Protein topography of the 40 S ribosomal subunit from rabbit reticulocytes shown by cross-linking with 2-iminothiolane
- PMID: 7275971
Protein topography of the 40 S ribosomal subunit from rabbit reticulocytes shown by cross-linking with 2-iminothiolane
Abstract
The small ribosomal subunit from rabbit reticulocytes was allowed to react with 2-iminothiolane under conditions that minimize the formation of 40 S subunit dimers. Reaction with 2-iminothiolane results in the formation of amidine-linked sulfhydryl derivatives of protein amino groups. Cross-linking between proximal sulfhydryl groups was promoted by mild oxidation of the modified ribosomal subunits. Protein extracted from cross-linked ribosomes was fractionated on the basis of charge by polyacrylamide-urea gel electrophoresis at pH 5.5. Cross-linked protein dimers in sequential slices of this gel were analyzed by diagonal polyacrylamide-sodium dodecyl sulfate gel electrophoresis. Constituent proteins of cross-linked pairs were located by their common mobility in the first dimension and by comparison of the sum of their molecular weights with that of the parent cross-linked species. They were unambiguously identified by extraction, radioiodination, two-dimensional polyacrylamide-urea gel electrophoresis and radioautography and comparison with nonradioactive markers. Thirty-six protein dimers were identified. Many proteins were found to occur in several cross-linked dimers and this facilitated representation of the results in a model showing the network of crosslinks. The results are discussed in relation to other structural and functional data on the 40 S ribosomal subunit.
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