Temperature-dependent and -independent apparent binding activities of [3H]glutathione in brain synaptic membranes.
An apparent binding activity of [3H]glutathione was examined by using synaptic membrane preparations of the rat brain. The activity was found to be more than two times as high at 30 degrees C as that found at 2 degrees C. At 2 degrees C, the apparent binding sites consisted of a single component with a Kd of 0.77 microM and a Bmax of 5.60 pmol/mg protein. In contrast, two independent separate sites with Kds of 0.56 and 12.6 microM and Bmaxs of 2.50 and 28.5 pmol/mg protein were observed at 30 degrees C. In vitro addition of Triton X-100 significantly inhibited the apparent binding activities detected at both temperatures, whereas pretreatment of the membranes with the detergent did not significantly affect both binding activities. Among 3 constituent amino acids of glutathione, L-cysteine induced a selective and irreversible potentiation of the apparent activities, which occurred independently of the incubation temperature. Scatchard analysis revealed that L-cysteine drastically increased the number of the low affinity sites without significantly altering their affinity. Apparent binding activities determined at both incubation temperatures were unevenly distributed in the central and peripheral structures. Distribution profile of the temperature-dependent activities was found to be closely related to that of the basal binding activity of [3H]L-glutamic acid, a putative central excitatory neurotransmitter. These results suggest that brain synaptic membranes may indeed contain specific binding sites of [3H]glutathione which have an interaction with the glutamate binding sites. Possible presence of two distinctly different apparent binding sites of [3H]glutathione, such as temperature-independent high affinity sites and temperature-dependent low affinity sites, is also suggested.
Ogita
K
K
Department of Pharmacology, Setsunan University, Osaka, Japan.
Yoneda
Y
Y
D000818:Animals; D001921:Brain / Q000187:drug effects / Q000378:metabolism*; D003545:Cysteine / Q000494:pharmacology; D005978:Glutathione / Q000378:metabolism*; D007700:Kinetics; D008297:Male; D051381:Rats; D011919:Rats, Inbred Strains; D013574:Synaptosomes / Q000378:metabolism*; D013696:Temperature
D005978:Glutathione; D003545:Cysteine