Non-mitochondrial oxidizing particles (microbodies) in rat liver and kidney and in $\text{Tetrahymena pyriformis}$☆

Glycolate oxidase, an FMN-dependent peroxisomal oxidase, plays an important role in plants, related to photorespiration, and in animals, where it can contribute to the production of oxalate with formation of kidney stones. The best studied plant glycolate oxidase is that of spinach; it has been expressed as a recombinant enzyme, and its crystal structure is known. With respect to animals, the enzyme purified from pig liver has been characterized in detail in terms of activity and inhibition, the enzyme from human liver in less detail. We describe here the purification and initial characterization of the recombinant human glycolate oxidase. Its substrate specificity and the inhibitory effects of a number of anions are in agreement with the properties expected from previous work on glycolate oxidases from diverse sources. The recombinant enzyme presents an inhibition by excess glycolate and by excess DCIP, which has not been documented before. These inhibitions suggest that glycolate binds to the active site of the reduced enzyme, and that DCIP also has affinity for the oxidized enzyme. Glycolate oxidase belongs to a family of l-2-hydroxy-acid-oxidizing flavoenzymes, with strongly conserved active-site residues. A comparison of some of the present results with studies dealing with other family members suggests that residues outside the active site influence the binding of a number of ligands, in particular sulfite.

Peroxisomes from Tetrahymena pyriformis contained catalase, d-amino acid oxidase, cyanide-insensitive fatty acyl-CoA oxidizing system, carnitine acetyltransferase, isocitrate lyase, leucine:glyoxylate aminotransferase and phenylalanine:glyoxylate aminotransferase. These activities, except carnitine acetyltransferase, were found at the highest levels in the light mitochondrial fraction, whereas the highest activity of carnitine acetyltransferase was found in the micotchondrial fraction. Sucrose density gradient centrifugation showed that the density of peroxisomes was approx. 1.228 g/ml and that of mitochondria was approx. 1.213 g/ml. When the light mitochondrial fraction was treated with deoxycholate or by freeze-thawing, most of the activities of catalase and isocitrate lyase were solubilized, whereas about half of the original activity of aminotransferase remained in the pellet fraction. Addition of fatty acid and clofibrate increased the activities of the cyanide-insensitive fatty acyl-CoA oxidizing system and isocitrate lyase in the peroxisomes. The activity of catalase was slightly increased by glucose and clofibrate; leucine:glyoxylate aminotransferase activity was significantly increased by clofibrate treatment.

• 1.

  1. Microbodies are present in Malpighian tubules, fat body and gut in a blowfly, A. grahami.

• 2.

  2. Those in Malpighian tubules are nucleoid type and contain urate oxidase and catalase. D-Amino acid oxidase and catalase are contained in the particles of fat body and gut.

• 3.

  3. The nucleoids in microbodies of Malpighian tubules seem to be loosened when the urate oxidase activity disappears during the metamorphosis.

An agarose derivative that is an affinity adsorbent for chromatography of urate oxidase was prepared by coupling 8-aminoxanthine with epoxy-activated Sepharose 4B. This affinity adsorbent was stable in 0.1 m borate buffer (pH 10.0) at 4°C since no decrease of effective ligand content in the gel was found for 5 months. Chromatography of crude urate oxidases prepared from bovine kidney, rat liver, and yeast on the affinity column resulted in 2- to 88-fold purifications.