Journal of Neuroscience, Vol 7, 302-309, Copyright © 1987 by Society for Neuroscience
Production, characterization, and immunohistochemical application of monoclonal antibodies to glutaminase purified from rat brain
T Kaneko, Y Urade, Y Watanabe and N Mizuno
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 micrograms 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.
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