Purification of Glutathione Reductase from Bovine Brain, Generation of an Antiserum, and Immunocytochemical Localization of the Enzyme in Neural Cells

Authors


  • Abbreviations used : AP, alkaline phosphatase ; DMEM, Dulbecco's modified Eagle's medium ; FITC, fluorescein isothiocyanate ; GalC, galactocerebroside ; GFAP, glial fibrillary acidic protein ; GR, glutathione reductase ; PAGE, polyacrylamide gel electrophoresis ; PBS, phosphate-buffered saline ; PPP, pentose phosphate pathway ; SDS, sodium dodecyl sulfate.

Address correspondence and reprint requests to Dr. R. Dringen at Physiologisch-chemisches Institut der Universität, Hoppe-Seyler-Str. 4, D-72076 Tübingen, Germany.

Abstract

Abstract : Glutathione reductase (GR) is an essential enzyme for the glutathione-mediated detoxification of peroxides because it catalyzes the reduction of glutathione disulfide. GR was purified from bovine brain 5,000-fold with a specific activity of 145 U/mg of protein. The homogeneity of the enzyme was proven by sodium dodecyl sulfate-polycrylamide gel electrophoresis and silver staining of the gel. The purified GR from bovine brain is a dimer of two subunits 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 brain cells. The antiserum was useful for the detection of GR by double-labeling immunocytochemical staining in astroglia-rich and neuron-rich primary cultures from rat brain. In homogenates of these cultures, no significant difference in the specific activities of GR was determined. However, not all cell types present in these cultures showed identical staining intensity for GR. In astrogliarich primary cultures, strong GR immunoreactivity was found in cells positive for the cellular markers galactocerebroside and C3b (antibody Ox42), indicating that oligodendroglial and microglial cells, respectively, contain GR. In contrast, only weak immunoreactivity for GR was found in cells positive for glial fibrillary acidic protein. In neuron-rich primary cultures, GAP43-positive cells stained with the antiserum against GR. These data demonstrate that, in cultures of neural cells, neurons, oligodendroglial cells, and microglial cells express high levels of GR.

Ancillary