Purification and kinetic characterization of the liverwort Pallavicinia lyelli (Hook.) S. Gray. cytosolic ascorbate peroxidase.
Ascorbate peroxidase (APX) of the liverwort Pallavicinia lyelli was extracted and purified through ammonium sulfate precipitation, Butyl-Toyopearl, DEAE-Cellulofine and Sephadex G-75 chromatography. The purification factor for APX was 285 with 7.9% yield. The enzyme was characterized for thermal stability, pH and kinetic parameters. The molecular mass of APX was approximately 28 kDa estimated by SDS-PAGE. The purity was checked by native PAGE, showing a single prominent band. The optimum pH was 6.0. The enzyme had a temperature optimum at 40 degrees C and was relatively stable at 60 degrees C, with 54% loss of activity. 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. Spectral analysis and inhibitor studies were consistent with the presence of a heme moiety. When compared with ascorbate peroxidase activity derived from ruptured intact chloroplasts, the purified enzyme was found to have a higher stability, a broader pH optimum for activity and the capacity to utilize alternate electron donors. p-Chloromercuribenzoate (pCMB), hydroxyurea and salicylic acid (SA) significantly inhibited APX activity. Ascorbate (AsA) and pyrogallol were found to be efficient substrates for Pallavicinia APX, considering the Vmax/Km ratio. We detected the activity of monodehydroascorbate reductase (MDHAR) involved in the regeneration of ascorbate, but failed to detect the dehydroascorbate reductase (DHAR) activity. The data obtained in this study may help to understand desiccation tolerance mechanism in the liverwort.
Sajitha Rajan
S
S
Plant Biochemistry and Molecular Biology Lab, Dept. of Botany, University College, Thiruvananthapuram, Kerala 695 034, India.
Murugan
K
K
D060387:Ascorbate Peroxidases; D001205:Ascorbic Acid / Q000378:metabolism; D055162:Biocatalysis / Q000187:drug effects; D003600:Cytosol / Q000201:enzymology*; D004591:Electrophoresis, Polyacrylamide Gel; D004791:Enzyme Inhibitors / Q000494:pharmacology; D004795:Enzyme Stability; D035926:Hepatophyta / Q000201:enzymology*; D006863:Hydrogen-Ion Concentration; D006918:Hydroxyurea / Q000494:pharmacology; D007700:Kinetics; D008970:Molecular Weight; D010544:Peroxidases / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism*; D010940:Plant Proteins / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism*; D011190:Potassium Cyanide / Q000494:pharmacology; D011748:Pyrogallol / Q000378:metabolism; D020156:Salicylic Acid / Q000494:pharmacology; D019810:Sodium Azide / Q000494:pharmacology; D013053:Spectrophotometry; D013379:Substrate Specificity; D013696:Temperature
D004791:Enzyme Inhibitors; D010940:Plant Proteins; D011748:Pyrogallol; D019810:Sodium Azide; D010544:Peroxidases; D060387:Ascorbate Peroxidases; D011190:Potassium Cyanide; D020156:Salicylic Acid; D001205:Ascorbic Acid; D006918:Hydroxyurea