Confounding of Alternate Respiration by Lipoxygenase Activity

Abstract

The initial burst of respiratory activity (Qo2) of Neither the biochemical nature nor the physiological significance of the cyanide-insensitive, alternate respiration is known, but its activity has now been demonstrated in a number of plant systems (2, 9) Alternate respiration is commonly defined or diagnosed as that portion of respiration which is insensitive to cyanide but inhibited by hydroxamic acids (2,8). This activity is thought to be due to an electron flow which does not involve Cyt oxidase (2, 10). We present data which show that initial in vivo 02 uptake by soybeans is not inhibited by KCN and is inhibited by hydroxamate; and that this apparent respiration may be due in part to the activity of lipoxygenase, an enzyme which catalyzes the dioxygenation of certain unsaturated fatty acids (5). emulsion in 10 mm phosphate buffer (pH 7.0) (11). A I mg/ml (3.56 mM) LA solution was used as substrate for the LOX assays. LOX was dissolved in 10 mm phosphate buffer (pH 7.0) and injected into the 02 electrode reaction vessel to give a reaction mixture which had 100 ,ug of LOX/ml. Assuming a mol wt of about 100,000 (1), LOX concentration was about I ,UM. MATERIALS AND METHODS RESULTS AND DISCUSSIONThe respiration of soybean particles was characterized by a burst of 02 consumption during the first few min of imbibition (6). This respiratory burst was not inhibited by KCN and was inhibited by SHAM (Fig. 1). The initial respiratory activity was also markedly inhibited by PG (Fig. 2), being about 10 times more sensitive to PG than to SHAM. If the normally alkaline KCN solution was strongly buffered (100 mM) to maintain the pH at neutrality, the small promotion of respiration seen in Figure 1 was no longer observed; and there was still no evidence of inhibition in the first few min. SHAM and PG sensitivities were not affected by buffering at pH 7.0.Suspecting that the SHAM inhibition might be related to LOX activity, we tested the cell-free enzyme's sensitivity to the hydroxamate and found (Fig. 3) that indeed it was, being 90%o inhibited by 5 mm SHAM. Autooxidation of LA (in the absence of LOX) was unaffected by SHAM. The LOX activity was not inhibited by KCN (data not shown). The enzyme was about 10 times more sensitive to PG than to SHAM, being 90%o inhibited at about 0.5 mM PG (data not shown).To investigate further the possible role of LOX in the apparent initial respiration, soybean seed particles were placed in buffered solution with or without SHAM and 02 uptake was measured with and without the addition of 50 jig/ml (0.18 mM) LA substrate (Fig. 4). In the absence of SHAM, when LA was added, there was an immediate 9-to 10-fold increase in respiratory rate and all of the 02 remaining in the solution was quickly consumed. When 5 mM SHAM was present, the initial 02 consumption was inhibited approximately 50%1b, and the addition of LA caused only a transient 2-to 3-fold increase in 02 consumption. (Added Tween 20 was without effect on the respiration rates in the presence of absence of hydroxamate.) The m...

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

“…The results presented here and by Siedow and Girvin (13) suggest that neither SHAM nor PG, a purported inhibitor of lipoxygenase (11), can be used to define the enzyme system(s) involved in cyanide-resistant 02 uptake in potatoes. However, a kinetic analysis using both inhibitors is used in this ' In some experiments, cyanide-resistant respiration was induced in the potato by aging slices from untreated potatoes in an aerated 0.1 mm CaSO4 solution.…”

“…Rich et al (12) have documented inhibition of tyrosinase and peroxidase by SHAM; however, both of these enzymes are inhibited by cyanide. Parrish and Leopold (11) have demonstrated that the cyanide-resistant 02 consumption by ground soybean endosperm which is inhibited by SHAM is attributable to lipoxygenase activity. Hence, the ability of SHAM to uniquely inhibit a single enzyme, i.e.…”

“…The supernatant was passed through a Sephadex G-50 column, and the pooled protein fraction was used for the lipoxygenase assay. Lipoxygenase was assayed at pH 7.0 polarographically as described by Parrish and Leopold (11). The washed mitochondrial fraction was also used to determine the lipoxygenase activity associated with it using the methodology of Siedow and Girvin (13).…”

Cytochrome and Alternative Pathway Respiration in Green Algae

“…High SHAM concentrations have also been reported to inhibit Cyt pathway activity (2,11). Other potential complications arising from the use of SHAM are inhibition of lipoxygenase or other 02-consuming enzymes (14,16), as well as the recent report that use of alternative pathway inhibitors may result in a small increase in electron flow through the cytochrome pathway (27). More specific inhibitors ofthe alternative pathway have been found, but they are not suitable for working with intact tissues (13).…”

Respiration of Gametangia of the Aquatic Phycomycete Allomyces macrogynus

“…More recently, alternative respiration has been shown to be inhibited by propyl gallate, also an inhibitor of lipoxygenase (12,16,18).…”