Light and electron microscopic localization of D-aspartate oxidase in peroxisomes of bovine kidney and liver: an immunocytochemical study.

Abstract

D-Aspartate oxidase (EC 1.4.3.1; D-ASPOX) specifically oxidizes the D-isomers of dicarboxylic amino acids such as aspartic or glutamic acid. Subcellular fractionation experiments in the past showed its association with peroxisome preparations in kidney cortex and liver. However, no information exists on the in situ localization and distribution of the enzyme in different cell types. We have purified the enzyme from the bovine kidney and raised an antibody against it in rabbits. The monospecificity of the antibody has been confirmed by Western blotting and it does not crossreact with D-amino, acid oxidase. Immunohistochemical localization of the antigen in bovine kidney and liver with the streptavidin-biotin-peroxidase technique revealed a punctate localization in the epithelial cells of proximal nephron tubules, particularly in the straight P-3 segment, as well as in hepatocytes. This is consistent with a localization in peroxisomes. Best results have been obtained with Carnoy-fixed material after paraffin embedding or after fixation with formaldehyde-glutaraldehyde in cryostat sections. Immunoelectron microscopy with protein A-gold confirms the peroxisomal localization of D-ASPOX. Gold particles are distributed over the matrix, suggestive of a peroxisomal matrix enzyme. This is the first report on the localization of D-ASPOX, a little-known peroxisomal enzyme. The techniques described and the antibody prepared will now allow systematic investigation of its tissue distribution.

Cellular and subcellular distribution of D‐aspartate oxidase in human and rat brain

“…A polyclonal antibody against D-ASPOX protein purified from bovine kidney cortex (anti-protein antibody) was raised in rabbit and has been characterized (Zaar, 1996). Furthermore, after D-ASPOX c-DNA from bovine kidney cortex was sequenced, an antibody (anti-peptide antibody) was raised in rabbit against the synthetic peptide CTYP-DTPIQKQKQWF mapping the amino acid sequence 55-68 of D-ASPOX protein (Pineda, Antikörper Service, Berlin, Germany).…”

“…The specificity of our anti-protein antibody against bovine kidney cortex D-ASPOX protein by Western blotting has been described previously (Zaar, 1996). To assess the cross-reactivity of this antibody with the D-ASPOX protein present in brain, an immunoblot analysis was performed.…”

“…In kidney epithelial cells and in hepatocytes of different species D-ASPOX has been localized to peroxisomes (Zaar et al, 1989;Van Veldhoven et al, 1991;Zaar, 1996). In the brain, only little information exists on its regional distribution, and nothing is known about the cellular and subcellular localization of the enzyme.…”

D‐Aspartate Oxidase: The Sole Catabolic Enzyme Acting on Free D‐Aspartate in Mammals

“…In mammals, the DDO activity is highest in the kidney, followed by the liver and brain, and is low in other peripheral tissues [31] [32]. Immunohistochemical analysis of the bovine kidney and liver with a specific anti-DDO antibody revealed that DDO is localized to the epithelial cells of proximal renal tubules in the kidney and to hepatocytes in the liver [35]. The intracellular localization of DDO in the peroxisome has been demonstrated by electron-microscopic analyses of the bovine and rat kidney, bovine liver, and rat and human brain [35] [36].…”

“…Immunohistochemical analysis of the bovine kidney and liver with a specific anti-DDO antibody revealed that DDO is localized to the epithelial cells of proximal renal tubules in the kidney and to hepatocytes in the liver [35]. The intracellular localization of DDO in the peroxisome has been demonstrated by electron-microscopic analyses of the bovine and rat kidney, bovine liver, and rat and human brain [35] [36]. DDO Activity has also been detected in purified peroxisome preparations from the bovine, rat, and sheep kidney, and rat and human liver [31] [37].…”