Purification and characterization of ascorbate peroxidase in Chlorella vulgaris.
Chlorella vulgaris contained only one isoform of ascorbate peroxidase (AsAP) as the hydrogen peroxide (H2O2)-scavenging system except for catalase at a specific activity of 3.3 +/- 0.2 units/mg protein. The activity of glutathione peroxidase was not detected in the extracts from cells grown in the absence and presence of sodium selenite. We detected the activity of monodehydroascorbate reductase involved in the regeneration of ascorbate, but we failed to detect the dehydroascorbate reductase activity. AsAP has been purified to electrophoretic homogeneity from Chlorella cells. The enzyme was a monomer with a molecular mass of 32 kDa using gel filtration and SDS-polyacrylamide gel electrophoresis. The enzyme showed higher specificity with ascorbate than with pyrogallol. The K(m) values of the enzyme for ascorbate and H2O2 were 111 +/- 8.9 and 20 +/- 2.5 microM, respectively. When the enzyme was diluted with the ascorbate-deleted medium, the half inactivation time was approximately 15 min. The absorption spectra of the purified enzyme and the inhibition by cyanide and azide showed that it is a hemoprotein. The enzyme was markedly inhibited by 0.2 mM p-chloromercuribenzoate. The enzyme cross-reacted by immunoblotting with the monoclonal antibody raised against Euglena cytosolic AsAP. The amino acid sequences in the N-terminal region of Chlorella AsAP showed no significant similarity to any other AsAPs from higher plants and algae.
Takeda
T
T
Department of Food and Nutrition, Faculty of Agriculture, Kinki University, Nara, Japan.
Yoshimura
K
K
Ishikawa
T
T
Shigeoka
S
S
D000595:Amino Acid Sequence; D060387:Ascorbate Peroxidases; D001205:Ascorbic Acid / Q000378:metabolism; D002708:Chlorella / Q000201:enzymology*; D002850:Chromatography, Gel; D002852:Chromatography, Ion Exchange; D007700:Kinetics; D008969:Molecular Sequence Data; D010544:Peroxidases / Q000737:chemistry / Q000302:isolation & purification* / Q000378:metabolism*; D011748:Pyrogallol / Q000378:metabolism; D016415:Sequence Alignment; D017386:Sequence Homology, Amino Acid; D013053:Spectrophotometry; D013379:Substrate Specificity
D011748:Pyrogallol; D010544:Peroxidases; D060387:Ascorbate Peroxidases; D001205:Ascorbic Acid