An Accounting of Horseradish Peroxidase Isozymes Associated with the Cell Wall and Evidence that Peroxidase Does Not Contain Hydroxyproline

Abstract

Isopycnic equilibrium centrifugation techniques were used to determine whether any horseradish (Amoracia lapathifolia) peroxidase isozymes were associated with hydroxyproline containing moieties. Purified peroxidase, horseradish root extracts, and peroxidase isozymes released from horseradish root cell walls were tested. In no case could any peak of peroxidase activity be found to band with hydroxyproline.A fluorimetric method for measurement of peroxidase activity was used to determine quantitatively the amount of total peroxidase located on horseradish root cell walls. Twenty per cent of the total peroxidase is found in the cell wall fraction after extraction; 93% of this cell wall associated peroxidase can be removed by washing with 2 m NaCl. Some peroxidase isozymes released by salt washing are not found in the cytoplasmic extract. This indicates that not all of the ionically bound peroxidase represents cytoplasmic contamination. The 1.4% of the total peroxidase activity can thus be considered tightly bound to the cell wall. Of this portion, 75% can be solubilized by treatment with a cellulase preparation. One isozyme is released which was not present in the original cytoplasmic extract.