Isolation of ;Vacuoplasts' from Poterioochromonas malhamensis

A method is reported for the isolation of ;vacuoplasts' from Poterioochromonas malhamensis. Vacuoplasts are separated mechanically by centrifugation on silica-sol gradients. They consist of the leucosin storage vacuole, a portion of the plasma membrane, and some cytoplasmic components. This method is suited to give a high yield of vacuoplasts.Vacuoles are the largest membrane-bound organelles in plant cells. Only a few methods exist for their large scale isolation and purification in an intact and physiologically active state. The high shear forces used to disrupt cell walls during tissue fractionation usually also disrupt the fragile vacuoles. Intact vacuoles have been isolated from root storage tissue of Beta vulgaris L. (14) by a slicing procedure or by lysing enzymically prepared protoplasts from yeast (12). Vacuoles from higher plants have been isolated by osmotic lysis of protoplasts (18), treatment of protoplasts with polybases (6, 7), or centrifugation of protoplasts at high g forces against a solution of Ficoll (16).The wall-less flagellate P. malhamensis is a fresh water organism with two unequal flagellae, belonging to the order Chrysomonadina, a group whose members characteristically store oil and leucosin. Leucosin (= chrysolaminarin) is stored in a posterior vacuole, which may be so large as to almost fill the cell. Leucosin is a polysaccharide composed of beta- (1-->3)-linked d-glucose residues. It has a degree of polymerization of about 34, is water soluble, and probably has a slightly branched structure (1).