Selective potentiation by l-cysteine of apparent binding activity of [ 3H]glutathione in synaptic membranes of rat brain

Abstract

Significant apparent binding activity of [ 3H]glutathione was detected in synaptic membranous preparations of the rat brain. In vitro addition of sucrose (50–1000 mM) and Triton X-100 (0.02–0.1%) significantly diminished the apparent binding activity, whereas pretreatment of the membranes with Triton X-100 (0.01–0.4%) did not affect the activity. A slight but statistically significant reduction of the apparent binding activity was induced by the in vitro addition (1 mM) of two constituent amino acids, l-glutamic acid and glycine. In contrast, another constituent amino acid, l-cysteine, potently enhanced the binding activity at a concentration higher than 0.1 mM. No prominent alteration of the activity occurred following the inclusion of structurally-related amino acids, dithiothreitol, dithio-erythritol and numerous other amino acids. Scatchard analysis revealed that the apparent binding consisted of two independent separate components with K d values of 0.76 and 11.0 μM, and B max values of 4.00 and 27.0 pmol/mg protein respectively. In vitro addition of 1 mM l-cysteine resulted in a single component with a K d of 8.5 μM and a B max of 105 pmol/mg protein. Pretreatment of the membranes with 1 mM l-cysteine potentiated the apparent binding, with a further addition of l-cysteine having no effect. The retina had the highest activity followed by the hypothalamus, striatum, spinal cord, midbrain, hippocampus, medulla-pons, cerebellum and cerebral cortex, which occurred independently of the incubation temperature. In peripheral organs examined, the pituitary possessed higher activity than the retina, with progressively lower activities in the adrenal, liver, spleen, skeletal muscle and heart. No significant activity was detected in the kidney. Addition of 1 mM l-cysteine significantly potentiated the activities at 30°, but not at 2°, in the hippocampus and cerebral cortex without affecting those in other central structures. In contrast, a profound inhibition of the activity was induced by the addition of l-cysteine in the pituitary, adrenal, intestinal mucosa, skeletal muscle and retina independently of the temperature. These results suggest that l-cysteine may selectively potentiate the apparent binding activity of [ 3H]glutathione in particular regions of the brain, while eliminating that in the peripheral excitable tissues.