Tritium exchange kinetics of 60 S and 40 S ribosomal subunits from Saccharomyces cerevisiae were studied using a rapid centrifugal, ultrafiltration procedure. This assay used commercially available disposable columns and microconcentrators. The tritium-labeled ribosome was separated from the tritiated solvent using a prepacked gel-filtration column. The labeled ribosome was applied to a microconcentrator and the exchange-out kinetics of the ribosome was measured by centrifugation of the ribosome solution and measurement of the amount of radioactivity present in the filtrate. One major advantage of this method is its simplicity and rapidity. With this method, the tritium exchange-out behavior of 60 S and 40 S ribosomal subunits and of subunits during reassociation were determined. The two subunits exhibited different exchange-out rates. Both subunits consisted of multiple classes of exchangeable protons. Considerable conformational changes in both subunits were evident during subunit reassociation, as additions of equal molar quantities of unlabeled 40 S subunits to labeled 60 S subunits caused an immediate increase in the exchange rate. Similarly, an increase in the exchange rate in the small subunits upon addition of unlabeled 60 S subunits was observed.
- Eukaryotic ribosome
- Ribosomal subunit conformation
- Ribosomal subunit interaction
- Tritium exchange
- Yeast ribosome
ASJC Scopus subject areas
- Structural Biology