ALTERNATIVE OXIDASE FROM ARUM AND SOYBEAN - ITS STABILIZATION DURING PURIFICATION

Complete purification of the alternative oxidase from plant mitochondr ia has not been achieved successfully, because of its instability on s olubilization. We report here that the addition of pyruvate to the iso lation medium stabilizes the activity of the solubilized enzyme. A pro cedure is described for the rapid isolation and partial purification o f the cyanide-insensitive alternative oxidase from both Arum maculatum and soybean cotyledon (Glycine max) mitochondria. The degree of purif ication was 16- and 74-fold for Arum and soybean enzyme, respectively. The specific activities increased from 1 300 to 20 300 nmol oxygen co nsumed mg(-1) protein min(-1) (using duroquinol as substrate) after pu rification for the Arum enzyme and from 6 to 445 nmol oxygen consumed mg(-1) protein min(-1) for the soybean enzyme. A turnover for the part ially purified Arum enzyme was estimated to be 47 electrons s(-1). The partially purified enzyme from both Arum and soybean cotyledon mitoch ondria was sensitive to alternative oxidase inhibitors such as salicyl hydroxamic acid, n-propyl gallate and octyl gallate, but not to myxoth iazol, KCN or antimycin A. The activity of the enzyme could be stimula ted by pyruvate, bur not by malate and succinate. The stability of the purified enzyme was also dependent on the continued presence of pyruv ate. In the absence of pyruvate, the enzyme activity was lost in a tim e-dependent manner and the ability of pyruvate to recover the activity was also irreversibly lost.