The use of proteolytic enzymes in the study of ribosomal structure

Abstract

1. 1. The effects of proteolytic enzymes (trypsin, pronase, chymotrypsin) on the basic proteins of purified rat liver ribosomes were analyzed by disk electrophoresis on polyacrylamide gel, combined with microdensitometric assay of the stained bands. With intact ribosomes the effects were highly selective; some proteins were fairly resistant, while others were altered at a rate far above the average. The pattern of selectivity was remarkably similar with different enzymes. In contrast, free ribosomal proteins were degraded rapidly and without the discrimination characteristic of the same proteins in the intact structures. 2. 2. After preincubation with increasing concentrations of proteolytic enzymes under the experimental conditions used, the ability of the particles to incorporate labeled amino acids into protein first increased, then gradually decreased. Polyuridylic acid-dependent phenylalanine incorporation was more sensitive to proteolysis than endogenous incorporation. Some dissociation of the particles occurred under the influence of the enzymes, especially pronase. 3. 3. The experimental data indicate that in general only a limited part of the protease-sensitive proteins was accessible to the enzymes. One particular protein (Band 10) apparently lacked chymotrypsin-specific bonds in the exposed part of the chain, although the molecule as a whole was readily degraded by this enzyme. The peptide fragments produced by the enzymes were fairly large, and remained attached to the ribosomes, temporarily shielded from further attack. It is inferred that in intact ribosomes basic proteins are to a great extent embedded inside the particles. Protease-sensitive proteins may only by fairly short, specific loops reach the ribosomal surface.