The Problem of Reduced Nicotinamide Adenine Dinucleotide Oxidation in Glyoxysomes

Abstract

NADH is generated in glyoxysomes both in the glyoxylate cvcle and in p-oxidation. No system has yet been described which would oxidize NADH in these organelles. A series of oxidants which might function by coupling NADH oxidation to 02 through endogenous carriers in the glyoxysomes was examined. Oxidation was brought about by ferricyanide or dichlorophenol-indophenol, but it was shown that this "dia-phorase" activity is probably a contaminant. Hydroxypyruvate reductase (NAD-linked) is present in the glyoxysomes, and at very high substrate concentrations (>10 mM) this enzyme can also transfer electrons from NADH to glyoxylate. However, it is most unlikely that this concentration of glyoxylate is ever approached in glyoxysomes, where the malate synthetase would compete on much superior terms. The maximum rates of NADH oxidation observed in the presence of ferricyanide or glyoxylate are only a fraction of those required to reoxidize NADH at the rate occurring in vivo. The reactions which are known to occur in glyoxysomes include two in which NADH is generated, the oxidation of malate in the glyoxylate cycle (2) and the oxidation of /3-hy-droxyacyl-CoA during ,B-oxidation of fatty acids (5, 9). The sustained operation of the overall sequence in the organelles requires reoxidation of NADH, and since the glyoxysomes themselves apparently do not have this capacity, the cooperation of the mitochondria has been invoked (5). In this paper we describe the ability of the glyoxysomes to transfer electrons from NADH to certain acceptors but give reasons for supposing that these are not the effective oxidants in vivo. MATERIALS AND METHODS Seeds of castor bean (Ricinus communis) were soaked overnight and germinated in moist vermiculite at 30 C for 5 days. Endosperm tissue was homogenized using the method and grinding medium described by Theimer and Beevers (12). After filtering the crude homogenate through four layers of cheesecloth and centrifuging at 270g for 10 min to remove intact cells and debris, the supernatant (supernatant I) was centrifuged at 10,800g for 20 min, yielding supernatant II and a pellet, the crude particulate fraction. The crude particulate pellet was gently resuspended in grinding medium and mito-'This work was supported by Grant GB 24961 from the National Science Foundation. chondria and glyoxysomes were separated by linear sucrose density gradient centrifugation and collected as described previously (7). Enzyme Assays. The procedures used for isocitrate lyase, malate synthetase, malate dehydrogenase, fumarase, and cit-rate synthetase have been outlined previously (4). Glycolate oxidase was assayed by the method of Feierabend and Beevers (6). Hydroxypyruvate reductase was determined by measuring the decrease in absorbance at 340 nm due to the oxidation of NADH or NADPH by hydroxypyruvate. The reaction mixture contained, in a final volume of 1.0 ml, 50 mM potassium phosphate , pH 6.5, 0.25 mm NADH or NADPH and enzyme. The reaction was started by adding hydroxypyruvate to a final concentration of 1 mm. To assay for glyoxylate reductase, lactate dehydrogenase and ferricyanide-dependent NADH oxidation, 50 mM glyoxylate, 10 mM pyruvate, or 0.1 mm potassium ferri-cyanide respectively were substituted for hydroxypyruvate. Where necessary, rates of NADH oxidation were corrected for endogenous NADH-oxidase activity or non-enzymic reaction. The a-oxidation activity was determined on the crude particulate pellet resuspended in grinding medium in which the sucrose concentration had been increased to 0.6 M. To minimize organelle breakage due to dilution in the reaction mixture , all reagents were prepared in 0.6 M sucrose. The reaction was followed by measuring NADH formation at 340 nm (5). Protein was determined by the method of Lowry et al. (11). RESULTS AND DISCUSSION As established previously, NADH is not oxidized by isolated glyoxysomes. The addition of FMN, FAD, cysteine, KNO,, glutathione or cytochrome c failed to bring about NADH oxidation. Ferricyanide and DCPIP did elicit NADH oxidation by the glyoxysomes, at rates of 0.12 and 0.06 umole per min per mg glyoxysomal protein respectively. The rate of ferri-cyanide-induced oxidation was not enhanced by flavins and was unaffected by cyanide. This enzyme activity is of the kind previously reported as a diaphorase (8). Presumably it reveals the potential for transfer of electrons from NADH to some endogenous carrier and is thus of possible significance to NADH oxidation in glyoxysomes in vivo. However, when the enzyme activity was measured across the gradient used to prepare the organelles, it was found that more than 90% was recovered in the mitochondria and coincided closely with the fumarase distribution; the small peak of activity in the glyoxy-some region, nevertheless, followed the isocitrate lyase. The possibility that the activity in the glyoxysomes represented a contaminant was examined by taking the whole fraction, adding sucrose to make the concentration 60%, and centrifuging in a flotation gradient (6). As expected, most of the protein was recovered in a peak of mean density 1.25 g/cm' and coincided with the distribution of isocitrate lyase. A peak of ferri-249