Isolation and Characterization of Vacuoles from Melilotus alba Mesophyll

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Abstract
Methods for the preparation of protoplasts and vacuoles from mesophyll tissues of sweet clover (M. alba Desr.) are described. Vacuoles are obtained using a new procedure which involves lysis of the plasmalemma during a brief centrifugation of protoplasts through a diethylaminoethyl dextran layer. This method combines the release of vacuoles and their purification in 1 step. The contamination of vacuole preparations was found to be low, as judged by enzymic markers and microscopic inspection. The method described is rapid and gives a good yield of vacuoles without causing changes in osmotic pressure. Several hydrolases were located in vacuoles from sweet clover, which were also examined for their amino content.