RESUMO
Ascorbate peroxidase (AP) is a key enzyme that scavenges potentially harmful H2O2 and thus prevents oxidative damage in plants, especially in N2-fixing legume root nodules. The present study demonstrates that the nodule endodermis of alfalfa (Medicago sativa) root nodules contains elevated levels of AP protein, as well as the corresponding mRNA transcript and substrate (ascorbate). Enhanced AP protein levels were also found in cells immediately peripheral to the infected region of soybean (Glycine max), pea (Pisum sativum), clover (Trifolium pratense), and common bean (Phaseolus vulgaris) nodules. Regeneration of ascorbate was achieved by (homo)glutathione and associated enzymes of the ascorbate-glutathione pathway, which were present at high levels. The presence of high levels of antioxidants suggests that respiratory consumption of O2 in the endodermis or nodule parenchyma may be an essential component of the O2-diffusion barrier that regulates the entry of O2 into the central region of nodules and ensures optimal functioning of nitrogenase.
Assuntos
Antioxidantes/metabolismo , Fabaceae/enzimologia , Regulação da Expressão Gênica de Plantas , Peroxidases/biossíntese , Plantas Medicinais , Ascorbato Peroxidases , Ácido Ascórbico/metabolismo , Difusão , Fabaceae/fisiologia , Regulação Enzimológica da Expressão Gênica , Medicago sativa , Oxigênio/metabolismo , Consumo de Oxigênio , Pisum sativum , Raízes de Plantas , Poaceae , Glycine maxRESUMO
Ascorbate peroxidase is a widespread plant enzyme that catalyzes the removal of potentially harmful H2O2. This enzyme is particularly important in legume root nodules due to their high potential for generating activated forms of oxygen. A cDNA clone of soybean nodule ascorbate peroxidase was used to construct an expression system in Escherichia coli. The recombinant protein had an N-terminal tag of six consecutive histidine residues to allow for purification by Ni(2+)-agarose affinity chromatography. Large amounts of recombinant peroxidase (about 27% of total soluble protein) were produced but most of the peroxidase was present in the apo-form (without heme). Addition of delta-aminolevulinic acid to the growth media resulted in an increase in production of holoprotein. Apoprotein was easily converted to the holo-form by in vitro reconstitution with hemin. The reconstituted protein was catalytically, spectrally, and immunologically indistinguishable from native ascorbate peroxidase.
Assuntos
Glycine max/enzimologia , Peroxidases/biossíntese , Sequência de Aminoácidos , Apoenzimas/biossíntese , Apoenzimas/genética , Apoenzimas/isolamento & purificação , Apoenzimas/metabolismo , Ascorbato Peroxidases , Sequência de Bases , Compartimento Celular , Clonagem Molecular , Coenzimas/biossíntese , Coenzimas/genética , Coenzimas/isolamento & purificação , Coenzimas/metabolismo , Citosol/enzimologia , Escherichia coli/genética , Histidina/genética , Dados de Sequência Molecular , Peroxidases/genética , Peroxidases/isolamento & purificação , Peroxidases/metabolismo , Proteínas Recombinantes/biossíntese , Proteínas Recombinantes/isolamento & purificação , Proteínas Recombinantes/metabolismo , Glycine max/genética , EspectrofotometriaRESUMO
All health care providers must share the responsibility for educating health professionals and the lay public about the need for donor organs and the process of donation. PAs should become familiar with the procedures involved, including the protocol for diagnosing brain death, the criteria by which potential organ donors are identified, the maintenance of organs in vitro, and the methods of preserving procured organs for transplantation. PAs are urged to become active in procurement/transplantation programs at their institutions or by working with a procurement agency to establish a program if one does not already exist. In addition, PAs should become familiar with the laws that govern organ donations in their states.
