Immunohistochemical studies of brain pyridoxine-5′-phosphate oxidase

A total of six hybridoma cell lines, which produce monoclonal antibodies (mAbs) against the sheep brain pyridoxine-5′-phosphate oxidase (PNP oxidase), were established. Isotype analysis revealed that all antibodies corresponded to the IgG 2B κ subclass. Immunoblotting with various tissue homogenates indicated that all the mAbs specifically recognize a single protein band of 30 kDa. They also appear to be extensively cross-reactive among different mammalian and avian sources. These results demonstrated that only one type of immunologically similar PNP oxidase is present in all of the mammalian tissues tested. When the purified PNP oxidase was incubated with the mAbs, the enzyme activity was inhibited up to a maximum of 81%. Furthermore, these antibodies were successfully applied in immunohistochemistry in order to detect PNP oxidase in various regions of rat brain tissues. The immunoreactive neurons in PNP oxidase were found in cerebellar cortex, hippocampus, amygdala, paraventricular nucleus, cerebral cortex and ependyma. This result suggests that PNP oxidase may play an important role in the neuronal metabolism.