A critical study of the intracellular distribution of ascorbate oxidase and a comparison of the kinetics of the soluble and cell-wall enzyme

Abstract

Ascorbate oxidase is present in both the soluble and cell-wall preparations from many plant tissues, and can be distinguished by its kinetic characteristics from other catalysts of ascorbate oxidation. The soluble and cell-wall ascorbate oxidases from cabbage leaves have the same Michaelis constant (10 -2 M) and optimal pH (6·0) but the cell-wall enzyme has a higher energy of acivation and is less sensitive to inhibition by 8-hydroxyquinoline, azide, and diethyldithiocarbamate than the soluble enzyme. The cell-wall enzyme cannot be efficiently released by incubation with 0·15 M NaCl, 0·15 M CaCl 2, 1·5 M sucrose or 5% deoxycholate. Incubation of the walls with cellulase releases some activity. Up to 50% of the ascorbate oxidase of barley root-tips is freely accessible to ascorbate in medium bathing the roots and is postulated to be located in vivo in the cell walls. It is concluded that the ascorbate oxidase activity of cell-wall and soluble fraction from cabbage leaves is attributable to the same enzyme, and that in vivo the majority of the ascorbate oxidase is located in the cell walls. The data do not exclude the possibility that the remainder, recovered in vitro in the supernatant, may in vivo be very loosely associated with some subcellular particle.