Glyoxylate cycle enzymes and catalase in digitonin-fractionated mitochondria inTurbatrix aceti

Abstract

The subcellular localization of the glyoxylate cycle enzymes, isocitrate lyase and malate synthase, and the peroxisomal marker, catalase, was examined in 14-day-old cultures of the nematodeTurbatrix aceti. Glyoxylate cycle enzymes co-sedimented with selected mitochondrial enzymes during rate sedimentation. Two separate enzyme peaks were resolved after isopycnic centrifugation on a sucrose gradient: glyoxylate cycle enzymes, isocitrate lyase and malate synthase, and the Krebs cycle markers, fumarase and citrate synthase, banded at a density of 1.214 g/cm3 while catalase consistently sedimented at 1.197 g/cm3. Electron microscopy revealed that mitochondria with similar morphology were the only recognizable organelles in both enzyme peaks. Notably, organelles resembling microbodies (glyoxysomes) were not present. The compartmentation of these enzymes within the mitochondria was examined by sequentially disrupting organelle membranes with varying concentrations of digitonin. The complete release of isocitrate lyase and malate synthase occurred concomitantly with the loss of matrix material from the mitoplasts. Citrate synthase was concurrently released, although to a lesser extent, while fumarase and catalase remained associated with disrupted membrane fragments at the highest concentration of digitonin used. Thus, glyoxylate cycle enzymes and catalase appear to be localized withinT. aceti mitochondria and not within glyoxysomes as generally found in other eucaryotes.