Production, characterization, and immunohistochemical application of monoclonal antibodies to glutaminase purified from rat brain

Abstract

Monoclonal antibodies were produced against phosphate-activated glutaminase (EC 3.5.1.2) as a marker for glutamatergic neurons: The enzyme was purified 1000-fold from rat brain mitochondria with a recovery of 27%. Upon SDS-PAGE the purified enzyme showed a single band up to 1.7 μg after the silver staining at molecular weight 62,000. Two monoclonal antibodies (IgMs) were produced; these absorbed more than 90% of glutaminase activity in rat brain homogenate. In immunoblotting after PAGE of the homogenate, the antibodies recognized only 1 protein band at the same position as that of the purified enzyme. Thus, the antibodies are specific and sufficient markers for glutaminase. Many neuronal cells in the rat brain were labeled immunohistochemically with these antibodies, but non-neuronal elements such as glial cells and vessels were not. Intense labeling was consistently observed in putative glutamatergic neurons such as pyramidal cells of layers V and VI in the cerebral neocortex. Intense staining was also seen in possible mossy fiber endings in the granular layer of the cerebellar cortex and in neurons giving off mossy fibers such as those in the pontine nuclei, pontine tegmental reticular nucleus of Bechterew, lateral reticular nucleus of the medulla oblongata, and external cuneate nucleus.