Electron microscopic cytochemical localization of?-hydroxyacid oxidase in rat kidney cortex

Abstract

The substrate specificity of alpha-hydroxyacid oxidase in the rat kidney has been investigated cytochemically by the cerium technique and biochemically with a luminometric assay applied to isolated renal peroxisomes. Rat kidneys were fixed by perfusion via the abdominal aorta with a low concentration (0.25%) of glutaraldehyde. Vibratome sections were 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 Pipes or 0.1 M Tris-maleate buffer both adjusted to pH 7.8. Ten aliphatic alpha-hydroxyacids with chain lengths between 2 and 8 carbon atoms and two aromatic substrates were tested. The alpha-hydroxyacid oxidase in the kidney exhibited a markedly different substrate specificity than the corresponding enzyme in the liver. Thus glycolate gave a negative reaction while two aromatic substrates, mandelic acid and phenyllactic acid, stained prominently. With aliphatic substrates a stronger reaction was obtained in Pipes than in the Tris-maleate buffered incubation media. The best reaction in the kidney was obtained with hydroxybutyric acid. These cytochemical findings were confirmed by the luminometric determination of the oxidase activity in isolated purified peroxisome fractions. By electron microscopy the electron dense reaction product of cerium perhydroxide was found in the matrix of peroxisomes in the proximal tubules. The intensity of reaction varied markedly in neighbouring epithelial cells but also in different peroxisomes within the same cell. Thus heavily stained particles were seen next to lightly reacted ones. These observations establish the substrate specificity of alpha-hydroxyacid oxidase in the rat kidney and demonstrate the marked heterogeneity in the staining of renal peroxisomes for this enzyme.

Iodixanol (Optiprep), an Improved Density Gradient Medium for the Iso-osmotic Isolation of Rat Liver Peroxisomes

“…Close inspection of the peroxisomal marker enzyme distributions suggests the occurrence of heterogeneity: compared to the distribution of catalase, that of gly-the enzyme content of the individual organelles has colate oxidase was slightly shifted to lower densities, been demonstrated (41)(42)(43). while that of urate oxidase was broader in all gradients Compared to Nycodenz (15), separations are cleaner, and contamination of peroxisomes by mitochondria and analyzed.…”

Immunohistochemical Localization of Peroxisomal Enzymes in Developing Rat Kidney Tissues

“…Peroxisome-specific enzymes have been localized specifically in each subcompartment by immunocytochemistry or enzyme histochemistry. Catalase, isozyme B of l-␣-hydroxyacid oxidase (HOX-B), acyl-CoA oxidase, enoyl-CoA hydratase/3-hydroxyacyl-CoA dehydrogenase bifunctional protein or multifunctional protein 1 (BP), and 3-ketoacyl-CoA thiolase are localized in the electron-dense peripheral matrix (Bendayan et al 1983;Yokota et al 1985;Angermüller et al 1986). D-Amino acid oxidase (DAOX) is confined to the central electron-lucent matrix (Usuda et al 1986;Angermüller and Fahimi 1988).…”

Molecular cloning and nucleotide sequence of cDNA encoding rat kidney long‐chain l‐2‐hydroxy acid oxidase

“…This peroxisomal enzyme is found together with isozyme A in hog, beef and sheep kidney (Robinson et al, 1962;McGroarty et al, 1974;Zaar and Fahimi, 1991), whereas it represents the only ahydroxy-acid-oxidizing activity in rat kidney (Blanchard et al, 1946;Nakano and Danowski, 1966;McGroarty et al, 1974;Angermiiller et al, 1986;Zaar and Fahimi, 1991).…”