Isolation and Biochemical Characterization of a «Cytosolic Fraction» from Sweet Clover Protoplasts

We describe a method for the isolation of cytosol from mesophyll protoplasts of sweet clover (Melilotus alba Desr.). Polybase induced lysis of protoplasts under gentle conditions, followed by centrifugations at increasing velocities resulted in the progressive sedimentation of intact subcellular organelles. The final supernatant obtained at 100,000 × g was assumed to be the cytosol. Its contamination was found to be low, according to the activities of marker enzymes, and mainly due to vacuolar sap (ca. 10 %) and soluble constituents of peroxisomes (ca. 20 %). Enzymes of the sucrose pathway are exclusively located in the cytosol and can therefore be used as markers of this subcellular compartment.