Large Scale Isolation and Characterization of Calf Thymocyte Plasma Membranes

Abstract

Abstract A large scale procedure for preparing plasma membranes from calf thymocytes has been devised. Kilogram quantities of frozen calf thymus were shredded in a high speed, motordriven vegetable shredder, followed by brief homogenization of the shredded tissue in 4 volumes of buffered 0.25 m sucrose using a Waring Blendor. The homogenate was subjected to differential centrifugation to remove nuclei and mitochondria and a crude membrane fraction was obtained which was subjected to centrifugation in a sucrose step gradient to yield a purified plasma membrane fraction. From each kilogram of calf thymus the procedure yielded 500 mg of purified thymocyte plasma membranes which were enriched 33-fold over the homogenate in 5'-nucleotidase activity (2.54 µm per hour per mg of protein) and 12-fold in sialic acid (36.8 nmoles per mg of protein). The membranes contained sialic acid, fucose, galactose, mannose, glucose, N-acetylglucosamine, and N-acetylgalactosamine in the ratio of 0.7:0.7:3.4:2.4:1.6:3.0:0.9. The purified membranes retained binding sites for all the phytohemagglutinins tested including those from kidney bean, lentil, mushroom, Ricinus communis, and also wheat germ agglutinin. The characteristics of the binding were very similar to those of intact calf thymocytes.