Alternative Pathway Respiration and Lipoxygenase Activity in Aged Potato Slice Mitochondria

Abstract

Mitochondrial preparations isolated from aged white potato (Solanum tuberosum L.) slices exhibited classical cyanide-insensitive 02 uptake which was inhibited by salicylhydroxamic acid and tetraethylthiuram disulfide (disulfiram). These mitochondria also possessed lipoxygenase activity, as determined by 02 uptake in the presence of 4 millimolar linoleic acid. Purification of the mitochondrial preparation on a continuous Percoll gradient resulted in a large decrease i lipoxygenase activity whereas cyanide-insensitive (disulfiram sensitive) 02 consumption was still observed. These data indicate that cyanide-insensitive 02 consumption in mitochondrial preparations isolated from aged white potato slices is of mitochondrial origin and not due to lipoxygenase contamination.It has long been known that the aging of tissue slices from various tubers in liquid-aerated media induces metabolic changes within the tissue (7,17). In potato slices, aging causes an increase in tissue respiration which becomes partially resistant to cyanide and carbon monoxide inhibition (5,16). This resistance to cyanide has been attributed to the development of an alternative electron transfer pathway in the mitochondria (1, 6). It has been shown, however, that potato tubers contain high levels of lipoxygenase (11) and that, upon slicing of potatoes, there is a rapid loss of phospho-and galactolipid indicative of membrane lipid breakdown (15).Because of the possible contribution of lipoxygenase in the assessment of mitochondrial cyanide-insensitive respiration, we have attempted to determine whether cyanide-insensitive 02 consumption exhibited by mitochondrial preparations from aged potato slices was indeed attributable to a development of the alternative path or was due to lipoxygenase contamination. [6]) and specific inhibition by substituted hydroxamic acids, including SHAM5 (12). Hydroxamates also inhibit the activity of lipoxygenase (10), which catalyzes oxidation of polyunsaturated fatty acids. Crude mitochondrial preparations from wheat seedlings (3), mung bean (13), and soybean (9) have all been shown to be contaminated by lipoxygenase. Although purification of wheat seedling mitochondria on a continuous Percoll gradient resulted in a total loss of lipoxygenase activity as well as a complete loss of cyanide-insensitive respiration (3), other workers have been unsuccessful in attempts to totally remove lipoxygenase activity from soybean and mung bean mitochondria on Percoll and sucrose gradients, respectively (9, 13). However, Miller and Obendorf (9) reported that disulfiram, an inhibitor ofcyanideinsensitive respiration in red sweet potato mitochondria (4), had no effect on lipoxygenase activity in soybean mitochondrial preparations and could be used to distinguish between residual lipoxygenase activity and cyanide-insensitive mitochondrial respiration. ' MATERIALS AND METHODS Plant Tissue. White potato tubers (Solanum tuberosum L.) and red sweet potatoes (Ipomoea batatas Lam.) were purchased at local markets and stored at ro...

Preparation and Properties of Mitochondria from Cowpea Nodules

“…This activity was not due to lipoxygenase, for the concentration of this enzyme was highest in preparations of nodule mitochondria, which did not show cya- nide resistant 02 uptake. The lipoxygenase contamination of cowpea hypocotyl mitochondria is similar to that reported for purified mung bean hypocotyl mitochondria (19). The lipoxygenase activity in nodule mitochondria was less than that reported for washed mitochondria from soybean axes and potato tubers, but was 3 to 4 times greater than the lipoxygenase activity in those preparations after they had been purified with Percoll (19).…”

“…Lipoxygenase acting on the fatty acids in a mitochondrial preparation can contribute to 02 uptake. Because lipoxygenase is not inhibited by cyanide, its presence as a contaminant may be interpreted as cyanide-insensitive or alternative respiration ( 19).…”

Structural Features Required for Inhibition of Soybean Lipoxygenase-2 by Propyl Gallate

“…Also mitochondria isolated from skunk cabbage and Peltandra virginica spadices showed significant levels of alternative pathway, but no measurable lipoxygenase activity (23). Similarly, most of the lipoxygenase activity present in aged potato tuber mitochondria prepared by differential centrifugation is removed when the organelles are purified by sucrose gradient centrifugation while the alternative pathway is usually unaffected (20). Clearly, the experimental evidence which demonstrates a distinction between lipoxygenase and the alternative oxidase is substantial.…”

Localization of Lipoxygenases 1 and 2 in Germinating Soybean Seeds by an Indirect Immunofluorescence Technique

“…However, Fritz et al [10] showed that, whereas lipoxygenase activity accounted for a large portion ofthe 02 absorption in crude extracts of corn seedlings, only 0.4% of 02 was actually chemically incorporated into the tissue during rapid respiration of the seedlings in vivo. Recently, Shingles et al (19) reported that lipoxygenase contamination of preparations of potato mitochondria does not contribute significantly to the cyanide-insensitive 02 consumption. Simultaneously, Dupont et aL (9) arrived at the same conclusion for mitochondria from potato, soybean, and arum.…”