β-Oxidation and Glyoxylate Cycle Coupled to NADH: Cytochrome c and Ferricyanide Reductases in Glyoxysomes

Abstract

ABSTRACIGlyoxysomes isolated from castor bean (Ricinus communis L., var Hale) endosperm had NADH:ferricyanide reductase and NADH:cytochrome c reductase activities averaging 720 and 140 nanomole electrons/per minute per milligram glyoxysomal protein, respectively. These redox activities were greater than could be attributed to contmination of the glyoxysomal fractions in which 1.4% of the protein was mitochondrial and 5% endoplasmic reticulum. The NADH:ferricyanide reductase activity in the glyoxysomes was greater than the palmitoyl-coenzyme A (CoA) oxidation activity which generated NADH at a rate Malate was also oxidized by glyoxysomes, if acetyl-CoA, ferricyanide, or cytochrome c was present. Glyoxysomal NADH:ferricyanide reductase activity has the capacity to support the combined rates of NADH generation by 8-oxidation and the glyoxylate cycle.

Ascorbate Free-Radical Reduction by Glyoxysomal Membranes

“…The rate of ascorbate free-radical reduction in glyoxysomes was similar to the rate of ferricyanide reduction; both were about 2000 nmol per min per mg protein (Table II). These rates ofNADH oxidation are equivalent to about 400 nmol electrons per min per mg glyoxysomal protein, which is similar to the rate of NAD+ reduction by fatty acid oxidation in glyoxysomes (8). AFR activity was not enhanced in purified membranes as was FCR (Table II), as the Na2CO3 treatment used in purification appeared to partially inactivate AFR.…”

“…The AFR and NADPH:CCR assays included 1 mm KCN to inhibit mitochondrial NADH oxidation. (8,9,14). In glyoxysomes, NADH consumption was dependent on ascorbate free-radical generated by the ascorbic acid/ascorbate oxidase system, and omission of ascorbate or ascorbate oxidase resulted in loss ofactivity (Table I).…”

“…A malate-aspartate shuttle has been proposed to serve this function (19), but the necessary movements of malate and aspartate between glyoxysomes and mitochondria have not been specifically demonstrated. We have hypothesized that intraglyoxysomal NADH could be oxidized by the membrane dehydrogenase, which could then transfer the electrons to an extraglyoxysomal acceptor (8,16).Ferricyanide or Cyt c can be used as artificial electron acceptors for the glyoxysomal membrane NADH dehydrogenase (1 1). Ascorbate free-radical may function as a cytosolic acceptor in this system because of its favorable reduction potential with respect to NADH (E'.…”

“…2 Present address: Wiley, Rein and Fielding, Attorneys, 1776 K Street, NW, Washington, DC 20006. mal ,3-oxidation (4,8) and the glyxoylate cycle, and additional energy is conserved in mitochondria by oxidation of succinate to oxaloacetate (3). The amount of energy released is greater than the energy required during subsequent gluconeogenesis from oxaloacetate (6).…”

“…A malate-aspartate shuttle has been proposed to serve this function (19), but the necessary movements of malate and aspartate between glyoxysomes and mitochondria have not been specifically demonstrated. We have hypothesized that intraglyoxysomal NADH could be oxidized by the membrane dehydrogenase, which could then transfer the electrons to an extraglyoxysomal acceptor (8,16).…”