Binding of Butyl Gallate to Isolated Mung Bean Mitochondria

Abstract

The binding of radioactively labeled butyl gailate to sucrose gradientpurified mung bean ( Vigna radiata L.) mitochondria was studied. propyl gallate and the hydroxamic acids inhibit the alternative pathway at the same (or at least spatially very similar) site. The structural features required for inhibition of the alternative pathway by propyl gallate have also been determined (24). Additional compounds which inhibit the alternative pathway at site(s) other than the site which is sensitive to gallates and hydroxamates have also been reported (7,11,20).Studies using the binding of radioactively labeled herbicides to isolated chloroplasts have proved remarkably successful in helping characterize the component known as 'B' on the photosynthetic electron transfer chain (15,16, 28). While binding studies using labeled inhibitors with animal mitochondria have also been reported (4, 12), almost no such experiments have appeared using isolated mitochondria from higher plants. In the present report, we synthesized a radioactive analog of propyl gallate, [14CJbutyl gallate, and studied its binding to isolated, purified mung bean mitochondria. The results serve to confirm independently several earlier observations obtained from kinetic studies ofpropyl gallate inhibition of the alternative pathway and allow, for the first time, an estimate of the absolute amount of alternative oxidase present in plant (in this case, mung bean) mitochondria.The cyanide-resistant, alternative pathway represents one of the most intensively studied aspects of the plant mitochondrial electron transfer chain yet many of the basic questions regarding the nature of the pathway remain unanswered. It is generally accepted that the cyanide-resistant electron flux to O2 involves a mitochondrial-associated oxidase, and that electrons branch off the main, cyanide-sensitive pathway onto the alternative pathway at the level of the ubiquinone pool (5, 17, 23). Several lines of evidence indicate that the alternative oxidase functions as a pdiphenol:oxygen oxidoreductase (2, 13). However, the nature of the oxidase remains to be elucidated, and in no case is the absolute amount of alternative oxidase present in the mitochondrial membrane known.Whereas progress toward characterization of the alternative oxidase has proceeded somewhat slowly, recent years have seen the appearance of a number of compounds which specifically inhibit electron transfer through the alternative pathway in isolated mitochondria, specificity being defined by a lack of effect on cyanide-sensitive electron transfer. In 1971, Schonbaum et al. (22) reported that aryl-substituted hydroxamic acids could completely inhibit the alternative pathway at concentrations which were without effect on the main pathway. More recently, Siedow and Girvin (25) found that the antioxidant n-propyl gallate could inhibit the alternative pathway, and kinetic studies showed that ' This investigation was supported by National Institute of General Medical Sciences Grant GM 26095 to J. N. Siedow. MATER...

Binding of Butyl Gallate to Plant Mitochondria

“…Butyl gallate bound specifically to gradient-purified mitochondria isolated from both cyanide-sensitive and -resistant sources (Table II) mum level of butyl gallate specifically bound (P,) was generally in the range of 1 to 2 nmol/mg protein. This latter value is considerably higher than the concentrations associated with individual protein electron carriers in the electron transport chain (0.2-0.4 nmol/mg protein) but also lower than the amount of ubiquinone present in the membrane (4-6 nmol/mg protein) (22). The observation that butyl gallate bound to mitochondria independent of a measurable alternative pathway was similar to results seen with disulfiram which bound to both cyanide-sensitive and -resistant mitochondria from white potato tubers (7).…”

“…Purified plant mitochondria were prepared from washed mitochondria on a two-step sucrose gradient modified from Douce et al (5) as described by Siedow and Bickett (22). With the exception of rat liver, purified mitochondria (resuspended in wash buffer minus BSA) were used in all binding studies.…”

“…The binding procedure was similar to that used previously by Siedow and Bickett (22) except that only 0.5 to 1.0 mg mitochondrial protein/ml was used in each assay and the concentration of unlabeled butyl gallate used to displace labeled butyl gallate was increased from 100 to 400…”

“…Mitochondrial electron transport was measured polarographically using a Clark-type 02 electrode and conditions described previously (21,22). Protein was measured according to the method of Lowry et al (13) with BSA as the standard.…”

“…Protein was measured according to the method of Lowry et al (13) with BSA as the standard. ['4C]Butyl gallate was synthesized and stock solutions made up and quantified in ethanol as before (22). Unlabeled 3 Abbreviations: PVPP, polyvinylpolypyrrolidone; SHAM, salicylhydroxamic acid.…”