Glycolic acid oxidase formation in greening leaves

Abstract

The glycolic acid oxidase activity in the sap from etiolated Thatcher wheat leaves is 10% or less of the activity from the corresponding green leaves. The activity of the enzyme in extracts from etiolated leaves may be increased the same 5-10-fold by addition of 10-4 M FMN, by grinding the leaves with glycolate, or by incubation of the inactive sap for 18 hours at 2[degree]C with a substrate for the enzyme. The enzyme activity is also increased by spraying etiolated leaves 24 hours prior to harvest with 1 of the substrates. After 1 ammonium sulfate fractionation of the proenzyme, the in vitro activation could only be accomplished by 10-4 M FMN. Although a very active glycolic acid oxidase was always found in extracts from green leaves, the activity may also be increased 2-5-fold by addition of 10-4 M FMN or by grinding the green leaves with glycolate. The total enzyme which can be revealed by excess FMN from green leaves was about 2 to 3 fold greater on a [image] basis than the total detectable enzyme from the etiolated leaves. During greening of the etiolated leaves there was no change in the total flavin content. However, the FAD concentration of the etiolated Thatcher wheat leaf was about double the FMN content while the FMN content of the green leaf was about double the FAD concentration. The total FMN rnolarity based upon volume of leaf extract was between 5 x 10-9 to 1 x 10-8 M, while 10-4 M FMN was needed to activate the enzyme in vitro. These results suggest that either considerable amount of proenzyme was present in the etiolated leaves or that a labile form of the holoenzyme was present and was destroyed during grinding of the leaves. However, neither interpretation fully explains all of the results.