title: Ascorbate peroxidase, a scavenger of hydrogen peroxide in glyoxysomal membranes.
abstract: Efficient destruction of hydrogen peroxide (H(2)O(2)) in peroxisomes requires the action of an anti-oxidant defense system, which consists of low molecular weight anti-oxidant compounds, such as ascorbic acid, along with protective enzymes, such as catalase and ascorbate peroxidase (APX). We investigated the contribution of the ascorbate enzyme system to the consumptions of H(2)O(2) and NADH within glyoxysomes of germinating castor beans (Ricinus communis). We solubilized the glyoxysomal membrane APX (gmAPX) using octyl-glucoside and purified its activity by gel filtration. The activity was associated with a 34kDa protein, as determined by SDS-gel electrophoresis and Western blotting. The enzymatic properties of gmAPX were studied and this enzyme was found to utilize ascorbic acid as its most effective natural electron donor but it would also use pyrogallol and guaiacol at a smaller extent. Cyanide and azide drastically inhibited gmAPX, as well as certain thiol-modifying reagents and some metal chelators. The inhibition by cyanide and azide of the enzyme combined with its absorption spectra confirmed that it is a hemoprotein. The apparent K(m) value of the enzyme for ascorbic acid was 300 microM while the K(m) for H(2)O(2) was 60 microM. APX in the glyoxysomal membrane can work in cooperation with monodehydroascorbate reductase to oxidize NADH, regenerate ascorbate, detoxify H(2)O(2), and protect the integrity of glyoxysomal proteins and membranes.
authors.affiliation: Department of Biological Sciences, George Washington University, Washington, DC, USA.
authors.forename: Konstantina
authors.initials: K
authors.lastname: Karyotou
authors.forename: Robert P
authors.initials: RP
authors.lastname: Donaldson
mesh_terms: D060387:Ascorbate Peroxidases; D001205:Ascorbic Acid / Q000378:metabolism; D015153:Blotting, Western; D002367:Ricinus communis / Q000378:metabolism; D002374:Catalase / Q000378:metabolism; D002614:Chelating Agents / Q000494:pharmacology; D002850:Chromatography, Gel; D003902:Detergents / Q000494:pharmacology; D004586:Electrophoresis; D004591:Electrophoresis, Polyacrylamide Gel; D005960:Glucosides / Q000737:chemistry; D020676:Glyoxysomes / Q000378:metabolism*; D006861:Hydrogen Peroxide / Q000737:chemistry / Q000378:metabolism*; D015151:Immunoblotting; D007425:Intracellular Membranes / Q000378:metabolism*; D007700:Kinetics; D008954:Models, Biological; D010100:Oxygen / Q000378:metabolism; D010544:Peroxidases / Q000378:metabolism / Q000494:pharmacology*; D011485:Protein Binding; D013053:Spectrophotometry
chemical_list: D002614:Chelating Agents; D003902:Detergents; D005960:Glucosides; C018619:octyl-beta-D-glucoside; D006861:Hydrogen Peroxide; D010544:Peroxidases; D060387:Ascorbate Peroxidases; D002374:Catalase; D001205:Ascorbic Acid; D010100:Oxygen