To prepare crude particulate fractions, samples were thawed, suspended in 0.1X buffer (buffer consists of NAcc, 140mM; potassium chloride, 1.5mM; calcium chloride, 2.0mM; magnesium sulfate, 1.0mM; and N-[2-hydroxyethyl]piperazine-N′-[2-ethanesulfonic acid], 25mM; pH, 7.5), homogenized using a motor-driven pestle, and centrifuged at 15 000g for 20 minutes. The supernatants were discarded, and the pellets were resuspended in fresh 0.1X buffer and again centrifuged. The resuspension/ centrifugation cycle was repeated 3 times. After the fourth centrifugation, the supernatant was discarded, fresh hypotonic buffer was added, and the samples were stored frozen as pellets until assay for tritium-labeled CP55,940 binding.
