MOLECULAR CHARACTERIZATION AND PHYSIOLOGICAL-ROLE OF A GLYOXYSOME-BOUND ASCORBATE PEROXIDASE FROM SPINACH

cDNAs encoding two cytosolic and two chloroplastic ascorbate peroxidas e: (AsAP) isozymes from spinach have been cloned recently [Ishikawa et al. (1995) FEES Lett, 367: 28, (1996) FEBS Lett. 384: 289]. We herein report the cloning of the fifth cDNA of an AsAP isozyme which localiz es in spinach glyoxysomes (gAsAP), The open reading frame of the 858-b ase pair cDNA encoded 286 amino acid residues with a calculated molecu lar mass of 31,507 Da. By determination of the latency of AsAP activit y 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. coil). The enzymatic properties of the partially purified recombinant gAsAP w ere consistent with those of the native enzyme from intact glyoxysomes . The recombinant enzyme utilized ascorbate (AsA) as its most effectiv e natural electron donor; glutathione (GSH) and NAD(P)H could not subs titute for AsA, The substrate-velocity curves with the recombinant enz yme showed Michaelis-Menten type kinetics with AsA and hydrogen peroxi de (H2O2); the apparent K-m values for AsA and H2O2 were 1.89 +/- 0.05 mM and 74 +/- 4.0 mu M, respectively. When the recombinant enzyme was diluted with AsA-depleted medium, the activity was stable over 180 mi n, We discuss the H2O2-scavenging system maintained by AsAP and the re generation system of AsA in spinach glyoxysome.