na+, K+, Mg+, and Ca2+ and was inhibited by Cd2+, Cu2+, Hg2+, and Zn2+. Amino acid composition analysis was performed, and the N-terminal sequence, Met-Lys-Val-Leu-Leu-Leu, was determined by microsequencing. The availability of the purified enzyme should permit investigation of its reaction mechanism as well as facilitate biochemical and molecular studies of the genetic defect in congenital erythropoietic porphyria.
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PMID:Purification and properties of uroporphyrinogen III synthase from human erythrocytes. 380 19

Porphobilinogen deaminase (hydroxymethylbilane synthase; EC 4.3.1.8), the third enzyme of the heme biosynthetic pathway, catalyzes the stepwise condensation of four porphobilinogen units to yield hydroxymethylbilane, which is in turn converted to uroporphyrinogen III by cosynthetase. We compared the apparent molecular mass of porphobilinogen deaminase from erythropoietic and from non-erythropoietic cells by sodium dodecyl sulfate/polyacrylamide gel electrophoresis and immune-blotting. The results indicate that two isoforms of porphobilinogen deaminase can be distinguished and differ by 2000 Da. Analysis of cell-free translation products directed by mRNAs from human erythropoietic spleen and from human liver demonstrates that the two isoforms of porphobilinogen deaminase are encoded by distinct messenger RNAs. We cloned and sequenced cDNAs complementary to the non-erythropoietic form of porphobilinogen deaminase encoding RNA. Comparison of these sequences to that of human erythropoietic mRNA [Raich et al. (1986) Nucleic Acids Res. 14, 5955-5968] revealed that the two mRNA species differ by their 5' extremity. From the mRNA sequences we could deduce that an additional peptide of 17 amino acid residues at the NH2 terminus of the non-erythropoietic isoform of porphobilinogen deaminase accounts for its higher molecular mass. RNase mapping experiments demonstrate that the two porphobilinogen deaminase mRNAs are distributed according to a strict tissue-specificity, the erythropoietic form being restricted to erythropoietic cells. We propose that a single porphobilinogen deaminase gene is transcribed from two different promoters, yielding the two forms of porphobilinogen deaminase mRNAs. Our present finding may have some relevance for further understanding the porphobilinogen deaminase deficiency in certain cases of acute intermittent porphyria with an enzymatic defect restricted in non-erythropoietic cells.
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PMID:Tissue-specific expression of porphobilinogen deaminase. Two isoenzymes from a single gene. 381 74

The treatment of rats with cis-platinum for 7 days caused a profound, and seemingly selective, decrease (70-80%) in the microsomal cytochrome P-450 levels in the testis. This decrease was accompanied by marked reductions (70-80%) in steroid 17 alpha-hydroxylase activity and in plasma testosterone concentration. The treatment of rats with human chorionic gonadotropin partially restored the cytochrome P-450 concentration and 17 alpha-hydroxylase activity and permitted the plasma testosterone level to approach control values. The effect of cis-platinum on the testicular cytochrome P-450 appeared unrelated to deficiencies in heme metabolic processes, in so far that neither was the activity of delta-aminolevulinate synthetase decreased, nor was that of heme oxygenase increased. These enzymes are rate-limiting in heme biosynthesis and degradation pathways, respectively. Also, the activities of uroporphyrinogen I synthetase, delta-aminolevulinate dehydratase, and ferrochelatase and the concentration of total porphyrins in the testis remained unchanged. The sodium dodecyl sulfate-gel electrophoresis of the microsomal preparation did not reveal a diminished level of apocytochrome; however, in this preparation, heme could not be detected in molecular weight regions corresponding to cytochrome P-450. The microsomal cytochrome b5 and the mitochondrial heme concentrations were not decreased in cis-platinum-treated rats. It is suggested that the mechanism of depletive action of cis-platinum on microsomal cytochrome P-450 involves an impairment of the effective assembly of heme and apoprotein moieties. It is further suggested that the anterior pituitary hormones control the factor(s) involved in this assembly, a process which is interrupted by cis-platinum.
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PMID:Inhibition of testicular cytochrome P-450-dependent steroid biosynthesis by cis-platinum. Reversal by human chorionic gonadotropin. 403 24

Porphyria cutanea tarda (PCT) is characterized biochemically by excessive hepatic synthesis and urinary excretion of uroporphyrin I. Clinical evidence has implicated iron in the pathogenesis of PCT. The synthesis of the normally occurring isomer of uroporphyrin, namely uroporphyrin III, from porphobilinogen (PBG) requires two enzymes; uroporphyrinogen I synthetase and uroporphyrinogen III cosynthetase (COSYN). In the absence of COSYN only uroporphyrinogen I is formed. These experiments were designed to study the effect of iron on porphyrin biosynthesis in porcine and human crude liver extracts and to measure COSYN activity in the presence of iron.Mitochondria-free crude liver extracts were prepared in 0.25 m sucrose at pH 7.4 by centrifugation at 37,000 g. Preparations were incubated with either 0.2 mm amino-levulinic acid (ALA) or 0.1 mm PBG. The addition of ferrous ion (either from ferritin iron [4 mug/ml] and cysteine [6.7 mm] or ferrous ammonium sulfate [0.3 mm Fe] and cysteine) significantly increased the rate of uroporphyrin synthesis from either ALA or PBG. The predominant porphyrin synthesized in the presence of ferrous ion was uroporphyrin I whereas coproporphyrin III predominated in its absence. Orthophenanthroline blocked these effects of ferrous ion.To investigate the effect of ferrous ion on COSYN, crude liver extracts were incubated with ferrous ammonium sulfate (0.3 mm Fe) and cysteine (6.7 mm) and the COSYN activity of the incubates was assayed directly. In both porcine and human extracts ferrous ion caused marked inhibition of COSYN activity. Orthophenanthroline blocked the inhibitory effect.Inactivation of COSYN by heating resulted in marked enhancement of porphyrin synthesis from PBG. The sole product was uroporphyrin I.Thus, inactivation of COSYN results in accelerated synthesis of uroporphyrin I. This effect of ferrous ion provides a possible biochemical explanation for the excess production and excretion of uroporphyrin I in patients with PCT and the reversal of this defect by phlebotomy.
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PMID:The role of iron in the pathogenesis of porphyria cutanea tarda. An in vitro model. 464 Sep 47

Acute intermittent porphyria is the major autosomal dominant form of acute hepatic porphyrias. The disease is due to mutations in the gene encoding for porphobilinogen deaminase (PBGD). Many different strategies have been developed to screen for mutations. However the high prevalence (0.6 per thousand) of PBGD gene defect, the large allelic heterogeneity of mutations (n = 130), and the limitations of the PBGD enzymatic assay for asymptomatic patients' detection, require for diagnosis an efficient and easy to handle strategy for locating mutations within the PBGD gene. In a recent study the sensitivity of the denaturing gradient gel electrophoresis (DGGE) technique was 100%. However DGGE requires the preparation of gradient gels and the use of primers with long GC-clamps; thus alternative methods should be preferable in the clinical laboratory. We have compared the detection rate of DGGE with heteroduplex analysis (HA) using 16 characterized PBGD gene mutations. Six different HA conditions were used to determine the efficiency of the method, including: (1) MDE (mutation detection enhancement) gel concentration; (2) addition of urea and sodium dodecyl sulfate (SDS); (3) radioactive labelling. The sensitivity of each HA condition varied from 31 to 81% vs. 100% in DGGE analysis. HA using 1 x MDE with 15% urea with or without 0.55% SDS was the most sensitive condition. This first comparative study of DGGE and HA mutation screening methods suggests that DGGE is a more sensitive screening assay than optimized HA. However, because of its simplicity HA should be considered as an efficient alternative mutation screening method.
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PMID:Evaluation of mutation screening by heteroduplex analysis in acute intermittent porphyria: comparison with denaturing gradient gel electrophoresis. 1006 25

Mutations in the human PBGD (porphobilinogen deaminase) gene cause the inherited defect AIP (acute intermittent porphyria). In the present study we report the structure of the human uPBGD (ubiquitous PBGD) mutant, R167Q, that has been determined by X-ray crystallography and refined to 2.8 A (1 A=0.1 nm) resolution (Rfactor=0.26, Rfree=0.29). The protein crystallized in space group P2(1)2(1)2 with two molecules in the asymmetric unit (a=81.0 A, b=104.4 A and c=109.7 A). Phases were obtained by molecular replacement using the Escherichia coli PBGD structure as a search model. The human enzyme is composed of three domains each of approx. 110 amino acids and possesses a dipyrromethane cofactor at the active site, which is located between domains 1 and 2. An ordered sulfate ion is hydrogen-bonded to Arg26 and Ser28 at the proposed substrate-binding site in domain 1. An insert of 29 amino acid residues, present only in mammalian PBGD enzymes, has been modelled into domain 3 where it extends helix alpha2(3) and forms a beta-hairpin structure that contributes to a continuous hydrogen-bonding network spanning domains 1 and 3. The structural and functional implications of the R167Q mutation and other mutations that result in AIP are discussed.
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PMID:Structure of human porphobilinogen deaminase at 2.8 A: the molecular basis of acute intermittent porphyria. 1920 7

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