Hog cerebellar D-amino acid oxidase and its histochemical and immunofluorescent localization.

Abstract

Abstract— Hog cerebellar d‐amino acid oxidase (d‐AAO; EC 1.4.3.3) has been purified to homogeneity. The enzyme was found to be indistinguishable from crystalline hog kidney d‐AAO by a number of criteria, including electrophoresis in both cationic and anionic discontinuous buffer systems, FAD and sulfhydryl contents, and monomer molecular weights of about 40,000 as determined by SDS disc gel electrophoresis. Both preparations exhibited similar specific activities (23 μmol d‐ala oxidized min−1 mg−1 protein), substrate specificities, and susceptibilities to competitive inhibitors.Rabbit antisera were prepared against each enzyme preparation. Double immunodiffusion revealed no antigenic differences between the two when antiserum against either preparation was used. Although a soluble protein, d‐AAO activity in cerebellum is particulate.Two methods were utilized to study the histological localization of d‐AAO activity in hog cerebellum: peroxidase‐coupled histochemistry and immunofluorescence. The histochemical procedure seems specific for d‐AAO since l‐amino acids are inert and known competitive inhibitors of the purified flavoenzyme prevent staining. Rabbit antiserum prepared against purified hog cerebellar d‐AAO was visualized indirectly with fluorescein‐labeled goat antirabbit IgG antiserum. Control experiments with serum from unimmunized rabbits were negative.The results of both techniques were identical in three respects: (1) d‐AAO was observed in many fibers emanating from cerebellar white matter; the white matter itself did not exhibit d‐AAO, despite presence of the oxidase by biochemical assay; (2) intense d‐AAO activity (or antigen) was found in mossy fiber rosettes (glomeruli) in the granular layer; (3) a peculiar and intense localization of d‐AAO was noted at the level of, but not within, the Purkinje cell soma. The molecular layer was essentially devoid of d‐AAO histochemical activity except for minimal staining near the pial surface. However, the more sensitive immunofluorescent technique revealed d‐AAO containing fibers in the molecular layer running parallel to each other and perpendicular to the pial surface; the relatively large size and small number of these fibers do not suggest identification as granule cell axons. No d‐AAO has been found in granule cell soma, Golgi Type‐II cells, or Purkinje cell soma. These results are discussed in terms of the localization of d‐AAO in mossy fibers and their terminals and in certain cell‐type(s) of cerebellar origin.