Cyanide-insensitive and Cyanide-sensitive O 2 Uptake in Wheat

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...

mentioning

confidence: 99%

Alternative Pathway Respiration and Lipoxygenase Activity in Aged Potato Slice Mitochondria

Shingles¹,

Arron²,

Hill³

1982

“…Siedow and Girvin (21), however, demonstrated that propyl gallate inhibits not only lipoxygenase but also the alternative pathway with a Ki' 5-to 10-fold lower than that found for SHAM. The similarities in sensitivity to SHAM, propyl gallate, and cyanide make it difficult to distinguish between the activity of lipoxygenase and the alternative pathway in plant tissues and have led to some speculation that the alternative pathway is somehow related to the presence of lipoxygenase (5,8,9). In an effort to determine if more subtle differences exist between lipoxygenase and the alternative pathway, studies of the inhibition of these two activities by propyl gallate were undertaken.…”

mentioning

confidence: 99%

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

Peterman¹,

Siedow²

1983

“…We conclude therefore that respiration was mediated mainly via the alternative respiratory pathway. It has beTn suggested that lipoxygenase is responsible for much of the cya'nide-resistant 02 uptake in some systems (7,8,20) but inasmuch as this enzyme is mainly associated with lipid degradation it is hard to conceive of lipoxygenase playing a significant role during exponential growth (26).…”

Section: Discussionmentioning

confidence: 99%

Cyanide-Resistant Respiration in Suspension Cultured Cells of Nicotiana glutinosa L

Horn¹,

Mertz²

1982

The respiration of dark-grown Nicotiava glutinosa L. cells in liquid suspension culture was found to be highly cyanide resistant and salicylhydroxamic acid (SHAM) sensitive, indicative of an active alternative respiratory pathway. This was especially true during the lag and logarithmic phases of the 14-day growth cycle. Mitochondria isolated from logarithmically growing cells exhibited active oxidation of malate, succinate, and exogenous NADH. Oxidation of all three substrates had an optimum pH of 6.5 and all were highly resistant to inhibition by cyanide and sensitive to SHAM. Respiratory control was exhibited by all three substrates but only if SHAM was present to block the alternative pathway and divert electrons to the phosphorylating cytochrome pathway. The cyanide-resistant oxidation of exogenous NADH has previously only been associated with Arum spadix mitochondria. Coemergence during evolution of the alternative respiratory pathway and the exogenous NADH dehydrogenase in plant mitochondria as a possible mechanism for removal of cytoplasmic NADH is proposed. Evidence is presented which suggests that mitochondrial assays should be performed at pH 6.5.Cyanide-resistant respiration has been described in a wide assortment of plant species (10,24). Most of the work has been performed with mitochondria isolated from intact plant tissues (mung bean shoots, skunk cabbage spadices, etc.). With the current interest in plant tissue culture as an experimental tool it was thought to be of importance to characterize the respiration of cultured plant cells. Wilson (28-30) has examined respiration of isolated mitochondria from cultured sycamore cells and found it to be partially resistant to inhibition by cyanide. RC2 was rather low with a ratio of 2.0 or less. With the discovery of the hydroxamates, inhibitors of the alternate respiratory pathway (22), it has been reported that they are effective in inhibiting respiration resistant to cyanide or antimycin A in cultured cells of tobacco (21) and soybeans (16). In the present investigation, using suspension cultured cells of Nicotiana glutinosa L. and isolated mitochondria from these cells, it has been established that respiration is sensitive to the hydroxamate SHAM and the sensitivity varies over the course of the growth cycle. The high sensitivity of respiration to SHAM and the low RC found in mitochondria from these cultures suggests that mitochondrial ATP production is limited. Preliminary studies showed that a final concentration of 1.6% alcohol caused no significant change in either the qualitative or quantitative activity of the mitochondria within a 10-min period.The reaction mixture for the respiratory measurements of whole cells consisted of 0.09 M sucrose, 50 mM Mes (pH 6.5). The buffer and pH were determined to be optimum for these cells (11).Mitochondrial Isolation. The mitochondrial isolation procedure was similar to that described by Charbonnier and Berville (4). The tissue was placed in a chilled mortar containing cold grinding medium (1:1, w/v) a...