Purification of glutathione reductase from bovine brain, generation of an antiserum, and immunocytochemical localization of the enzyme in neural cells

Glutathione reductase (GR) is an essential enzyme for the glutathione-media ted detoxification of peroxides because it catalyzes the reduction of gluta thione disulfide. GR was purified from bovine brain 5,000-fold with a speci fic activity of 145 U/mg of protein. The homogeneity of the enzyme was prov en by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and silver staining of the gel. The purified GR from bovine brain is a dimer of two su bunits that have an apparent molecular mass of 55 kDa. The purified GR was used to generate a rabbit antiserum with the intention to localize GR in br ain cells. The antiserum was useful for the detection of GR by double-label ing immunocytochemical staining in astroglia-rich and neuron-rich primary c ultures from rat brain. In homogenates of these cultures, no significant di fference in the specific activities of GR was determined. However, not all cell types present in these cultures showed identical staining intensity fo r GR. In astroglia-rich primary cultures, strong GR immunoreactivity was fo und in cells positive for the cellular markers galactocerebroside and C3b ( antibody Ox42), indicating that oligodendroglial and microglial cells, resp ectively, contain GR, In contrast, only weak immunoreactivity for GR was fo und in cells positive for glial fibrillary acidic protein. In neuron-rich p rimary cultures, GAP43-positive cells stained with the antiserum against GR , These data demonstrate that, in cultures of neural cells, neurons, oligod endroglial cells, and microglial cells express high levels of GR.