ANTISERA TO GLUTATHIONE - CHARACTERIZATION AND IMMUNOCYTOCHEMICAL APPLICATION TO THE RAT CEREBELLUM

Rabbits were immunized with reduced glutathione (gamma-glutamyl-cystei nyl-glycine) coupled to bovine serum albumin by glutaraldehyde or a mi xture of glutaraldehyde and formaldehyde. The antisera that were forme d were tested qualitatively, by screening them against more than 50 am ino acids and peptide conjugates that had been immobilized on cellulos e discs (spot test), and quantitatively, by immunogold analysis of tes t conjugates that had been embedded in an epoxy resin. It was shown th at the antisera selectively recognized the reduced and oxidized forms of glutathione and that they did not exhibit any significant crossreac tivity with glutamate, cysteine, glycine, gamma-glutamyl-cysteine or c ysteinyl-glycine. Immunocytochemistry of Vibratome sections of rat cer ebellum suggested that glutathione occurs in glial cells as well as in neurons. This was confirmed by electron microscopic, immunogold cytoc hemistry of tissue from rat cerebellum that had been freeze-substitute d and embedded in Lowicryl under low temperature. Gold particles were concentrated over Golgi epithelial cells and perivascular glial proces ses, but also occurred over several types of neuronal profile includin g Purkinje and granule cell bodies, and messy fibre terminals. At the subcellular level, glutathione-like immunoreactivity was found in the cytoplasmic matrix, mitochondria and nuclei. The immunolabelling inten sity was strongly reduced in animals that had been pretreated with but hionine sulphoximine, which is known to depress the level of glutathio ne by inhibiting gamma-glutamyl-cysteine synthetase. The availability of antisera to glutathione is likely to further our understanding of t he physiological and pathophysiological roles of this tripeptide.