Polyribosomes from Peas: V. An Attempt to Characterize the Total Free and Membrane-bound Polysomal Population

Attempts were made to isolate and characterize the total population of free and membrane-bound polysomes from the elongating region of dark-grown pea stems (Pisum sativum L.). Partial separation of free from membrane-bound polysomes was achieved by relatively low speed centrifugation of the homogenate. Complete separation was not achieved. Based on analysis of the rRNA content of various subcellular fractions, fractionated tissue yielded greater than 95% of the rRNA found in whole tissue. Approximately 45% of the ribosomal material was membrane-bound (released by detergent) and was found in the "wall" (13%), the "nuclear" pellet (2%), and the "mitochondrial" pellet (29%). The remaining 55%, consisting primarily of free polysomes, could be recovered free from membranous material by sedimentation through a dense (700 mg/ml) sucrose pad for 90 hours. The advantages and disadvantages of using sucrose pads for the separation of free and membrane-bound polysomes are discussed.