Cyanide-insensitive and Cyanide-sensitive O₂ Uptake in Wheat

Abstract

The mitochondrial fraction isolated from durum wheat seedlings by differential centrifugation demonstrated antimycin A-or It has become apparent that the distribution of "high energy" metabolites within a cell is under tight intracellular regulation. In particular, experimental evidence accumulating on the existence of numerous and highly specific transport and shuttle systems (8,20) indicates that there may be regulatory systems involving chloroplastic, mitochondrial, and cytoplasmic components. A necessary prerequisite for studying such systems is the ability to separate the various subcellular components in order to assess their individual functions and capabilities.Cyanide-insensitive respiration has often been measured by researchers primarily interested in mitochondrial electron transport and the energetics of ATP production (2, 19). It now seems clear (5, 13) that lipoxygenase activity may account for a substantial portion of total 02 consumption at certain times during development in soybean and wheat. Here, we report a series of respiratory studies carried out on conventionally isolated mitochondria and highly purified mitochondria from germinating wheat seedlings 12 through 64 h after imbibition. Data are presented which demonstrate the possible confusion that can arise when attempting to assay mitochondrial cyanide-insensitive res-

Inhibition of O2 Consumption Resistant to Cyanide and Its Development by N-Propyl Gallate and Salicylhydroxamic Acid

“…These results do, however, support the suggestion made earlier that PG may be acting on different sites in slices and isolated mitochondria. It has been reported by Goldstein et al (6) that lipoxygenase activity (linoleic acid as substrate) is associated with a washed mitochondrial fraction from wheat, which also apparently contains an operational alternative pathway. Of the two mitochondrial fractions separable by Percoll density gradient centrifugation, one fraction contained a linoleic acid oxidizing component while the other did not.…”

“…A controversy arises concerning present, past, and future use of a nonpurified mitochondrial fraction for alternative pathway studies. Also, the possibility presented by Goldstein et aL (6), that in a washed mitochondrial fraction lipoxygenase activity may be contributing to 02 consumption in the absence of added fatty acid, is intriguing. However, in potato we feel that the difference in the K, of PG for lipoxygenase and mitochondrial alternative oxidase is indicative of the fact that the same enzyme is not responsible for both types of 02 uptake.…”

Cyanide-insensitive and Cyanide-sensitive O2 Uptake in Wheat

“…Protein content was estimated by the Bio-Rad method (1). Electron microscopy techniques have been previously described (6).…”

“…We have discussed previously the initial separation of the differential centrifugation fraction into two bands of mitochondrial activity on a linear PDG (PVP-coated silica sol, Pharmacia) (6). To purify further the mitochondria present in PDG The bands which collected at the interfaces were harvested, washed, and assayed polarographically as previously described (6) with the following exceptions.…”

“…To purify further the mitochondria present in PDG The bands which collected at the interfaces were harvested, washed, and assayed polarographically as previously described (6) with the following exceptions. Each step-gradient fraction consisted of material from six linear gradient bands (representing 3.6 g scutellar tissue).…”