MATERIALS AND METHODS

Crude Plasma Membranes. Approximately 0.5 g of frozen tissue was pulverized and homogenized in 5 mL of ice-cold 20 mM sodium phosphate buffer, pH 7.5, 10 mM EDTA, and 1 mM phenylmethanesulfonyl fluoride. The samples were centrifuged at approximately lOOOg for 5 min at 4 C to sediment nuclei and debris, and the supernatant was centrifuged at 30000g for 45 min at 4 C. The pellet was resuspended in 5 mL of ice-cold 10 mM sodium acetate buffer (pH 3.5)/150 mM NaCl using a syringe and a 21-gauge needle with the orifice opposed to the wall of the glass container in order to obtain a fine suspension. The samples were centrifuged at 30000g for 20 min at 4 C, and the supernatants were discarded. This step removed endogenously bound folates in the samples.(Additional acid washes did not expose any more [3H] folic acid binding sites.) The membranes were washed with 5 mL of 10 mM sodium phosphate buffer (pH 7.5)/150 mM NaCl by resuspending and centrifuging as above. The resulting membrane pellets were dissolved in 1 mL of 10