Subcellular localization of glyoxylate cycle enzymes in Ascaris suum larvae.
Evidence is presented on the particulate nature of glyoxylate cycle enzymes in metazoa with the use of 15-day old larvae of the nematode Ascaris suum. Homogenization procedures were developed to disrupt the resistant nematode cuticle. Malate synthase and isocitrate lyase, key enzymes of the glyoxylate cycle, consistently sedimented with mitochondrial enzymes in differential pellets while catalase, a major peroxisomal enzyme, was always soluble. Isopycnic sucrose gradient centrifugation of the differential pellet yielded two protein peaks: one at 1.18 g/cm3 (characteristic for mitochondria), and another at 1.23 g/cm3 (common for glyoxysomes and peroxisomes). Electron microscopy of these fractions revealed that the lighter peak consisted primarily of mitochondria, while the heavier band contained proteinaceous bodies termed "dense granules" morphologically resembling microbodies. SIgnificantly, both malate synthase and isocitrate lyase cosedimented with the mitochondrial marker enzymes in the lighter peak (1.18 g/cm3) and not with the dense granules. Further purification of mitochondria, accomplished by separating dense granules with a step gradient before isopycnic centrifugation, substantiated the evidence that microbodies (glyoxysomes) do not occur in these nematode larvae. Rough-surfaced membranes were alternatively considered as the subcellular site, but the evidence tends to favor localization of the glyoxylate bypass enzymes in the mitochondria.
D000818:Animals; D001200:Ascaris / Q000201:enzymology* / Q000648:ultrastructure; D002374:Catalase / Q000032:analysis; D002458:Cell Fractionation; D002950:Citrate (si)-Synthase / Q000032:analysis; D005649:Fumarate Hydratase / Q000032:analysis; D007814:Larva / Q000201:enzymology; D008291:Malate Dehydrogenase / Q000032:analysis*; D008928:Mitochondria / Q000201:enzymology*; D009940:Organoids / Q000201:enzymology; D007652:Oxo-Acid-Lyases / Q000032:analysis*
D008291:Malate Dehydrogenase; D002374:Catalase; D002950:Citrate (si)-Synthase; D007652:Oxo-Acid-Lyases; D005649:Fumarate Hydratase