RESUMO
Enzymes that bear a nonnative or artificially introduced metal center can engender novel reactivity and enable new spectroscopic and structural studies. In the case of metal-organic cofactors, such as metalloporphyrins, no general methods exist to build and incorporate new-to-nature cofactor analogs in vivo. We report here that a common laboratory strain, Escherichia coli BL21(DE3), biosynthesizes cobalt protoporphyrin IX (CoPPIX) under iron-limited, cobalt-rich growth conditions. In supplemented minimal media containing CoCl2, the metabolically produced CoPPIX is directly incorporated into multiple hemoproteins in place of native heme b (FePPIX). Five cobalt-substituted proteins were successfully expressed with this new-to-nature cobalt porphyrin cofactor: myoglobin H64V V68A, dye decolorizing peroxidase, aldoxime dehydratase, cytochrome P450 119, and catalase. We show conclusively that these proteins incorporate CoPPIX, with the CoPPIX making up at least 95% of the total porphyrin content. In cases in which the native metal ligand is a sulfur or nitrogen, spectroscopic parameters are consistent with retention of native metal ligands. This method is an improvement on previous approaches with respect to both yield and ease-of-implementation. Significantly, this method overcomes a long-standing challenge to incorporate nonnatural cofactors through de novo biosynthesis. By utilizing a ubiquitous laboratory strain, this process will facilitate spectroscopic studies and the development of enzymes for CoPPIX-mediated biocatalysis.
Assuntos
Metaloporfirinas/química , Porfirinas/biossíntese , Porfirinas/química , Biocatálise , Cobalto/química , Cobalto/metabolismo , Escherichia coli/metabolismo , Proteínas de Escherichia coli/metabolismo , Heme/metabolismo , Ferro , Metais/química , Mioglobina/química , Protoporfirinas/biossíntese , Protoporfirinas/químicaRESUMO
Porphyrins are intermediate metabolites in the biosynthesis of vital molecules, including heme, cobalamin, and chlorophyll. Bacterial porphyrins are known to be proinflammatory, with high levels linked to inflammatory skin diseases. Propionibacterium species are dominant skin commensals and play essential roles in defending against pathogens and in triggering an inflammatory response. To better understand how the inflammatory potential of the skin microbiome may vary depending on its propionibacterial composition, we compared the production levels of porphyrins among Propionibacterium acnes, Propionibacterium granulosum, Propionibacterium avidum, and Propionibacterium humerusii strains. We found that porphyrin production varied among these species, with P. acnes type I strains producing significantly larger amounts of porphyrins than P. acnes type II and III strains and other Propionibacterium species. P. acnes strains that are highly associated with the common skin condition acne vulgaris responded to vitamin B12 supplementation with significantly higher porphyrin production. In contrast, vitamin B12 supplementation had no effect on the porphyrin production of health-associated P. acnes strains and other propionibacteria. We observed low-level porphyrin production in most Propionibacterium strains harboring the deoR repressor gene, with the exception of P. acnes strains belonging to type I clades IB-3 and IC. Our findings shed light on the proinflammatory potential of distinct phylogenetic lineages of P. acnes as well as other resident skin propionibacteria. We demonstrate that the overall species and strain composition is important in determining the metabolic output of the skin microbiome in health and disease.IMPORTANCE Porphyrins are a group of metabolites essential to the biosynthesis of heme, cobalamin, and chlorophyll in living organisms. Bacterial porphyrins can be proinflammatory, with high levels linked to human inflammatory diseases, including the common skin condition acne vulgaris. Propionibacteria are among the most abundant skin bacteria. Variations in propionibacteria composition on the skin may lead to different porphyrin levels and inflammatory potentials. This study characterized porphyrin production in all lineages of Propionibacterium acnes, the most dominant skin Propionibacterium, and other resident skin propionibacteria, including P. granulosum, P. avidum, and P. humerusii We revealed that P. acnes type I strains produced significantly more porphyrins than did type II and III strains and other Propionibacterium species. The findings from this study shed light on the proinflammatory potential of the skin microbiome and can be used to guide the development of effective acne treatments by modulating the skin microbiome and its metabolic activities.
Assuntos
Porfirinas/biossíntese , Propionibacterium/metabolismo , Pele/microbiologia , Humanos , Microbiota , Filogenia , Propionibacteriaceae/metabolismo , Propionibacterium/classificação , Propionibacterium acnes/metabolismoRESUMO
BACKGROUND: Increasing antibiotic resistance among pathogens has raised the demands for new treatment methods such as antimicrobial photodynamic therapy (aPDT) and phototherapy (PT). Experiments for investigating the effects of these methods are often performed in vitro, but the procedures for cultivation of microbes vary between different studies. The aim of this study has been to elucidate how the profile of endogenously produced porphyrins differs by changing the variables of bacteria culturing conditions. METHODS: Two oral pathogens, Aggregatibacter actinomycetemcomitans and Porphyromonas gingivalis, were selected as model organisms. The contents of porphyrins and heme in the bacteria were analysed with liquid chromatography-tandem mass spectrometry when bacteria was cultivated for different lengths of time (3-9 days), upon passaging as well as when growth medium were supplemented with or without horse blood. RESULTS: Both porphyrin and heme content in A. actinomycetemcomitans are highly affected by the age of the culture, and that the porphyrin profiles changes during cultivation. When cultivated colonies of A. actinomycetemcomitans were passaged onto a new, fresh growth medium a large change in porphyrin content occurred. Additional porphyrins were detected; uroporphyrin and 7-carboxylporphyrin, and the total porphyrin content increased up to 28 times. When P. gingivalis was grown on blood containing medium higher concentrations of protoporphyrin IX (2.5 times) and heme (5.4 times) were quantified compared to bacteria grown without blood. CONCLUSIONS: This study demonstrate that there is a need for more standardized culturing protocols when performing aPDT and PT experiments in vitro to avoid large variations in porphyrin profiles and concentrations, the aPDT/PT target compounds, depending on the culturing conditions.
Assuntos
Aggregatibacter actinomycetemcomitans/metabolismo , Técnicas de Cultura de Células/métodos , Porfirinas/biossíntese , Porphyromonas gingivalis/metabolismo , Heme/biossíntese , HumanosRESUMO
Various diseases have been linked to the human microbiota, but the underlying molecular mechanisms of the microbiota in disease pathogenesis are often poorly understood. Using acne as a disease model, we aimed to understand the molecular response of the skin microbiota to host metabolite signaling in disease pathogenesis. Metatranscriptomic analysis revealed that the transcriptional profiles of the skin microbiota separated acne patients from healthy individuals. The vitamin B12 biosynthesis pathway in the skin bacterium Propionibacterium acnes was significantly down-regulated in acne patients. We hypothesized that host vitamin B12 modulates the activities of the skin microbiota and contributes to acne pathogenesis. To test this hypothesis, we analyzed the skin microbiota in healthy subjects supplemented with vitamin B12. We found that the supplementation repressed the expression of vitamin B12 biosynthesis genes in P. acnes and altered the transcriptome of the skin microbiota. One of the 10 subjects studied developed acne 1 week after vitamin B12 supplementation. To further understand the molecular mechanism, we revealed that vitamin B12 supplementation in P. acnes cultures promoted the production of porphyrins, which have been shown to induce inflammation in acne. Our findings suggest a new bacterial pathogenesis pathway in acne and provide one molecular explanation for the long-standing clinical observation that vitamin B12 supplementation leads to acne development in a subset of individuals. Our study discovered that vitamin B12, an essential nutrient in humans, modulates the transcriptional activities of skin bacteria, and provided evidence that metabolite-mediated interactions between the host and the skin microbiota play essential roles in disease development.
Assuntos
Acne Vulgar/microbiologia , Acne Vulgar/patologia , Microbiota/genética , Pele/microbiologia , Transcriptoma/genética , Vitamina B 12/farmacologia , Adulto , Estudos de Casos e Controles , Suplementos Nutricionais , Regulação para Baixo/efeitos dos fármacos , Feminino , Perfilação da Expressão Gênica , Humanos , Masculino , Redes e Vias Metabólicas/efeitos dos fármacos , Microbiota/efeitos dos fármacos , Modelos Biológicos , Óperon/genética , Porfirinas/biossíntese , Propionibacterium acnes/efeitos dos fármacos , Propionibacterium acnes/genética , Transcrição Gênica/efeitos dos fármacos , Transcriptoma/efeitos dos fármacos , Vitamina B 12/biossíntese , Adulto JovemRESUMO
In a previous paper, we pointed out that the capability to synthesize glycine from serine is constrained by the stoichiometry of the glycine hydroxymethyltransferase reaction, which limits the amount of glycine produced to be no more than equimolar with the amount of C 1 units produced. This constraint predicts a shortage of available glycine if there are no adequate compensating processes. Here, we test this prediction by comparing all reported fl uxes for the production and consumption of glycine in a human adult. Detailed assessment of all possible sources of glycine shows that synthesis from serine accounts for more than 85% of the total, and that the amount of glycine available from synthesis, about 3 g/day, together with that available from the diet, in the range 1.5-3.0 g/day, may fall significantly short of the amount needed for all metabolic uses, including collagen synthesis by about 10 g per day for a 70 kg human. This result supports earlier suggestions in the literature that glycine is a semi-essential amino acid and that it should be taken as a nutritional supplement to guarantee a healthy metabolism.
Assuntos
Colágeno/biossíntese , Metabolismo Energético , Glicina/biossíntese , Adulto , Animais , Isótopos de Carbono/metabolismo , Dieta , Glutationa/metabolismo , Glicina Hidroximetiltransferase/metabolismo , Glioxilatos/metabolismo , Humanos , Estrutura Molecular , Porfirinas/biossíntese , Serina/metabolismoRESUMO
INTRODUCTION: Acne vulgaris is a multifactorial disease of the pilosebaceous unit characterized by the development of inflammatory (papules, pustules, cysts) and/or non inflammatory lesions (open and closed comedones) that may progress to scars. The increase of bacterial resistances, adverse effects and teratogenicity of retinoids and lack of response to usual therapies have led to investigate new therapeutic alternatives for acne. MATERIAL AND METHOD: We studied 36 patients with mild to moderate acne vulgaris. We performed treatment every 4 weeks using pulsed dye laser therapy with a wavelength of 585 nm and pulse duration of 350 microseconds. RESULTS: At twelve weeks of treatment a decrease of 27 % of non inflammatory lesions and of 57 % of active lesions was observed. Treatment was well tolerated and considered positive, in terms of healing, in 25 patients. CONCLUSIONS: Pulse dye laser therapy mainly improves inflammatory lesions of acne with few adverse effects.
Assuntos
Acne Vulgar/terapia , Terapia com Luz de Baixa Intensidade , Propionibacterium acnes/efeitos da radiação , Acne Vulgar/microbiologia , Adolescente , Adulto , Feminino , Humanos , Masculino , Aceitação pelo Paciente de Cuidados de Saúde , Porfirinas/biossíntese , Porfirinas/efeitos da radiação , Propionibacterium acnes/metabolismo , Tolerância a Radiação , Resultado do TratamentoRESUMO
5-Aminolevulinic acid (ALA) or its derivative methyl 5-aminolevulinate (MAL) combined with folic acid was applied in nude mice bearing human colon adenocarcinoma. The aim of the study is to see whether folic acid may increase biosynthesis of porphyrins in tumor tissue after systemic or topical administration of ALA or MAL. The production of porphyrins was determined by spectrofluorometric measurements with an optical fibre probe. It was found that the porphyrin production after i.p injection of 200 mg kg(-1) ALA or MAL was significantly increased by i.p injection of 100 mg kg(-1) folic acid. However, in the case of topically applied 20% ALA, folic acid had no effect. In the case of topically applied 20% MAL, folic acid (i.p or topically applied) reduced the porphyrin synthesis. This might be used for the protection of normal skin against photosensitization. The effects of folic acid were similar in tumors and normal skin. Two mechanisms may explain the results: enhancement of the efficiency of the rate-limiting enzyme porphobilinogen deaminase by folic acid or interference of folic acid with the transport of ALA and MAL to and into the cells synthesizing porphyrins in the tissues. The present data seem to favour the latter mechanism. Folic acid may have a role as an adjuvant in photodynamic therapy with systemically administered ALA and its derivatives.
Assuntos
Adenocarcinoma/tratamento farmacológico , Ácido Aminolevulínico/análogos & derivados , Ácido Aminolevulínico/administração & dosagem , Neoplasias do Colo/tratamento farmacológico , Ácido Fólico/administração & dosagem , Porfirinas/biossíntese , Pele/efeitos dos fármacos , Adenocarcinoma/metabolismo , Ácido Aminolevulínico/química , Animais , Neoplasias do Colo/metabolismo , Feminino , Ácido Fólico/química , Ácido Fólico/farmacocinética , Humanos , Injeções Intraperitoneais , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Nus , Fotoquimioterapia/métodos , Porfirinas/química , Sensibilidade e Especificidade , Pele/metabolismo , Espectrometria de Fluorescência , Fatores de Tempo , Transplante Heterólogo , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
BACKGROUND/AIMS: 5-Aminolevulinic acid (ALA), a precursor of porphyrins is used for photodynamic diagnosis and therapy within topical or systemic applications. A potential toxic effect on the human liver is of major interest and therefore we investigated the impact of a repeated application of ALA without illumination on cultures of human hepatocytes. METHODS: After ALA treatment of hepatocytes in vitro the porphyrin synthesis, albumin secretion, liver-specific enzyme release, and malondialdehyde levels were determined. In order to reduce levels of reactive oxygen substances, mannitol and the antioxidant enzymes superoxide dismutase and catalase were supplemented. RESULTS: Porphyrin biosynthesis by human hepatocytes in vitro was repeatedly stimulated by ALA (0.001-1.0 mM), which was indicated by an accumulation of protoporphyrin IX. A repetitive treatment (up to four times) of hepatocytes with ALA resulted in an impairment of the hepatic function and viability, depending on the ALA concentration (0.1-1.0 mM) and frequency of application (2-3 times). This was also accompanied by increased malondialdehyde levels indicating enhanced lipid peroxidation. Only superoxide dismutase was able to reduce cellular damage and prevent specific function. CONCLUSIONS: Repeated, not single, ALA treatment without illumination may cause deleterious effects to the liver, which are mediated by oxygen radicals and inhibited by an antioxidant.
Assuntos
Ácido Aminolevulínico/farmacologia , Hepatócitos/efeitos dos fármacos , Hepatócitos/metabolismo , Fármacos Fotossensibilizantes/farmacologia , Antioxidantes/farmacologia , Sobrevivência Celular/efeitos dos fármacos , Células Cultivadas , Relação Dose-Resposta a Droga , Hepatócitos/citologia , Humanos , Técnicas In Vitro , Peroxidação de Lipídeos/efeitos dos fármacos , Masculino , Porfirinas/biossíntese , Espécies Reativas de Oxigênio/metabolismo , Superóxido Dismutase/farmacologiaRESUMO
Current models for regulation of heme synthesis during erythropoiesis propose that the first enzyme of the pathway, 5-aminolevulinate synthase (ALAS), is the rate-limiting enzyme. We have examined cellular porphyrin excretion in differentiating murine erythroleukemia cells to determine in situ rate-limiting steps in heme biosynthesis. The data demonstrate that low levels of coproporphyrin and protoporphyrin accumulate in the culture medium under normal growth conditions and that during erythroid differentiation the level of excretion of coproporphyrin increases approximately 100-fold. Iron supplementation lowered, but did not eliminate, porphyrin accumulation. While ALAS induction is necessary for increased heme synthesis, these data indicate that other enzymes, in particular coproporphyrinogen oxidase, represent down-stream rate-limiting steps.
Assuntos
Células Precursoras Eritroides/metabolismo , Heme/biossíntese , Ácido Aminolevulínico/metabolismo , Animais , Inibidores Enzimáticos/farmacologia , Eritropoese , Heptanoatos/farmacologia , Ferro/metabolismo , Cinética , Leucemia Eritroblástica Aguda , Camundongos , Porfirinas/biossíntese , Células Tumorais CultivadasRESUMO
The use of 5-aminolaevulinic acid (ALA) is gaining increasing attention for photosensitization in photodynamic therapy of superficially localized tumours. The aim of this work was to determine the kinetics of porphyrin generation in tissues after topical application of ALA delivered in different vehicles on the skin overlying the tumour and normal skin of mice. Maximal accumulation was found in tumour 3 h after ALA application in both cream and lotion preparations. Normal and overlying tumour skin tissues showed different kinetic patterns, reflecting histological changes when the latter is invaded by tumour cells. Liver, kidney, spleen and blood porphyrins also raised from basal levels, showing that ALA and/or ALA-induced porphyrins reach all tissues after topical application. During the first 24 h of ALA topical application, precursors and porphyrins are excreted by both urine and faeces. ALA lotion applied on the skin overlying the tumour induced higher accumulation of tumoural porphyrins than cream, and lotion applied on normal skin appeared to be the most efficient upon inducing total body porphyrins. This work has demonstrated the great influence of the formulation of ALA vehicle on penetration through the skin. Knowledge of the kinetics of porphyrin generation after different conditions of ALA application is needed for the optimization of diagnosis and phototherapy in human tumours.
Assuntos
Ácido Aminolevulínico/farmacologia , Veículos Farmacêuticos/farmacologia , Fármacos Fotossensibilizantes/farmacologia , Porfirinas/biossíntese , Adenocarcinoma/tratamento farmacológico , Adenocarcinoma/metabolismo , Administração Tópica , Ácido Aminolevulínico/administração & dosagem , Ácido Aminolevulínico/farmacocinética , Animais , Cinética , Masculino , Neoplasias Mamárias Experimentais/tratamento farmacológico , Neoplasias Mamárias Experimentais/metabolismo , Camundongos , Camundongos Endogâmicos BALB C , Transplante de Neoplasias , Veículos Farmacêuticos/administração & dosagem , Fármacos Fotossensibilizantes/administração & dosagem , Fármacos Fotossensibilizantes/farmacocinética , Porfirinas/sangue , Distribuição TecidualRESUMO
BACKGROUND: Topical photodynamic therapy with endogenous porphyrins consists of irradiation of a tumor with visible light after the application of exogenous 5-aminolevulinic acid. OBJECTIVE: To assess the effectiveness of this modality, patients with precancerous conditions and various skin cancers were treated. METHODS: Thirteen patients with 70 skin lesions were enrolled. Standard treatment involved the topical application of 20% 5-aminolevulinic acid in an oil-in-water emulsion. The emulsion was applied under an occlusive dressing for 4 to 8 hours before exposure to photoactivating light. RESULTS: We observed a complete response after a single treatment for all 9 solar keratoses, 5 of 6 early invasive squamous cell carcinomas, and 36 of 37 superficial basal cell carcinomas. Only 1 of 10 nodulo-ulcerative basal cell carcinomas completely resolved. Eight cutaneous metastases of malignant melanoma were therapeutic failures. CONCLUSION: Topical photodynamic therapy with endogenous porphyrins is effective for superficial epithelial skin tumors.
Assuntos
Ácido Aminolevulínico/administração & dosagem , Ceratose/tratamento farmacológico , Fotoquimioterapia/métodos , Porfirinas/biossíntese , Neoplasias Cutâneas/tratamento farmacológico , Administração Cutânea , Adulto , Idoso , Idoso de 80 Anos ou mais , Ácido Aminolevulínico/metabolismo , Carcinoma Basocelular/tratamento farmacológico , Carcinoma Basocelular/patologia , Carcinoma de Células Escamosas/tratamento farmacológico , Carcinoma de Células Escamosas/patologia , Feminino , Seguimentos , Humanos , Ceratose/patologia , Masculino , Pessoa de Meia-Idade , Porfirinas/fisiologiaRESUMO
The interrelationship between the effect of serum on the induction of porphyrin synthesis, intracellular porphyrin accumulation and photodynamic sensitization of human K562 cells is described. Endogenous porphyrins, synthesized from supplemented 5-amino levulinic acid (5-ALA), were shown to accumulate in the cells, while an addition of serum triggered porphyrin translocation from the cell to the serum. In order to enhance porphyrin accumulation in the cells themselves, they were further stimulated by EDTA, which in combination with 5-ALA reduces Fe++ cellular content. The higher porphyrin cellular content under EDTA and 5-ALA induction was exploited to photoinactivate the human leukemic cells by more then 3 orders of magnitude.
Assuntos
Ácido Aminolevulínico/farmacologia , Ácido Edético/farmacologia , Luz , Porfirinas/metabolismo , Raios Ultravioleta , Sangue , Linhagem Celular , Sobrevivência Celular/efeitos dos fármacos , Sobrevivência Celular/efeitos da radiação , Meios de Cultura , Humanos , Cinética , Leucemia Mielogênica Crônica BCR-ABL Positiva , Porfirinas/biossínteseRESUMO
5-Aminolevulinic acid, porphyrin and chlorophyll contents as well the activities of 5-aminolevulinic acid dehydratase and PBG deaminase were studied in selenium treated mung bean seedlings. Selenium had no effect on 5-aminolevulinic acid synthetic ability but inhibited 5-aminolevulinic acid dehydratase and PBG deaminase activities. Further, it was observed that selenium induced accumulation of protoporphyrin-IX and Mg-protoporphyrin ester and decreased chlorophyll levels in both light and dark-grown seedlings. The results suggest the possible regulatory role of selenium on chlorophyll synthesis by interacting with sulfhydryl containing enzymes 5-aminolevulinic acid dehydratase and porphobilinogen deaminase.
Assuntos
Ácido Aminolevulínico/metabolismo , Clorofila/biossíntese , Fabaceae/metabolismo , Plantas Medicinais , Porfirinas/biossíntese , Selênio/fisiologia , Hidroximetilbilano Sintase/metabolismo , Sintase do Porfobilinogênio/metabolismoRESUMO
Experiments were designed to determine the role of heme and the importance of other factors in the growth of Campylobacter pylori. Campylobacter pylori strains were tested for their ability to synthesize porphyrin, for their ability to grow and be maintained on basal medium and basal medium supplemented with blood or blood products, and for the influence of bovine serum albumin and catalase on viability. Results indicated that Campylobacter pylori does not require heme as a source of porphyrin. Growth of Campylobacter pylori could not be sustained on media containing starch or hemoglobin, but was sustained on media containing erythrocytes, serum, bovine serum albumin or catalase. The ability to grow on media containing bovine serum albumin and catalase suggests that protection from toxic fatty acids and the prevention of toxic product formation may be important factors in the growth and survival of Campylobacter pylori in vitro. Both bovine serum albumin and catalase combined provide the minimum requirements which allow the spectrum of Campylobacter pylori present in a single culture to grow on blood-free media.
Assuntos
Campylobacter/crescimento & desenvolvimento , Porfirinas/biossíntese , Técnicas Bacteriológicas , Sangue , Campylobacter/metabolismo , Catalase , Contagem de Colônia Microbiana , Meios de Cultura , Heme/metabolismo , Humanos , Soroalbumina Bovina , Especificidade da Espécie , Fatores de TempoRESUMO
A new model for experimental studies of substances influencing porphyrin metabolism has been created. The model is formed by yeast Saccharomyces cerevisiae (RIBM-75) grown semiaerobically. The main advantages of this model include simple evocation of porphyria (intracellular accumulation of porphyrins in semiaerobic conditions) and direct measurement of experimental values (intracellular and extracellular concentrations of porphyrins). The porphyrinostatic effects of drugs can be assessed on the basis of sum of experimental values. The ratio of experimental criteria enables us to compare the influence of drugs on porphyrin permeation across the cellular membrane. Antimalarials, chloroquine and pyrimethamine, used or tested for therapy of chronic liver porphyria, have been tested on the model. The experiments showed that both chloroquine and pyrimethamine inhibited porphyrin synthesis. This effect can represent the proper therapeutical action of both drugs, which has not been known so far. Chloroquine releases the intracellular porphyrins in yeast similarly as it does in hepatocytes. However, pyrimethamine causes intracellular accumulation of porphyrins booth in yeast and hepatocytes.
Assuntos
Cloroquina/farmacologia , Porfirinas/biossíntese , Pirimetamina/farmacologia , Avaliação Pré-Clínica de Medicamentos/métodos , Heme/biossíntese , Saccharomyces cerevisiae/metabolismoRESUMO
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.
Assuntos
Amônia-Liases/metabolismo , Hidroximetilbilano Sintase/metabolismo , Plantas/enzimologia , Sintase do Porfobilinogênio/metabolismo , Porfirinas/biossíntese , Oxirredutases do Álcool/metabolismo , Centrifugação com Gradiente de Concentração , Fabaceae/enzimologia , Hidroxipiruvato Redutase , Malato Desidrogenase/metabolismo , Plantas Medicinais , Frações Subcelulares/enzimologia , Tripsina/farmacologiaRESUMO
The obligately aerobic soybean root nodule bacterium Rhizobium japonicum produces large amounts of heme (iron protoporphyrin) only under low oxygen tensions, such as exist in the symbiotic root nodule. Aerobically incubated suspensions of both laboratory-cultured and symbiotic bacteria (bacteroids) metabolize delta-aminolevulinic acid to uroporphyrin, coproporphyrin, and protoporphyrin. Under anaerobic conditions, suspensions of laboratory-cultured bacteria form greatly reduced amounts of protoporphyrin from delta-aminolevulinic acid, whereas protoporphyrin formation by bacteroid suspensions is unaffected by anaerobiosis, suggesting that bacteroids form protoporphyrin under anaerobic conditions more readily than do free-living bacteria. Oxygen is the major terminal electron acceptor for coproporphyrinogen oxidation in cell-free extracts of both bacteroids and free-living bacteria. In the absence of oxygen, ATP, NADP, Mg2+, and L-methionine are required for protoporphyrin formation in vitro. In the presence of these supplements, coproporphyrinogenase activity under anaerobic conditions is 5 to 10% of that observed under aerobic conditions. Two mechanisms for coproporphyrinogen oxidation exist in R. japonicum: an oxygen-dependent process and an anaerobic oxidation in which electrons are transferred to NADP. The significance of these findings with regard to heme biosynthesis in the microaerophilic soybean root nodule is discussed.
Assuntos
Porfirinas/biossíntese , Protoporfirinas/biossíntese , Rhizobium/metabolismo , Aerobiose , Ácido Aminolevulínico/metabolismo , Anaerobiose , Coproporfirinogênio Oxidase/metabolismo , Coproporfirinogênios/metabolismo , Coproporfirinas/biossíntese , Porfobilinogênio/biossíntese , Uroporfirinas/biossínteseRESUMO
Heme a was not detected either in mitochondria isolated from copper-deficient yeast or in the intact cells. Nevertheless, the intracellular concentration of free porphyrins indicated that the pathway of porphyrin and heme synthesis was not impaired in copper-deficient cells. The immunoprecipitated apo-oxidase from copper-deficient cells revealed an absorption spectrum with maxima at 645, 592, 559, 519 and 423 nm, similar to that of purified porphyrin a. When solubilized mitochondria from [3H]leucine and delta-amino[14C]levulinic acid-labeled copper-deficient yeast cells were incubated with rabbit antiserum against cytochrome c oxidase, a precipitate was obtained. Sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis of this immunoprecipitate showed [3H]leucine associated with six bands and delta-amino[14C]levulinic acid resolved in a single band. HCl fractionation of copper-deficient mitochondria labeled with delta amino[14C]levulinic acid showed a high specific radioactivity in the fraction extracted by 20% HCl, a solvent which extracts porphyrin a. Thin-layer chromatography of the radioactivity found in 20% HCl showed an RF value identical to that of purified porphyrin a. When delta-amino[3H]levulinic acid-labeled, copper-deficient yeast cells are grown in copper-supplemented medium, the porphyrin a accumulated in copper-deficient cells was converted into heme a, and this conversion was prevented by cycloheximidine. These observations suggest that porphyrin a is present in the apo-oxidase of copper-deficient cells, but that the conversion to heme a does not occur. This conversion reaction appears to be a point in the biosynthetic pathway of cytochrome c oxidase which is blocked by copper deficiency.
Assuntos
Apoenzimas/metabolismo , Apoproteínas/metabolismo , Cobre/deficiência , Complexo IV da Cadeia de Transporte de Elétrons/metabolismo , Heme/análogos & derivados , Porfirinas/isolamento & purificação , Saccharomyces cerevisiae/enzimologia , Ácido Aminolevulínico/metabolismo , Cromatografia em Camada Fina , Complexo IV da Cadeia de Transporte de Elétrons/imunologia , Eletroforese em Gel de Poliacrilamida , Heme/metabolismo , Mitocôndrias/análise , Peso Molecular , Porfirinas/biossíntese , EspectrofotometriaRESUMO
Twenty-two Haemophilus cultures of types prevalent in swine and of different geographic origins were subjected to biochemical and cultural examinations. Three subgroups were identified: One was unrease-positive, produced porphyrin from delta-aminolevulinic acid, and grew on infusion mediums supplemented only with V factor; the 2nd was unrease-negative, porphyrin-positive, and grew only on serum-enriched mediums with added V factor; and the 3rd was unrease-negative, porphyrin-negative, and grew only on serum-enriched mediums with added V and X factors. The groups generally corresponded to Haemophilus parahaemolyticus, Haemophilus parasuis, and Haemophilus suis, respectively. By means of the unrease and porphyrin tests, it was possible to assign, presumptively, porcine haemophilus cultures to 1 of the 3 species. Other tests, such as beta-galactosidase, hemolysis, and fermentation of carbohydrates were of secondary value in differentiating between these species.