ISOLATION AND CHARACTERIZATION OF CONCANAVALIN A-LABELED PLASMA-MEMBRANES OF CARROT PROTOPLASTS

The plasma membranes of protoplasts released from carrot suspension culture cells were labeled with [14C]acetyl-concanavalin A. After homogenization a single labeled membrane fraction was isolated in a continuous isopycnic Renografin gradient. The labeled membranes peaked at an apparent density of 1.14 g/cm3 between the Golgi fraction at a density of 1.11 g/cm3 as determined by latent IDPase activity and the mitochondria at a density of 1.16 g/cm3 as determined by the cytochrome c oxidase activity. This method provided a very discrete peak of putative plasma membrane. On discontinuous Renograffin gradients a relatively pure fraction of labeled plasma membranes could be readily isolated at the 1.122-1.146 g/cm3 interface. The labeled fraction was enriched in both an ATPase (pH 6.5) and a glucan synthetase with a pH optimum of 6.5 whose activity was promoted by Mg and cellobiose. Enzyme activities were not altered by the membrane label.