Surface localization of sites of reduction of nitroxide spin-labeled molecules in mitochondria.

Abstract

The relative rates of reduction of several spin-labeled molecules that partition differently across the hydrophobic-interface of inner membranes from rat liver mitochondria were investigated. Spin labels localized either deep in the hydrophobic region or in the aqueous phase are only slowly reduced; however, a spin-labeled analogue of the cationic detergent cetyltrimethylammonium bromide that partitions at the interface is rapidly reduced by coupled electron transport. Chemical studies on the reduction and oxidation of the spin label show that loss of signal is due to reduction and not destruction ofthe label. No evidence was found for flip-flop of the label in submitochondrial preparations.Spin reduction of respiring mitochondria, mitoplasts, or in- (4)(5)(6)(7)(8). By manipulation of available 02, with inhibitors specific for particular "sites" and uncouplers of respiration, it is sometimes possible to deduce the particular "site" at which the substance is being reduced. On the other hand, the test substance, e.g., a spin label, may have physicochemical properties ensuring that it will come to rest in a particular region of the mitochondrial structure. When both of these circumstances are realized, one can logically conclude that a transfer of reducing equivalents from the electron transport chain to the spin label goes on in the particular region of the mitochondrial structure where the spin-labeled molecule is localized. Our paper illustrates the foregoing strategy. To apply this strategy, we had to find a spin-labeled substance with the right properties (an analogue of cetyltrimethylammonium bromide, CDTAB) to show that after it was intercalated into the mitochondrial structure its signal decayed due to respiratory chain activity, and finally to show by manipulation of this activity that the reductant in the chain most likely to be affecting the spin label was ubiquinone.MATERIALS AND METHODS Preparations. Rat liver mitochondria were prepared in a 0.25 M sucrose, 1 mM Tris-HCl and 1 mM EDTA medium at pH 7.4 as described (9). Mitoplasts were also prepared (10). Submitochondrial preparations (SMP) were made following a method described in ref. 11. Protein concentrations were determined by a standard method (12).Spin Labels. The nitroxide spin-labeled molecules were provided by R. J. Mehlhorn (of this laboratory) and have the structures shown in Fig. 2 RESULTSChoosing an appropriate spin label We first surveyed the behavior of various classes of spin labels (Fig. 2) toward the respiratory chain activity. CDTAB, a cation 570 Abbreviations: CDTAB, spin-labeled analogue of cetyltrimethylammonium bromide; SMP, submitochondrial preparation.

Noninvasive Assessment of the Brain Redox Status after Transient Middle Cerebral Artery Occlusion Using Overhauser-Enhanced Magnetic Resonance Imaging

“…To examine its reduction at the mitochondria, methoxycarbonyl-PROXYL was mixed with the mitochondrial fraction. Two segments of the electron transport chain are primarily responsible for the reduction of nitroxyl radicals, complex I and complex II (Chen et al, 1988;Quintanilha and Packer, 1977). Only a small signal decay appeared when glutamate/malate was used, as a substrate for complex I, in both the hemispheres, whereas the addition of succinic acid, as a substrate for complex II, resulted in the clear decay of the methoxycarbonyl-PROXYL ESR signal.…”

“…Glutathione also affects the reduction of nitroxyl radicals indirectly (Bobko et al, 2007), through their interaction between ascorbic acid and GSH as a redox couple (Mehlhorn, 1991;Meister, 1994;Winkler et al, 1994). In addition, nitroxyl radicals can be reduced enzymatically by complexes I and II in the electron transport chain of the mitochondria (Chen et al, 1988;Quintanilha and Packer, 1977). In this study, we used the nitroxyl radicals to investigate whether the level of antioxidants or the activity of redox enzymes is altered in the brain after ischemia-reperfusion.…”

Interaction of Nitroxide Spin Labels with Chloroplasts

“…Because the active, paramagnetic nitroxide is readily reduced to the nonparamagnetic hydroxylamine (which cannot be detected with ESR), spin labels can also be used to probe redox reactions. The depths of sites of oxidation and reduction in the bilayer of mitochondrial membranes have been determined using spin labels (8).…”

Heterogeneity of Regional Redox Status and Relation of the Redox Status to Oxygenation in a Tumor Model, Evaluated Using Electron Paramagnetic Resonance Imaging

“…It has been reported that microsomes (11), mitochondria (12), and sulfhydryl-related systems (6) contribute to nitroxyl reduction in a body. Additionally, inhibition experiments with ascorbate oxidase suggested that ascorbic acid is a main cytosolic reducing agent for pyrrolidine nitroxyl radicals in biological systems (10,27,28).…”

“…The nitroxyl radicals are reduced to their corresponding hydroxylamine in an animal body. Numerous in vivo and in vitro studies have clarified that small-molecule antioxidants such as ascorbic acid and GSH, as well as enzymatic systems, contribute to the reduction (6,(9)(10)(11)(12). This reduction is reversible (13) and enhanced under hypoxic conditions in vivo, as well as in vitro (14,15).…”