RESUMEN
Accumulation of protoporphyrin IX (PPIX) and Zn-PPIX, are the clinical hallmarks of protoporphyria. Phenotypic expression of protoporphyria is due to decreased activity of ferrochelatase (FECH) or to increased activity of aminolevulinic acid synthase (ALAS) in red blood cells. Other genetic defects have been shown to contribute to disease severity including loss of function mutations in the mitochondrial AAA-ATPase, CLPX and mutations in the Iron-responsive element binding protein 2 (IRP2), in mice. It is clear that multiple paths lead to a common phenotype of excess plasma PPIX that causes a phototoxic reaction on sun exposed areas. In this study we examined the association between mitochondrial iron acquisition and utilization with activity of FECH. Our data show that there is a metabolic link between the activity FECH and levels of MFRN1 mRNA. We examined the correlation between FECH activity and MFRN1 mRNA in cell lines established from patients with the classical protoporphyria, porphyria due to defects in ALAS2 mutations. Our data confirm MFRN1 message levels positively correlated with FECH enzymatic activity in all cell types.
Asunto(s)
Proteínas de Transporte de Catión/genética , Ferroquelatasa/metabolismo , Linfocitos/enzimología , Mitocondrias/fisiología , Proteínas Mitocondriales/genética , Protoporfiria Eritropoyética/enzimología , Protoporfiria Eritropoyética/genética , 5-Aminolevulinato Sintetasa/genética , Línea Celular Transformada , Ferroquelatasa/análisis , Humanos , Fenotipo , Protoporfirinas/genética , Protoporfirinas/metabolismo , ARN MensajeroRESUMEN
A continuous spectrofluorimetric assay for determining ferrochelatase activity has been developed using the physiological substrates ferrous iron and protoporphyrin IX under strictly anaerobic conditions. In contrast to heme, the product of the ferrochelatase-catalyzed reaction, protoporphyrin IX is fluorescent, and therefore the progress of the reaction can be monitored by following the decrease in protoporphyrin fluorescence intensity (with excitation and emission wavelengths at 505 and 635 nm, respectively). This continuous fluorimetric assay detects activities as low as 0.01 nmol porphyrin consumed min(-1), representing an increase in sensitivity of up to two orders of magnitude over the currently used, discontinuous assays. The determination of the steady-state kinetic parameters of ferrochelatase yielded K(m)(PPIX)=1.4+/-0.2 microM, K(m)(Fe(2+))=1.9+/-0.3 microM, and k(cat)=4.0+/-0.3 min(-1). In addition to its applicability for acquisition of kinetic data to characterize ferrochelatase and recombinant variants, this new method should permit detection of low concentrations of ferrochelatase in biological samples.
Asunto(s)
Ferroquelatasa/análisis , Fluorometría/métodos , Protoporfirinas/análisis , Anaerobiosis , Animales , Cationes Bivalentes , Ferroquelatasa/metabolismo , Compuestos Ferrosos/química , Humanos , Cinética , Protoporfirinas/química , Proteínas Recombinantes/análisis , Reproducibilidad de los Resultados , LevadurasRESUMEN
La protoporfiria eritropoyética (PPE) es una enfermedad de origen genético provocada por el déficit de la enzima ferroquelatasa. Es poco frecuente y las características clínicas más frecuentes son las manifestaciones derivadas de la fotosensibilidad. Sin embargo, muchas veces los pacientes con PPE únicamente refieren sensaciones de prurito y quemazón tras la exposición al sol sin lesiones clínicas objetivables, lo que dificulta el diagnóstico. Describimos cuatro casos de PPE que hemos estudiado en la Unidad de Fotobiología de nuestro servicio y repasamos los aspectos más importantes de esta enfermedad (AU)
Asunto(s)
Humanos , Masculino , Femenino , Preescolar , Niño , Adolescente , Protoporfiria Eritropoyética/diagnóstico , Trastornos por Fotosensibilidad/complicaciones , Ferroquelatasa/análisis , Púrpura/etiología , Radiación Solar/efectos adversosRESUMEN
Workers in the diesel fuel distribution trade are intensively exposed to fuel vapours. Diesel fuel presents the main source of air pollution by benzene at a marine diesel fuel terminal. Levels of benzene are used to evaluate the external exposure to diesel fuel. Since benzene causes alterations in porphyrin metabolism, and some of these may lead to the generation of tumours, heme synthesis is proposed as a biomarker of early health effects of diesel fuel. A group of 20 workers exposed to diesel fuel and a group of 20 unexposed persons were examined and interviewed using structured questionnaires. The levels of 5-aminolevulinic acid (ALA) and protoporphyrin (PP), activities of ALA synthase and ferrochelatase, as well as levels of PP associated with DNA were determined in lymphocytes spectrophotometrically. Amounts of the metals Cd, Mn, Zn, Cu and Ca were measured in blood plasma by flame atomic absorption spectrometry method. Both ALA and PP levels were significantly increased in marine terminal workers: 3.0 +/- 0.4 vs. 0.8 +/- 0.2 nmol/10(6) lymphocytes: and 511 +/- 164 vs. 389 +/- 77 pmol/10(6) lymphocytes in exposed and control individuals, respectively. ALA-synthase activity was 2.5 fold higher in lymphocytes of workers exposed to diesel fuels (P < 0.01). At the same time ferrochelatase activity was decreased and protoporphyrin level was accordingly elevated. The amount of porphyrin associated with DNA increased 1.4 fold in exposed workers (P = 0.05). Among all investigated metals in blood plasma of exposed workers only zinc levels were statistically significantly increased (P < 0.05). The disturbances of heme metabolism in lymphocytes and zinc level in blood plasma caused by diesel fuel exposure seems to be a useful biomarkers for carcinogenic risk assessment.
Asunto(s)
Hemo/metabolismo , Exposición Profesional , Emisiones de Vehículos/efectos adversos , 5-Aminolevulinato Sintetasa/análisis , 5-Aminolevulinato Sintetasa/metabolismo , Adulto , Ácido Aminolevulínico , Biomarcadores/análisis , ADN/análisis , Femenino , Ferroquelatasa/análisis , Ferroquelatasa/metabolismo , Humanos , Exposición por Inhalación , Linfocitos/efectos de los fármacos , Linfocitos/metabolismo , Masculino , Persona de Mediana Edad , Neoplasias/inducido químicamente , Medición de Riesgo , Volatilización , Zinc/sangreRESUMEN
Ferrochelatase is the terminal enzyme of haem biosynthesis, catalysing the insertion of ferrous iron into the macrocycle of protoporphyrin IX, the last common intermediate of haem and chlorophyll synthesis. Its activity has been reported in both plastids and mitochondria of higher plants, but the relative amounts of the enzyme in the two organelles are unknown. Ferrochelatase is difficult to assay since ferrous iron requires strict anaerobic conditions to prevent oxidation, and in photosynthetic tissues chlorophyll interferes with the quantification of the product. Accordingly, we developed a sensitive fluorimetric assay for ferrochelatase that employs Co(2+) and deuteroporphyrin in place of the natural substrates, and measures the decrease in deuteroporphyrin fluorescence. A hexane-extraction step to remove chlorophyll is included for green tissue. The assay is linear over a range of chloroplast protein concentrations, with an average specific activity of 0.68 nmol x min(-1) x mg of protein(-1), the highest yet reported. The corresponding value for mitochondria is 0.19 nmol x min(-1) x mg of protein(-1). The enzyme is inhibited by N-methylprotoporphyrin, with an estimated IC(50) value of approximately 1 nM. Using this assay we have quantified ferrochelatase activity in plastids and mitochondria from green pea leaves, etiolated pea leaves and pea roots to determine the relative amounts in the two organelles. We found that, in all three tissues, greater than 90% of the activity was associated with plastids, but ferrochelatase was reproducibly detected in mitochondria, at levels greater than the contaminating plastid marker enzyme, and was latent. Our results indicate that plastids are the major site of haem biosynthesis in higher plant cells, but that mitochondria also have the capacity for haem production.
Asunto(s)
Ferroquelatasa/metabolismo , Hemo/biosíntesis , Fotosíntesis/fisiología , Pisum sativum/enzimología , Cloroplastos/metabolismo , Sulfato de Cobre/farmacología , Ferroquelatasa/análisis , Concentración de Iones de Hidrógeno , Cinética , Mitocondrias/metabolismo , Pisum sativum/citología , Plantas/enzimología , Plastidios/enzimología , Porfirinas/farmacología , Protoporfirinas/metabolismoRESUMEN
In order to study effects of environmental contamination, a suite of biomarkers were measured over the period 1996 to 1999 in livers of flounder (Platichthys flesus) from two urban embayments and one non-urban reference site of the Gulf of Finland in the vicinity of Tallinn, Estonia. Total cytochrome P450 (CYP) level, aryl hydrocarbon hydroxylase (AHH), 5-aminolevulinic acid synthetase (ALA-S), and heme synthetase (HEM-S) activities were quantified by means of spectrophotometry. These data were compared to results obtained in 1994 for the same biomarkers at one of the urban embayments and the non-urban site, as measured by the same protocols. For the flounder collected from the non-urban site, changes occurred in AHH activity and the total CYP level, which were significantly lower in 1996 and 1999 compared with 1994 (p < 0.05). Activity of ALA-S decreased slightly over this same period. The activity of HEM-S increased between 1996 and 1999. In the urban site first investigated in 1994, the activities of AHH and ALA-S, as well as the total level of CYP in flounder liver were significantly higher compared with 1999 (p < 0.05). HEM-S activities did not show any significant changes over this time period. AHH activities of flounder collected in another urban site decreased slightly between 1996 and 1999, in contrast to data on the total CYP level which diminished drastically over these years (p < 0.05). Activities of HEM-S increased significantly (p < 0.05) during the period investigated, while activities of ALA-S remained unchanged. These findings suggest that contamination of the marine environments by PAHs has gone down everywhere in the Tallinn area during the last 3 to 5 years. However, the results indicate that the area is still contaminated, as indicated by elevated heme synthesis enzymes and the total CYP content, and that monitoring of contaminants and their effects should be continued in this region.
Asunto(s)
5-Aminolevulinato Sintetasa/farmacología , Hidrocarburo de Aril Hidroxilasas/farmacología , Biomarcadores/análisis , Carcinógenos/análisis , Sistema Enzimático del Citocromo P-450/farmacología , Exposición a Riesgos Ambientales , Ferroquelatasa/farmacología , Lenguado/fisiología , Contaminantes Químicos del Agua/análisis , 5-Aminolevulinato Sintetasa/análisis , Animales , Hidrocarburo de Aril Hidroxilasas/análisis , Carcinógenos/efectos adversos , Ciudades , Sistema Enzimático del Citocromo P-450/análisis , Ferroquelatasa/análisis , Hígado/enzimología , Contaminantes Químicos del Agua/efectos adversosRESUMEN
Apocytochrome C550 was detected in the periplasm of a new mutant of Paracoccus denitrificans, HN48, that is pleiotropically lacking c-type cytochromes, produces reduced levels of siderophores and carries a Tn5 insertion in the ccmF gene for which sequence data, along with that for the contiguous ccmH, are reported. A counterpart to the ccmF gene was found in an archaebacterium but could not be located in the yeast genome, whereas mitochondrial haem lyases in the latter were not present in an archaeobacterial or in eubacterial genomes. A topological analysis for CcmF is presented which indicates at least eleven transmembrane helices, suggesting a role as a transporter; evidence against the substrate being haem is presented but sequence similarity with Escherichia coli gamma-aminobutyric acid transporter was identified. Analysis by pulse-chase methodology has shown that, in this and another cytochrome-c-deficient mutant, the apo form of P. denitrificans cytochrome C550 is much less stable than the holo form, directly demonstrating the presence of a periplasmic degradation system in P. denitrificans that removes non-functional proteins. A variety of phenotypes are observed for P. denitrificans mutated in different ccm genes, thus indicating that the stability of the ccm gene products does not require assembly of a complex of all the Ccm proteins.
Asunto(s)
Apoproteínas/metabolismo , Proteínas Bacterianas/genética , Proteínas Bacterianas/metabolismo , Grupo Citocromo c/metabolismo , Proteínas de la Membrana/genética , Proteínas de la Membrana/metabolismo , Paracoccus denitrificans/genética , Paracoccus denitrificans/metabolismo , Sideróforos/metabolismo , Secuencia de Aminoácidos , Archaea/genética , Proteínas Bacterianas/análisis , Proteínas Portadoras/genética , Proteínas Portadoras/metabolismo , Mapeo Cromosómico , Clonación Molecular , Elementos Transponibles de ADN , ADN Bacteriano/análisis , ADN Bacteriano/genética , Escherichia coli/genética , Escherichia coli/metabolismo , Ferroquelatasa/análisis , Ferroquelatasa/metabolismo , Genoma Bacteriano , Genoma Fúngico , Hemo/metabolismo , Proteínas de la Membrana/análisis , Datos de Secuencia Molecular , Mutagénesis Insercional , Paracoccus denitrificans/enzimología , Periplasma/enzimología , Periplasma/metabolismo , Plásmidos , Reacción en Cadena de la Polimerasa , Alineación de Secuencia , Análisis de Secuencia de ADN , Levaduras/genética , Ácido gamma-Aminobutírico/metabolismoRESUMEN
Promastigotes of Leishmania donovani (Dd-8 strain) showed presence of important key enzymes of heme synthesizing (delta-aminolevulinic acid synthase and ferrochelatase) and degrading (heme oxygenase and biliverdin reductase) systems, classical leishmanicidal drugs viz allopurinol, amphotericin B, pentamidine and CDRI compound 93/202 inhibited the heme oxygenase activity of the parasite, whereas, delta-aminolevulinic acid synthase activity practically remained unaffected. The Km, Vmax and pH values of heme oxygenase of promastigotes were found to be 1666 microM hemin, 625 nmol of bilirubin formed h-1 mg protein-1 and 7.5 respectively. The findings suggest the presence and importance of heme metabolism in the de novo synthesis of different hemoproteins of the Leishmania parasite as well as the detoxification and its defence against biological insults.
Asunto(s)
5-Aminolevulinato Sintetasa/análisis , Ferroquelatasa/análisis , Hemo Oxigenasa (Desciclizante)/análisis , Leishmania donovani/enzimología , Oxidorreductasas actuantes sobre Donantes de Grupo CH-CH , Proteínas Protozoarias/análisis , Animales , Concentración de Iones de Hidrógeno/efectos de los fármacos , Oxidorreductasas/metabolismoRESUMEN
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.
Asunto(s)
Escherichia coli/metabolismo , Hemo/biosíntesis , 5-Aminolevulinato Sintetasa/genética , 5-Aminolevulinato Sintetasa/metabolismo , Ácido Aminolevulínico/metabolismo , Animales , Coproporfirinógeno Oxidasa/análisis , Retroalimentación , Ferroquelatasa/análisis , Regulación Enzimológica de la Expresión Génica , Ácido Glutámico/metabolismo , Hidroximetilbilano Sintasa/metabolismo , Ratones , Porfirinas/biosíntesis , Pirroles/metabolismo , Ratas , Proteínas Recombinantes/metabolismo , TetrapirrolesRESUMEN
Porphyrin metabolism was studied in 21 children of both sexes suffering from acute lymphoblastic leukaemia (ALL) and 34 adult patients of different ages and sexes suffering from ALL (n = 14), non-Hodgkin's lymphoma (NHL), n = 14, or Hodgkin's disease (HD), n = 6. In addition, two groups of healthy children (n = 14), and adults (n = 17) were studied for comparison. It was apparent from this study that the activity of uroporphyrinogen-1-synthetase (URO-1-S, E.C. 4.3.1.8) was highly significantly activated in the blood of children, while the activities of blood 5-aminolevulinic acid dehydrase (E.C. 4.2.1.24) and ferrochelatase (E.C. 4.99.1.1.), as expressed by protoporphyrin/haem ratio, were inhibited in those children. Also, free erythrocyte total porphyrins were increased, while the haem content was reduced. The concentrations of 5-aminolevulinic acid, coproporphyprin and uroporphyrin were increased in the urine of children with ALL. On the other hand, some dramatic changes were found in porphyrin metabolism in adult patients suffering from ALL, NHL and HD. The aforementioned disturbances were discussed in the light of some factors which may affect the enzymatic activities in the synthesis of porphyrins.
Asunto(s)
Enfermedad de Hodgkin/metabolismo , Linfoma no Hodgkin/metabolismo , Porfirinas/metabolismo , Leucemia-Linfoma Linfoblástico de Células Precursoras/metabolismo , Adulto , Ácido Aminolevulínico/orina , Niño , Coproporfirinas/orina , Eritrocitos/enzimología , Femenino , Ferroquelatasa/análisis , Hemo/biosíntesis , Humanos , Hidroximetilbilano Sintasa/análisis , Masculino , Porfobilinógeno/orina , Porfobilinógeno Sintasa/análisis , Uroporfirinas/orinaRESUMEN
Protoporphyria is a hereditary disorder characterized by a marked decrease in the activity of ferrochelatase, the terminal enzyme in the heme biosynthetic pathway. We have prepared specific polyvalent antibodies against bovine ferrochelatase in rabbits. The specificity of the antibody preparation against ferrochelatase was demonstrated by western blot analysis and immunoprecipitation of ferrochelatase activity. The antibody also cross-reacted weakly with ferrochelatase from human mitochondria. To quantify immunoreactive ferrochelatase in tissue samples, a kinetic-based enzyme-linked immunosorbent assay (k-ELISA) was developed. Ferrochelatase activity and the level of immunoreactive protein were measured in hepatic mitochondria isolated from six normal and nine protoporphyric (homozygous) cattle. Ferrochelatase activity was less than 10% of normal in mitochondria from protoporphyric animals; the amount of immunoreactive material was equivalent to that from normal animals. Similar studies were performed with samples from three normal and two protoporphyric (heterozygous) humans. Ferrochelatase activity was decreased in protoporphyric samples (about 17% of normal, but there was no concomitant decrease in immunoreactive material. These data demonstrate that a normal amount of ferrochelatase protein is present and suggest that bovine and human protoporphyria result from point mutations in the gene encoding ferrochelatase.
Asunto(s)
Ferroquelatasa/genética , Hepatopatías/genética , Mutación , Porfirias/genética , Protoporfirinas/metabolismo , Animales , Western Blotting , Bovinos , Ensayo de Inmunoadsorción Enzimática , Ferroquelatasa/análisis , Humanos , Hepatopatías/enzimología , Hepatopatías/metabolismo , Microsomas Hepáticos/enzimología , Porfirias/enzimología , Porfirias/metabolismo , Protoporfiria Eritropoyética , Succinato Deshidrogenasa/análisisAsunto(s)
Eritropoyetina/farmacología , Hemo/biosíntesis , Hidroximetilbilano Sintasa/biosíntesis , 5-Aminolevulinato Sintetasa/análisis , Animales , Médula Ósea/patología , Hipoxia de la Célula , Células Cultivadas , Inducción Enzimática/efectos de los fármacos , Células Precursoras Eritroides/efectos de los fármacos , Células Precursoras Eritroides/metabolismo , Ferroquelatasa/análisis , Regulación de la Expresión Génica/efectos de los fármacos , Hemo/genética , Hemoglobinas/biosíntesis , Hidroximetilbilano Sintasa/análisis , Hiperplasia , Policitemia/metabolismo , Policitemia/patología , Ratas , Estimulación Química , Transaminasas/análisisRESUMEN
1. Baboon ferrochelatase was purified to apparent homogeneity. 2. The pH optimum was 7.85 and the pI 5.3. 3. The estimated molecular weight was 205 K made up by two 50 + 60 K heterodimers. 4. The Km values for proto- and mesoporphyrin were 18.5 and 10.8 microM with iron as co-substrate. With cobalt as co-substrate the Km values were 34.5 and 10.4 microM, respectively. The mean Km value for iron was 2.2 microM while cobalt acted as a complete inhibitor. 5. Lead played a dual role that of both pseudo substrate and inhibitor. As shown by inhibitor kinetics, Pb acted as a two-step two-site parabolic competitive inhibitor. The mean Ki value at low Pb levels was 0.65 mM and at high levels 0.17 mM. 6. Substrate inhibition occurred above 36 microM for proto- and 44 microM for mesoporphyrin with iron as co-substrate. For iron, with mesoporphyrin as co-substrate it occurred above 29 microM.
Asunto(s)
Ferroquelatasa/análisis , Liasas/análisis , Proteínas de la Membrana/análisis , Mitocondrias Hepáticas/enzimología , Animales , Cromatografía de Afinidad , Electroforesis en Gel de Poliacrilamida , Activación Enzimática , Ferroquelatasa/antagonistas & inhibidores , Ferroquelatasa/aislamiento & purificación , Concentración de Iones de Hidrógeno , Punto Isoeléctrico , Plomo/farmacología , Proteínas de la Membrana/aislamiento & purificación , Metales/metabolismo , Peso Molecular , Oxígeno/farmacología , Papio , Solubilidad , Especificidad por SustratoRESUMEN
Intraperitoneal heme treatment (5 mg/kg body wt) reversed the effects of a preceding (2 h) intraperitoneally injected Na2 S (150 mumol/kg body wt) dose on blood delta-amino levulinic acid synthase and heme synthase activities. The sulfide dosing caused decreased activity of both enzymes of which that of heme synthase was overcorrected above controls by the heme treatment 25 h after the sulfide dose. Heme alone caused a transient induction in the heme oxygenase activity in liver 23 h after the injection. The effects on heme synthesis in the sulphide-dosed rats support the role of the inactivation of the heme-controlled translational inhibitor of protein synthesis by the supply of exogenous heme.
Asunto(s)
Hemo/metabolismo , Sulfuros/toxicidad , 5-Aminolevulinato Sintetasa/análisis , Animales , Ferroquelatasa/análisis , Hemo/farmacología , Masculino , Ratas , Ratas EndogámicasRESUMEN
Protoporphyrinogen oxidase, the penultimate enzyme in the haem biosynthetic pathway has been purified to apparent homogeneity from bovine liver mitochondria, by a published method (Dailey, H.A. and Fleming, J.E., (1983)), with an additional ion-exchange chromatography step, using a Mono Q column on an FPLC-system. This gave a product with a 68% yield and 870-fold purification. Protoporphyrinogen oxidase (EC 1.3.3.4) has an apparent Mr of 57,000 and the Km for protoporphyrinogen IX was 16.6 microM. Activity of the isolated enzyme was increased by 66% in the presence of oleic acid, and evidence was obtained for a FAD prosthetic group. Ferrochelatase (EC 4.99.1.1) was purified and antibodies were raised in rabbits against ferrochelatase and protoporphyrinogen oxidase, respectively. Anti-protoporphyrinogen oxidase IgG showed marked cross-reactivity with ferrochelatase and anti-ferrochelatase IgG cross-reacted with protoporphyrinogen oxidase. In addition, radiolabelled peptides of both enzymes, generated by chymotrypsin, demonstrated common peptides when analysed by two-dimensional chromatography.
Asunto(s)
Ferroquelatasa/análisis , Liasas/análisis , Oxidorreductasas actuantes sobre Donantes de Grupo CH-CH , Oxidorreductasas/aislamiento & purificación , Animales , Bovinos , Quimotripsina , Reacciones Cruzadas , Epítopos , Ferroquelatasa/inmunología , Cinética , Mitocondrias Hepáticas/enzimología , Oxidorreductasas/inmunología , Oxidorreductasas/metabolismo , Mapeo Peptídico , Protoporfirinógeno-Oxidasa , Análisis EspectralRESUMEN
A rapid, reliable, sensitive and reproducible HPLC method was developed for the assay of ferrochelatase activity in rat liver. The assay was carried out aerobically with Zn2+ and mesoporphyrin or protoporphyrin IX as substrates. Zn-porphyrins formed were extracted with dimethyl sulphoxide/methanol (30:70, v/v) containing Zn-deuteroporphyrin as the internal standard for separation and quantification by reversed-phase chromatography. The Km for mesoporphyrin was 5.9 microM, for protoporphyrin IX 8.8 microM and for zinc 6.0 microM. The specific activities were 33.1 +/- 5.0 nmol Zn-mesoporphyrin or 13.4 +/- 2.0 nmol Zn-protoporphyrin formed per hour per mg of protein for mitochondria and 12.3 +/- 2.2 nmol Zn-mesoporphyrin or 4.6 +/- 0.9 nmol Zn-protoporphyrin per hour per mg of protein for liver homogenate.
Asunto(s)
Ferroquelatasa/análisis , Hígado/análisis , Liasas/análisis , Animales , Cromatografía Líquida de Alta Presión/métodos , Femenino , Concentración de Iones de Hidrógeno , Cinética , Porfirinas/análisis , Ratas , Ratas EndogámicasRESUMEN
Purified ferrochelatase (protoheme ferrolyase; EC 4.99.1.1) from the bacterium Rhodopseudomonas sphaeroides was examined to determine the roles of cationic and sulfhydryl residues in substrate binding. Reaction of the enzyme sulfhydryl residues with N-ethylmaleimide or monobromobimane resulted in a rapid loss of enzyme activity. Ferrous iron, but not porphyrin substrate, had a protective effect against inactivation by these two reagents. Quantitation with 3H-labeled N-ethylmaleimide revealed that inactivation required one to two sulfhydryl groups to be modified. Modification of arginyl residues with either 2,3-butanedione or camphorquinone 10-sulfonate resulted in a loss of ferrochelatase activity. A kinetic analysis of the modified enzyme showed that the Km for ferrous iron was not altered but that the Km for the porphyrin substrate was increased. These data suggested that arginyl residues may be involved in porphyrin binding, possibly via charge pair interactions between the arginyl residue and the anionic porphyrin propionate side chain. Modification of lysyl residues had no effect on enzyme activity. We also examined the ability of bacterial ferrochelatase to use various 2,4-disubstituted porphyrins as substrates. We found that 2,4-bis-acetal- and 2,4-disulfonate deuteroporphyrins were effective substrates for the purified bacterial enzyme and that N-methylprotoporphyrin was an effective inhibitor of the enzyme. Our data for the ferrochelatase of R. sphaeroides are compared with previously published data for the eucaryotic enzyme.
Asunto(s)
Arginina/fisiología , Ferroquelatasa/análisis , Liasas/análisis , Rhodobacter sphaeroides/enzimología , Compuestos de Sulfhidrilo , Diacetil/farmacología , Cinética , Especificidad por SustratoRESUMEN
Cobalt protoporphyrin generated from 5-amino[4-14C]laevulinate by homogenates or primary cultures of chick embryo liver exposed to CoCl2 was found to be radioactivity unextractable by acid/acetone, when extra protein was added. The activity of ferrochelatase was required for formation of cobalt protoporphyrin since inhibition of ferrochelatase with 3,5-diethoxycarbonyl-1,4-dihydrocollidine (in the presence of cycloheximide) inhibited formation of cobalt protoporphyrin and resulted in accumulation of protoporphyrin. Cobalt protoporphyrin was detected spectrophotometrically in hepatocyte cultures exposed to the combination of 2-allyl-2-isopropylacetamide and CoCl2: (1) as the pyridine haemochrome of the protein pellet remaining after acid-acetone extraction of the cells, or (2) as the material extracted from the protein pellet with acetic acid-pyridine-chloroform. The amount of cobalt protoporphyrin increased with increasing CoCl2 concentration as cellular haem declined. The decrease in haem was about equal to the amount of cobalt protoporphyrin that accumulated. 2-Allyl-2-isopropylacetamide and polychlorinated biphenyls were both powerful inducers of 5-aminolaevulinate synthase. The former led to protoporphyrin accumulation, whereas with the latter, uroporphyrin accumulated, probably due to a concomitant decrease in activity of uroporphyrinogen decarboxylase. The decrease in activity of 5-aminolaevulinate synthase produced by administration of CoCl2 was greater after treatment with 2-allyl-2-isopropylacetamide than after treatment with allylisopropylacetamide and 3,4,3',4'-tetrachlorobiphenyl. We conclude: (a) that cobalt protoporphyrin is readily formed in cultured hepatocytes, and (b) that its formation accounts for the action of cobalt on 5-aminolaevulinate synthase.
Asunto(s)
5-Aminolevulinato Sintetasa/antagonistas & inhibidores , Cobalto/metabolismo , Hígado/metabolismo , Porfirinas/metabolismo , Protoporfirinas/metabolismo , Animales , Embrión de Pollo , Cobalto/farmacología , Dicarbetoxidihidrocolidina/farmacología , Ferroquelatasa/análisis , Técnicas In VitroRESUMEN
A radiochemical assay for heme synthase (ferrochelatase) activity is described in this report. The principle of the assay is to measure the incorporation of 59Fe, added to the reaction flask with a known amount of iron, into heme. Iron is maintained in the ferrous state, and oxygen is excluded from the flask during the incubation of the substrates with enzyme. Labeled heme product is extracted by solvent partitioning and counted in a gamma scintillation spectrometer. The assay can detect picomole amounts of product and is most useful when low levels of heme synthase activity are present.
Asunto(s)
Ferroquelatasa/análisis , Liasas/análisis , Animales , Fibroblastos , Congelación , Técnicas In Vitro , Hierro/metabolismo , Radioisótopos de Hierro , Hígado/metabolismo , Masculino , Mitocondrias Hepáticas/enzimología , Porfirinas/metabolismo , Ratas , Ratas EndogámicasRESUMEN
The activities of 5 enzymes of the haem biosynthetic pathway and the protoporphyrin concentrations have been measured in peripheral red blood cells of 23 patients having a preleukaemic syndrome with refractory sideroblastic anaemia. A decreased delta-aminolaevulinic acid synthase (ALA-S) activity, an increased uroporphyrinogen I synthase activity and an increased red cell protoporphyrin concentration were consistent findings. Patients with abnormal leucocyte and/or platelet counts in the peripheral blood as well as patients with an excess of blast cells in the bone marrow had the lowest ALA-S activities. A further decrease in ALA-S activity was observed in 3 patients after leukaemic change in the disease. Patients having cytogenetic abnormalities showed no unique enzyme abnormalities. These results indicate that enzymatic disturbances of haem synthesis cannot be used as prognostic indicator of leukaemic transformation in refractory sideroblastic anaemia, but a very low ALA-S activity appears to accompany the development of a leukaemia in such patients.