Electron microscopic cytochemical localization of?-hydroxyacid oxidase in rat liver

Abstract

The substrate specificity and the intraperoxisomal localization of alpha-hydroxyacid oxidase in rat liver has been investigated cytochemically by the cerium technique and biochemically with a luminometric assay. Rat liver is fixed by perfusion with a low concentration (0.25%) of glutaraldehyde and vibratome sections are incubated for 60 min at 37 degrees C in a medium containing 3 mM CeCl3, 100 mM NaN3 and 5 mM of an alpha-hydroxyacid in 0.1 M of one of the following buffers: Pipes, Mops, Na-cacodylate, Tris-maleate, all adjusted to pH 7.8. Ten different alpha-hydroxyacids with a chain length between 2 and 8 carbon atoms were tested. The best results were obtained with glycolic, argininic and L-alpha-isocaproic acids. These cytochemical findings were confirmed also biochemically using purified peroxisomal fractions isolated by gradient centrifugation in metrizamide. The pattern of the intraperoxisomal localization of the enzyme was influenced markedly by the type of buffer used for the cytochemical incubation. Whereas in the Tris-maleate medium both the cores and the matrix stained with the same intensity, with all other buffers the reaction in cores was more prominent. The staining of cores was abolished by pretreating sections in Tris-maleate (pH 7.8) or alkaline pyrophosphate buffers. These observations establish the substrate specificity of alpha-hydroxyacid oxidase in rat liver and demonstrate the delicate association of this enzyme with the crystalline cores and the matrix of peroxisomes in rat liver.

Reconstruction of human hepatocyte glyoxylate metabolic pathways in stably transformed Chinese-hamster ovary cells

“…In fact, too much oxalate is detrimental because of the low solubility of its calcium salt, which can readily crystallize out as calculi in the kidney and urinary tract [1]. The most important precursor of oxalate in humans is the intermediary metabolite glyoxylate, which is itself synthesized from glycolate catalysed by the liver-specific [2] peroxisomal [3] enzyme GO (glycolate oxidase; EC 1.1.3.15) (Scheme 1). The source of glycolate is unclear, but it most probably arises directly from the diet or from carbohydrate dietary precursors [4].…”

Rat kidney L-2-hydroxyacid oxidase. Structural and mechanistic comparison with flavocytochrome b2 from bakers' yeast

“…In rat kidney, it is the only a-hydroxy acid oxidizing activity present (Blanchard et al, 1946;Nakano & Danowski, 1966;McGroarty et al, 1974). In pig kidney peroxisomes, it is found together with glycolate oxidase (short-chain hydroxyacid oxidase, EC 1.1.3.1) (Robinson et al, 1962); only the latter is present in mammalian liver and leaf peroxisomes (Tolbert, 1981;Angermuller et al, 1986b). Rat kidney hydroxyacid oxidase has been purified by various groups (Blanchard et al, 1945;Nakano & Danowski, 1966; Cromartie & Walsh, 1975a; ).…”

Current Cytochemical Techniques for the Investigation of Peroxisomes: A Review

“…Somewhat better results were reported by others in kidney and central nervous system (Arnold et al 1979; Hand 1979; Arnold and Holtzman 1980). Almost a decade later, Angermüller and Fahimi (1986) reported the appropriate processing conditions for fixation and visualization of the oxidase activities in different vertebrate tissues, consisting of a short perfusion-fixation with 0.25% glutaraldehyde in PIPES buffer, pH 7.4, followed by incubation in 3 mM cerium chloride with specific substrates (e.g., urate, glycolate, or D -proline) in appropriate buffers in the presence of 100 mM sodium azide (Angermüller and Fahimi 1986, 1988a, b; Angermüller et al 1986a,b). The latter is necessary to inhibit the peroxisomal catalase activity that otherwise would destroy the H 2 O 2 generated by the oxidases.…”