Molecular Characterization and Physiological Role of a Glyoxysome-Bound Ascorbate Peroxidase from Spinach

Abstract

cDNAs encoding two cytosolic and two chloroplastic ascorbate peroxidase (AsAP) isozymes from spinach have been cloned recently [Ishikawa et al. (1995) FEBS Lett. 367: 28, (1996) FEBS Lett. 384: 289]. We herein report the cloning of the fifth cDNA of an AsAP isozyme which localizes in spinach glyoxysomes (gAsAP). The open reading frame of the 858-base pair cDNA encoded 286 amino acid residues with a calculated molecular mass of 31,507 Da. By determination of the latency of AsAP activity in intact glyoxysomes, the enzyme, as well as monodehydroascorbate (MDAsA) reductase, was found to be located on the external side of the organelles. The cDNA was overexpressed in Escherichia coli (E. coli). The enzymatic properties of the partially purified recombinant gAsAP were consistent with those of the native enzyme from intact glyoxysomes. The recombinant enzyme utilized ascorbate (AsA) as its most effective natural electron donor; glutathione (GSH) and NAD(P)H could not substitute for AsA. The substrate-velocity curves with the recombinant enzyme showed Michaelis-Menten type kinetics with AsA and hydrogen peroxide (H2O2); the apparent Km values for AsA and H2O2 were 1.89 +/- 0.05 mM and 74 +/- 4.0 microM, respectively. When the recombinant enzyme was diluted with AsA-depleted medium, the activity was stable over 180 min. We discuss the H2O2-scavenging system maintained by AsAP and the regeneration system of AsA in spinach glyoxysome.

Trypanosoma cruzi expresses a plant-like ascorbate-dependent hemoperoxidase localized to the endoplasmic reticulum

“…For TcAPX, a K M value for ascorbate could be determined when using saturating concentrations of H 2 O 2 , indicating that this enzyme displays kinetics similar to the monomeric APXs (Fig. 2) (35,35,46). Further evidence that TcAPX is monomeric can be inferred from the sequence; TcAPX lacks many of the charged residues present in P. sativum cytosolic APX that have been implicated in homodimer formation (Fig.…”

“…For those that exist predominantly as homodimers, such as the cytosolic APXs from peas and soybean, pseudo first-order kinetics have been observed (33,34). In contrast, monomeric forms such as the peroxisomal APX from spinach and APXs from various unicellular algae obey Michaelis-Menten kinetics (35,36), which has led to the suggestion that the kinetics displayed by the homodimeric enzymes may be due to variation in the oligomeric form of the protein, especially when ascorbate concentrations are low (33). For TcAPX, a K M value for ascorbate could be determined when using saturating concentrations of H 2 O 2 , indicating that this enzyme displays kinetics similar to the monomeric APXs (Fig.…”

Responses of Photosynthetic Efficiency and Ascorbate Peroxidase Induced by Salt Stress in Rice (Oryza sativa L.)

“…APX can easily eliminate H2O2 with high affinity in the cytosol, chloroplasts (stroma and thylakoid membrane) and other subcellular locations (microbody) [9,16,37]. These APX isoforms have been separated from spinach chloroplasts, tea leaves and plastids of tobacco, followed by many studies on their enzymatic/molecular properties [6,29,35].…”

“…When plants are exposed to abiotic stresses, they experience functional disorder of metabolism including oxidative stress, osmotic stress and ionic imbalance [13,20,32]. Salinity induces oxidative stresses in plants by increasing reactive oxygen species (ROS) such as superoxide, hydrogen peroxide and hydroxyl radical [11,26] APX can easily eliminate H2O2 with high affinity in the cytosol, chloroplasts (stroma and thylakoid membrane) and other subcellular locations (microbody) [9,16,37]. These APX isoforms have been separated from spinach chloroplasts, tea leaves and plastids of tobacco, followed by many studies on their enzymatic/molecular properties [6,29,35].…”

In silico comparative analysis and expression profile of antioxidant proteins in plants

“…The chloroplastic APXs, including thylakoid-bound and stromal isoforms, scavenge the H 2 O 2 produced during photosynthesis. In comparison to the cytosolic and peroxisomal isoforms, chloroplastic enzymes are more labile and are rapidly inactivated in the absence of ascorbate (Miyake et al, 1993;Ishikawa et al, 1998;Shigeoka et al, 2002).…”