Cyanide-insensitive and Cyanide-sensitive O(2) Uptake in Wheat: II. GRADIENT-PURIFIED MITOCHONDRIA LACK CYANIDE-INSENSITIVE RESPIRATION.

Enriched mitochondrial fractions isolated from durum wheat seedlings via differential centrifugation exhibited classical cyanide- or antimycin A-insensitive O(2) uptake which was inhibited by either salicylhydroxamic acid or propyl gallate. Further purification of this fraction using Percoll density gradients resulted in two discrete bands which were essentially homogeneous mitochondrial populations, as verified by electron microscopy. Respiratory O(2) uptake in these two fractions was completely inhibited by cyanide or antimycin A. Addition of linoleic acid to a third-step gradient band, which was shown to contain virtually no mitochondria, resulted in demonstrable cyanide-insensitive O(2) uptake. This O(2) consumption was completely inhibited by propyl gallate or salicylhydroxamic acid, two known lipoxygenase inhibitors. In contrast, addition of linoleic acid to the two purified mitochondrial fractions did not stimulate O(2) uptake. These data indicate that lipoxygenase oxygenation, the enzyme physically separable from the mitochondria, is responsible for the cyanide-insensitive component of O(2) uptake that was observed in subcellular fractions isolated from etiolated wheat seedlings.