先天性红细胞生成性卟啉症患者酶活性的研究

先天性红细胞生成性卟啉症（ｃｏｎｇｅｎｉｔａｌｅｒｙ－ｔｈｒｏｐｏｉｅｔｉｃｐｏｒｐｈｙｒｉａ,ＣＥＰ）是Ｇｕｎｔｈｅｒ于１９１１年首先提出并加以描述,有时亦称Ｇｕｎｔｈｅｒ病．该病是因遗传性缺陷所致卟啉代谢中有关酶的异常造成的卟啉代谢紊乱而发生的一...     

The influence of carbamazepine on the heme biosynthetic pathway

Carbamazepine, a drug which is widely used in neurological diseases, has a porphyrogenic effect in chick embryo liver cells in culture. It increased the concentration of cellular porphyrins by 80-fold and delta-aminolevulinate synthase activity by 4-fold. The increase in the accumulation of porphyrins preceded that of ALAS activity. Measurements of the activities of aminolevulinate dehydrase, porphobilinogen deaminase, and uroporphyrinogen decarboxylase showed that C inhibits UROD up to nearly 50% and PBGD activity up to 20%, but does not affect the activity of ALAD. The pattern of accumulation of porphyrins, mainly uro- and heptacarboxylporphyrin, is compatible with an inhibition of UROD. We may, therefore, conclude that the porphyrogenic effect of C in monolayers of chick embryo liver cells is the result of its inhibitory effect on the activity of UROD.     

Evidence for the interference of aluminum with bacterial porphyrin biosynthesis

Aluminum (0.74 mm) was found to retard bacterial growth, and enhance porphyrin formation and excretion in Arthrobacter aurescens RS-2. Coproporphyrin III was shown to be the main porphyrin excreted by aluminum-exposed A. aurescens RS-2 cultures and by RS-2 cultures grown under anoxic conditions. Synthesis and excretion of porphyrins in A. aurescens RS-2 increased in a dose-dependent manner when the bacteria were exposed to increasing aluminum concentrations. Incubation of A. aurescens RS-2 with -aminolevulinic acid (-ALA, 1.2 mm) brought about the intense formation and excretion of porphyrins by the cells, in the presence or absence of aluminum. -ALA slightly enhanced the toxicity of aluminum towards RS-2 bacteria. Furthermore, the intracellular concentration of heme was reduced by 63.9 ± 8.67% in aluminum-exposed RS-2 bacteria when compared with control cultures. The results are discussed in light of the recent finding concerning aluminum toxicity and porphyrin biosynthesis in microorganisms.     

Influence of cesium on tetrapyrrole biosynthesis in etiolated and greening barley leaves

Cesium chloride (CsCl) treatment of greening primary leaves of barley for 8 h inhibited chlorophyl] accumulation in a concentration-dependent manner and led to the accumulation of excessive amounts of uroporphyrin(ogen) III (URO[gen]) and to a minor extent of heptacarboxylporphyrin(ogen). When dark-grown leaves were incubated with CsCl, accumulation of URO(gen) was observed only after feeding of the tetrapyrrole precursor 5-aminolevulinic acid. Western blot analysis showed no apparent difference in content of uroporphyrinogen decarboxylase (EC 4.1.1.37, UROD) or selected proteins involved in tetrapyrrole biosynthesis in extracts of CsCl-incubated (15 m M ) versus control leaves. UROD activity was drastically decreased upon CsCl treatment in leaves incubated in the dark or in the light (44 and 86%, respectively). Selected preceding enzymes of the tetrapyrrole biosynthetic pathway, 5-aminolevulinic acid dehydratase (EC 4.2.1.24, ALAD) and porphobilinogen deaminase (EC 4.3.1.8, PBGD), were influenced only to a minor extent under standard incubation conditions (15 m M CsCl). Furthermore, the ALA synthesizing capacity did not differ in leaves incubated with and without Cs⁻ cations. UROD activity of crude homogenates from control plants and after partial purification was reduced to 56 and 80%, respectively, upon addition of 10 m M CsCl. Equal concentrations of KCl were not inhibitory. Enzyme assays of the same barley extract in the presence of CsCl yielded no effect on ALAD and a minor loss of PBGD activity. The initial visible cytotoxic effect of CsCl appeared to be a selective inhibition of UROD resulting in accumulation of photosensitizing URO (gen). Consequences of the diminished UROD activity on early steps of the tetrapyrrole biosynthesis and its functional and regulatory significance for the porphyrin synthesis are discussed.     

Accelerated heme synthesis and degradation in transformed fibroblasts

Various parameters of the heme biosynthetic pathway were studied in two cell lines, one nontransformed and the other malignantly transformed (MLV/MS), both replicating at the same rate. Using the above system enabled us to distinguish between phenomena characteristic of the malignant transformation per se and those due to accelerated growth rate. Heme synthesis and degradation as well as the activities of ALAS, ALAD, PBGD, and FC were found to be increased in the transformed cells. However, the concentration of intracellular heme was markedly reduced from 30.4 +/- 4.4 pmole/mg protein in nontransformed cells to 10.5 +/- 2.6 pmole/mg protein in transformed cells. These observations show that malignant transformation leads to changes in heme metabolism unrelated to growth rate in this cell line.     

Subcellular localization of two porphyrin-synthesis enzymes in Pisum sativum (pea) and Arum (cuckoo-pint) species.

The subcellular location of the two porphyrin-synthesis enzymes 5-aminolaevulinate dehydratase (ALAD) and porphobilinogen deaminase (PBGD) was investigated in Pisum sativum (pea) leaves and spadices of Arum (cuckoo-pint). Throughout the tissue-fractionation procedures the distribution of the two enzymes paralleled that of the plastid marker enzyme (ADP-glucose pyrophosphorylase), even in Arum, a tissue where the synthesis of non-plastid haem is predominant. The distribution of cytosolic marker enzyme (lactate dehydrogenase) was significantly different from that of ALAD and PBGD and, although purified mitochondria from both species had some residual activity, this was always less than contaminating plastid marker enzyme. The results suggest that ALAD and PBGD are exclusively plastid enzymes. The significance of this for the role of plastids in cellular porphyrin synthesis is discussed.     

Production of uroporphyrinogen III, which is the common precursor of all tetrapyrrole cofactors, from 5-aminolevulinic acid by Escherichia coli expressing thermostable enzymes

Uroporphyrinogen III (urogen III) was produced from 5-aminolevulinic acid (ALA), which is a common precursor of all metabolic tetrapyrroles, using thermostable ALA dehydratase (ALAD), porphobilinogen deaminase (PBGD), and urogen III synthase (UROS) of Thermus thermophilus HB8. The UROS-coding gene (hemD ₂ ) of T. thermophilus HB8 was identified by examining the gene product for its ability to produce urogen III in a coupled reaction with ALAD and PBGD. The genes encoding ALAD, PBGD, and UROS were separately expressed in Escherichia coli BL21 (DE3). To inactivate indigenous mesophilic enzymes, the E. coli transformants were heated at 70 °C for 10 min. The bioconversion of ALA to urogen III was performed using a mixture of heat-treated E. coli transformants expressing ALAD, PBGD, and UROS at a cell ratio of 1:1:1. When the total cell concentration was 7.5 g/l, the mixture of heat-treated E. coli transformants could convert about 88 % 10 mM ALA to urogen III at 60 °C after 4 h. Since eight ALA molecules are required for the synthesis of one porphyrin molecule, approximately 1.1 mM (990 mg/l) urogen III was produced from 10 mM ALA. The present technology has great potential to supply urogen III for the biocatalytic production of vitamin B₁₂.     

Characterization of a new mutation (R292G) and a deletion at the human uroporphyrinogen decarboxylase locus in two patients with hepatoerythropoietic porphyria

Summary A deficiency in the activity of uroporphyrinogen decarboxylase (UROD), the fifth enzyme of the haem biosynthetic pathway, is found in familial porphyria cutanea tarda (F-PCT) and hepatoerythropoietic porphyria (HEP). A new mutation (R292G) and a deletion have been found in a pedigree with two HEP patients (two sisters). The R292G mutation was not detected in 13 unrelated affected patients with F-PCT, so it appears to be uncommon. The possibility that the arginine 292 may participate at the active site of the enzyme is discussed. A summary of the 7 mutations/deletions found in the UROD gene with their frequency is presented.     

Mechanism of 5-aminolevulinate synthase and the role of the protein environment in controlling the cofactor chemistry.

5-Aminolevulinate synthase, a pyridoxal 5'-phosphate-dependent enzyme of the alpha-oxoamine synthase family, catalyzes the first step of the heme biosynthetic pathway in mammalian cells. This reaction entails the condensation of glycine with succinyl-coenzyme A to yield 5-aminolevulinate, carbon dioxide and CoA. Mutations in the erythroid aminolevulinate synthase gene lead to a defective enzyme and are associated with the erythropoietic disorder X-linked sideroblastic anemia. In the past few years, rapid scanning-stopped-flow spectroscopy and chemical quenched-flow studies of the ALAS reaction, under single- and multi-turnover conditions, have provided important results for the interpretation of the catalytic mechanism. In particular, the role of the protein scaffold in modulating the chemical reactivity of the pyridoxal 5'-phosphate cofactor and, thus, the catalytic pathway of ALAS has been investigated in our laboratory using transient kinetics and global analysis of the kinetic data.     

Bildung kristallinen Nicotinblaus beim Abbau von Nicotin durchArthrobacter oxydans

Zusammenfassung Es wird einArthrobacter oxydans beschrieben, derl-,DL- undd-Nicotin über 6-Hydroxynicotin abbaut und sogenanntes Nicotinblau bildet. Dieser Farbstoff kann unter Verwendung eines speziellen Mediums in Form brillantgrüner Kristalle isoliert werden, die sich auf Agarplatten in den Zentren der Kolonien abscheiden. Einige Eigenschaften des kristallinen Nicotinblaus werden angegeben.

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Formation of crystalline nicotine blue by a nicotine degradingArthrobacter oxydans

Summary A strain ofArthrobacter oxydans is described which degradesl-,DL-, andd-nicotine, forming nicotine blue. Under specified growth conditions this pigment is deposited as brillant green crystals in the center of colonies on agar. Some properties of the isolated crystalline nicotine blue are described.

     

Decrease of hepatic delta-aminolevulinate dehydratase activity in an animal model of fatigue

Fatigue can be defined physiologically as inability to maintain the expected power output. At present, no standard of fatigue are yet available. In order to find biomarkers of fatigue, we investigated the level of delta-aminolevulinic acid (ALA), the first intermediate metabolite in the heme biosynthetic pathway, in the plasma and urine of an animal model of fatigue. To prepare fatigued animals, we kept rats for 5 days in a cage filled with water to a height of 1.5 cm. As a result, the plasma and urinary ALA levels were increased in the fatigued animals as compared with those in the control animals. One day after the rats had been returned to their normal cages, these increased levels were restored to the control ones. We also examined the activity of the enzyme ALA dehydratase (ALAD), which is the second enzyme in the heme biosynthetic pathway, and ALAD gene expression during the fatigue and its recovery sessions. The ALAD activity, as well as its gene expression, in the liver of the fatigued animals was decreased as compared with those of the control animals. Both activity and gene expression of ALAD were recovered to their respective control levels after the rats had been allowed to rest in their normal cages for 1 day. Furthermore, the activity of ALA synthase (ALAS), the rate-limiting enzyme in the heme biosynthesis, in the liver was increased after the fatigue session for 5 days. Although this level of increase in the plasma concentration of ALA may not induce fatigue, increase in plasma and urinary ALA levels can be biomarkers of fatigue.     

Haem biosynthesis and human porphyria cutanea tarda: effects of alcohol intake

The review describes the structural and biochemical properties of the haem biosynthetic enzyme, uroporphyrinogen decarboxylase (UROD), which sequentially catalyzes the removal of the four carboxyl groups from the acetate side chains of octacarboxylic uroporphyrinogen to form coproporphyrinogen, and the possible biochemical mechanism of the genesis of porphyria cutanea tarda (PCT). The disease is caused when the activity of UROD is significantly reduced. PCT is a multifactorial disease where both inherent and environmental factors such as alcohol, estrogens, halogenated aromatic hydrocarbons and viral infection (mainly hepatitis C) are involved in biochemical and clinical expression. In PCT, hepatic iron plays a key role. Alcohol intake could induce mobilization of iron from protein-bound ferritin. PCT should be managed by avoidance of these toxins and removal of iron by vigorous phlebotomy. Such iron-reduction therapy would provide additional benefit for hepatitis C patients by interferon therapy.     

Purification and properties of glutamine synthetase from Nocardia asteroides

Glutamine synthetase (GS, EC 6.3.1.2) from Nocardia asteroides was purified to homogeneity by ammonium sulfate precipitation, Sephadex G-150, and DEAE-Sepharose chromatography. The native molecular weight of the purified enzyme was determined to be 720 kDa. SDS-PAGE analysis of the purified preparation revealed a single band corresponding to 59 kDa, indicating the possible presence of 12 identical subunits. The divalent cations Mn^2- and Mg²⁺ were found to be essential for optimal transferase and biosynthetic activity, respectively. The optimal pH and temperature for both activities of the enzyme were found to be 7.2 and 50°C. Amino acids such as l-alanine, glycine, and aspartate inhibited the GS activity. The K _m values for the substrates of the biosynthetic reaction ATP, glutamate, and ammonium chloride were found to be 400 m, 7.7mm, and 200 m, respectively. Addition of ammonium chloride to the nitrogen-limited culture resulted in a decrease of GS transferase and biosynthetic activities. Phosphodiesterase treatment of the extract from ammonia-shocked cultures showed an increase in GS transferase activity. The results indicate the possible regulation of GS by covalent modification.     

Correction of the biochemical defect in porphobilinogen deaminase deficient cells by non-viral gene delivery

Porphobilinogen deaminase (PBGD), the third enzyme in the biosynthesis of heme, is deficient in acute intermittent porphyria (AIP). AIP is a genetic disease characterized by neurovisceral and psychiatric disturbances. Despite a palliative treatment, it may still be lethal. An initial step towards gene therapy was recently taken by showing that PBGD could be expressed to correct the enzyme deficiency in AIP fibroblasts. The aim of the present study was to investigate whether the biochemical defect can be corrected by using non-viral gene delivery. The biochemical defect in human and mouse PBGD deficient fibroblasts was demonstrated by analyzing synthesis of the heme precursor, protoporphyrin (PP), after addition of 5-aminolevulinic acid (ALA). Human AIP fibroblasts synthesized 21% and mouse PBGD deficient fibroblasts only 11% of the PP amount synthesized in respective control cells. Gene delivery increased the PBGD activity 88–200 fold in human AIP fibroblasts and synthesis of PP was increased from 21–152% of normal after ALA incubation. Similar results were obtained in mouse PBGD deficient cells, although the PP levels were several-fold lower as compared to human cells. HPLC analysis confirmed that PP was the main porphyrin intermediate that was formed. Addition of porphobilinogen (PBG) resulted in 3–7 fold lower synthesis of PP as compared to ALA addition. These results show that non-viral gene delivery of plasmids encoding PBGD results in a high expression of functional PBGD shown by induced synthesis of PP in PBGD deficient cells after supplementation of ALA and PBG.     

Enhanced stability of an L-hydantoinase mediated by its corresponding polyclonal antibody

Summary Polyclonal antibodies were produced against the highly purified L-5-aryl-alkyl-hydantoinase and hydantoin-racemase of Arthrobacter aurescens DSM 3747. Serological interaction of the hydantoinase antibody with its corresponding antigenic fraction let to a remarkable improvement of the stability of the hydantoinase. The immobilization of the catalytic active enzyme-antibody complex onto a Sepharose 4B-support underlined this antibody-mediated stabilization effect, while control experiments with preimmunsera or the hydantoinracemase antibodies gave no or even a negative effect.     

The IsdG‐family of haem oxygenases degrades haem to a novel chromophore

Enzymatic haem catabolism by haem oxygenases is conserved from bacteria to humans and proceeds through a common mechanism leading to the formation of iron, carbon monoxide and biliverdin. The first members of a novel class of haem oxygenases were recently identified in Staphylococcus aureus (IsdG and IsdI) and were termed the IsdG‐family of haem oxygenases. Enzymes of the IsdG‐family form tertiary structures distinct from those of the canonical haem oxygenase family, suggesting that IsdG‐family members degrade haem via a unique reaction mechanism. Herein we report that the IsdG‐family of haem oxygenases degrade haem to the oxo‐bilirubin chromophore staphylobilin. We also present the crystal structure of haem‐bound IsdI in which haem ruffling and constrained binding of oxygen is consistent with cleavage of the porphyrin ring at the β‐ or δ‐meso carbons. Combined, these data establish that the IsdG‐family of haem oxygenases degrades haem to a novel chromophore distinct from biliverdin.