Student Theses and Dissertations

Date of Award

1972

Document Type

Thesis

Degree Name

Doctor of Philosophy (PhD)

Abstract

Several aspects of the relationship between free and membrane-bound
ribosomes and of the ribosome-membrane interaction were investigated
in a rat liver cell-free system.

a) When nascent polypeptide chains were terminated by incubating
rough microsomes in a medium optimal for amino acid incorporation, a
subsequent incubation in a solution containing 0.5 M RCl and 0.0025 M
MgCl^ resulted in a partial detachment of ribosomes, as 40 S and 60 S
particles.

b) Solutions containing high concentrations of monovalent ions (1 M RCl) and no magnesium ions detached essentially all membrane-bound ribosomes as unfolded subunits, while the peptidyl tRNA molecules remained associated with the microsomal vesicles.

c) An exchange of small subunits between free and membrane-bound ribosomes was found to occur in vitro upon release of polypeptide chains under conditions thought to approximate the physiological one. The exchange resulted in the transfer of tritium-labeled subunits to rough microsomes and, vice versa, the detachment of labeled subunits from rough microsomes upon addition of competing, unlabeled subunits. Up to 60% of the membrane-associated small subunits exchanged with excess
added small subunits obtained from free polysomes when polypeptide chains of membrane-bound ribosomes were released by puromycin. The exchange required a macromolecular fraction of the cell sap, was stimulated by ATP or GTP, and occurred at low concentrations of magnesium ions. Addition of large subunits to the system caused a transfer of small subunits of membrane-bound ribosomes into a newly formed pool of free monomers. However, in the time period studied, membrane-bound large subunits did not exchange efficiently with added large subunits obtained from either free or bound ribosomes. These results were confirmed by analyzing the material bound to membranes after incubation for exchange.

d) The binding of ribosomes to rough microsomes stripped of their ribosomes was studied at low ionic strength. The binding reached a maximal value after incubation for five minutes at 37° C. The RNA to protein and RNA to phospholipid ratios in reconstituted rough microsomes were 65% and 55% of untreated rough microsomes respectively. The binding of ribosomes to smooth microsomes treated for stripping was half that observed for stripped rough microsomes, and binding to similarly treated erythrocyte ghosts was virtually nil. The capacity of heat-treated stripped rough microsomes to bind ribosomes was reduced approximately sevenfold with respect to untreated controls.

e) Comparison of the buoyant densities of ribosomal subunits by CsCl density gradient centrifugation revealed no differences between subunits obtained from free and bound ribosomes. Sodium dodecyl sulfate polyacrylamide gel electrophoresis also showed close similarities in the protein patterns of subunits from free and bound ribosomes, with the exception of one protein band which was more Intense in free large subunits.

These findings are discussed in relation to possible mechanisms of the assembly of membrane-bound ribosomes.


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