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The phosphorylation of the ribosomal proteins of Krebs II ascites cells

Elsevier B.V.
Publication Date
DOI: 10.1016/0005-2787(77)90203-9
  • Biology


Abstract The ribosomal proteins which were labelled when resting Krebs II ascites cells were incubated with [ 32P] orthophosphate were analysed using several systems of polyacrylamide gel electrophoresis. When protein was extracted from the total ribosomal population and subjected to electrophoresis in gels containing sodium dodecyl sulfate a variable number of radioactive bands was observed, indicating contamination of the ribosomes by non-ribosomal phosphoproteins. Ribosomes were therefore purified by dissociation into their subunits at high ionic strength and temperature, removing non-ribosomal material. Protein extracted from these subunits consistently gave a total of five labelled bands in gels containing sodium dodecyl sulphate. This was far fewer than other workers had observed when ribosomes were phosphorylated using protein kinases in vitro. Sodium dodecyl sulphate gel electrophoresis of the 40 S ribosomal proteins revealed two, relatively large, phosphoproteins and these migrated as basic proteins during acid urea gel electrophoresis. They were identified by two-dimensional gel electrophoresis to be ribosomal proteins S2 and S6, although the extent of their phosphorylation was rather low. Sodium dodecyl sulphate gel electrophoresis of the 60 S ribosomal protein indicated that most of the radioactivity was associated with two, relatively small, barely resolved, phosphoproteins, while a third protein was also slightly phosphorylated. Acid urea gel electrophoresis showed a single strongly labelled band migrating as if it were somewhat more acidic than most of the other ribosomal proteins. The phosphoproteins of the 60 S ribosomal subunit were located on two-dimensional gel electropherograms after reducing the pH of the first dimension from 8.6 to 6.6. Although their positions coincided with stained protein spots these latter did not correspond to any previously defined eukaryotic ribosomal proteins.

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