ISOLATION OF PROTOPLASTS FROM TOMATO FRUIT (LYCOPERSICON-ESCULENTUM) - 1ST UPTAKE STUDIES

Protoplasts were liberated from pericarp and placental tissue of immature green tomato fruits by treatment with macerocym, pectolyase and several cellulases. Using a flotation technique with percoll, clean protoplasts could be collected which were stable for at least 24 h. Viability was determined by cytoplasmic streaming and by accumulation of fluorescein in the cytoplasm. Exclusion of Evan's blue and uptake of neutral red in the vacuole indicated the retention of selective membrane permeability during the isolation procedure. At the plasma membrane, glucose is transported preferentially. The apparent K(m) is 0.5 and 1.3 mM for protoplasts from pericarp and placental tissue, respectively, V(max) amounts 37 and 48 nmol.mg protein-1.h-1.2-Deoxyglucose and, to a lesser extent, fructose compete with glucose at the carrier binding site. The uptake is inhibited by CCCP. Fructose and sucrose permeate into pericarp protoplasts with lower affinity than glucose (apparent K(m) being 12 mM and 7 mM, respectively). After 15 min [C-14]glucose uptake, for pericarp protoplasts about 65% of the radioactivity can be found in sucrose, for placental protoplasts about 35%.