Free, cytoskeletal-bound and membrane-bound polysomes isolated from MPC-11 and Krebs II ascites cells differ in their complement of poly(A) binding proteins.
A three-step detergent/salt extraction procedure (Vedeler et al., Mol Cell Biochem 100: 183-193, 1991) was used to isolate free polysomes (FP), cytoskeletal-bound polysomes (CBP) and membrane-bound polysomes (MBP) from MPC-11 and Krebs II ascites cells. Polysomes were pelleted, washed with high salt buffer and re-pelleted. Proteins in the dialysed high-salt extracts were subjected to poly(A) Sepharose chromatography and poly(A) binding and non-binding proteins were separated by SDS-PAGE. In MPC-11 cells the FP fraction contains thirteen poly(A) binding proteins and four non-poly(A) binding proteins while the corresponding fraction in Krebs II ascites cells has four poly(A) binding proteins and six proteins which do not bind poly(A). The CBP fraction isolated from MPC-11 cells has a complement of ten poly(A) binding proteins, four which are non-poly(A) binding, and a protein of 105 kDa which has both poly(A) binding and non-poly(A) binding properties. In the CBP fraction prepared from Krebs II ascites cells a protein band at 32 kDa exhibits both poly(A) binding and non-poly(A) binding properties. In this fraction there are six poly(A) binding proteins and an additional eight which do not bind poly(A). Of the total number of proteins eight of these have a molecular weight below 40 kDa. The MBP fraction in MPC-11 cells contains three poly(A) binding proteins and eleven with non-poly(A) binding properties. In contrast this fraction in Krebs II ascites cells has a complement of thirteen poly(A) binding and ten non-poly(A) binding proteins.(ABSTRACT TRUNCATED AT 250 WORDS)
Department of Biochemistry and Molecular Biology, University of Bergen, Norway.
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Moss
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Pryme
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Vedeler
D016475:3T3 Cells; D000818:Animals; D001201:Ascites; D002455:Cell Division; D002462:Cell Membrane / Q000737:chemistry*; D002469:Cell Separation; D002474:Cell-Free System; D002846:Chromatography, Affinity; D003599:Cytoskeleton / Q000737:chemistry*; D051379:Mice; D039102:Poly(A)-Binding Proteins; D011132:Polyribosomes / Q000737:chemistry*; D016601:RNA-Binding Proteins / Q000032:analysis*; D014407:Tumor Cells, Cultured
D039102:Poly(A)-Binding Proteins; D016601:RNA-Binding Proteins