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 (F P), cytoskeletal-bound polysomes (CBP) and membrane-bound polysomes (M BP) from MPC-11 and Krebs II ascites cells. Polysomes were pelleted, w ashed 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 nonbinding proteins were separated by SDS-PAGE . In MPC-11 cells the FP fraction contains thirteen poly(A) binding pr oteins and four non-poly(A) binding proteins while the corresponding f raction in Krebs II ascites cells has four poly(A) binding proteins an d six proteins which do not bind poly(A). The CBP fraction isolated fr om 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 bot h poly(A) binding and non-poly(A) binding properties. In the CBP fract ion prepared from Krebs II ascites cells a protein band at 32 kDa exhi bits both poly(A) binding and non-poly(A) binding properties. In this fraction there are six poly(A) binding proteins and an additional eigh t 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-pol y(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. The results show differences in the poly(A) bindin g properties of the proteins in tl;e three polysome fractions and that the complements of polysome-associated proteins are different in the two cell lines. This may be related to the differences in the growth c haracteristics of MPC-11 and Krebs II ascites cells.