| Reactive oxygen species (ROS), such as superoxide, hydrogen peroxide (H2O2) and hydroxyl radical, are by-products of metabolism, yet are toxic to cells. Organelles such as chloroplasts and peroxisomes produce and destroy ROS. Efficient destruction of ROS requires the action of an antioxidant defense system, consisting of antioxidant compounds, such as ascorbic acid, along with protective enzymes, such as catalase and ascorbate peroxidase (APX). The contribution of the ascorbate-utilizing enzyme system to the consumption of H2O2 and NADH within glyoxysomes of germinating castor beans (Ricinus communis) was investigated.; APX is an H2O2-scavenging peroxidase that can rapidly catalyze the H2O2-reduction into water, using ascorbate as a one-electron donor generating monodehydroascorbate radical. Various APX isozymes have been identified in chloroplasts and cytosol. Recently, a novel type of APX was found in peroxisomes. In order to explore the physiological role of this APX isoform in the reactive oxygen species metabolism of plant peroxisomes, we hereby describe the identification and enzymatic characterization of glyoxysomal membrane APX (gmAPX).; GmAPX was solubilized using octyl-glucoside and its activity was purified by gel filtration. We used an anti-APX polyclonal antibody, which had been shown to recognize a protein with a sequence similarity to APX. Although this antibody was shown to cross-react with glyoxysomal membrane proteins of cucumber, sunflower, castor bean and cotton, APX activity was not demonstrated. Here we demonstrate that the antibody made against the cucumber peroxisomal membrane protein recognizes a 34 kD castor bean glyoxysomal membrane protein that has APX activity.; The gmAPX was found to utilize ascorbic acid as its main electron donor but would also use pyrogallol and guaiacol. Cyanide and azide inhibited gmAPX, as well as certain thiol-modifying reagents and some metal chelators. The inhibition of the enzyme by cyanide and azide, combined with its absorption spectra revealed that it is a hemoprotein. The apparent Km value of the enzyme for ascorbic acid was 300 muM while the Km for H2O2 was 60 muM. The cooperation of this enzyme with the monodehydroascorbate reductase (MDAR) NAD+/ascorbate-regenerating system and their role in the protection of the integrity of glyoxysomal membranes is also discussed. |
