Rat 3‐Hydroxyanthranilic Acid Oxygenase: Purification from the Liver and Immunocytochemical Localization in the Brain

Abstract

3-Hydroxyanthranilic acid oxygenase (3HAO; EC 1.13.11.6). the biosynthetic enzyme of the endogenous excitotoxin quinolinic acid, was purified to homogeneity from rat liver and partially purified from rat brain. The pure enzyme is a single subunit protein with a molecular weight of 37–38.000. Kinetic analyses of both pure liver and partially purified brain 3HAO revealed an identical K_m of 3 μM for the substrate 3-hydroxyanthranilic acid. Evidence for the identity of liver and brain 3HAO was further provided by physicochemical (electrophoretic behavior, heat sensitivity) and biochemical (pH dependency, activation by Fe²⁺) means. Antibodies were produced against the pure liver enzyme and the identity of liver and brain 3HAO substantiated immunologically in immunotitration and Ouchterlony double-diffusion experiments. Immuhohistochemical studies using purified anti-rat 3HAO antibodies were performed on tissue sections of perfused brains and demonstrated a preferential staining of astroglial cells. Notably, the cellular localization of 3HAO in the brain appears to be in part distinct from that of quinolinic acid phosphoribosyltransferase, the catabolic enzyme of quinolinic acid. Pure rat 3HAO and its antibodies can be expected to constitute useful tools for the further elucidation of the brain's quinolinic acid system.