The Use of Metrizamide as a Density-Gradient Medium in Studies of Rat-Liver Polysomes

Abstract

The behavior of rat liver cytoplasmic ribonucleoproteins in metrizamide [N-(2,4,6-tri-iodo-3-N-methylacetylamino-benzoyl)-glucosamine] was studied to determine whether the iodinated compound would offer any advantage over other centrifugation media for studies of polysome structure and function. Polysomes had a density of 1.295-1.300 g/cm3 in metrizamide gradients which was also the density of glycogen, but it reached its equilibrium more rapidly than the polysomes. A short centrifugation of a polysome suspension from non-starved rats over a 40% metrizamide cushion was sufficient to eliminate more than 85% of the glycogen with a polysome yield of about 75%. Ribosomal subunits had neighboring densities (1.23 and 1.20 g/cm3 for large and small EDTA-derived subunits; 1.23 and 1.21 g/cm3 for large and small KCl/puromycin-derived subunits, respectively). Polysomal messenger ribonucleoproteins were heterogeneously distributed (.rho. = 1.12-1.23 g/cm3) and overlapped with subunits in a similar manner as in sucrose gradients. Analysis of a post-mitochondrial supernatant in metrizamide showed a clear separation of free polysomes, rough membranes and the soluble phase.