Abstract
Optimal conditions for the isolation and in vitro translation of structurally and functionally intact polyribosomes and poly(A)-containing messenger-RNA (mRNA) from adult rat heart muscle are described. Using these conditions, 1 g heart muscle (wet weight) yields 865 μg polyribosomes, 719 μg microsomal RNA and 17.7 μg poly(A)-containing mRNA. The isolated polyribosomes, microsomal RNA and mRNA were structurally characterized by sedimentation analysis on continuous, 10 to 40% sucrose gradients (polyribosomes) and 5 to 40% sucrose gradients (microsomal RNA and mRNA). The polyribosomal profiles showed most of the polyribosomes sedimentating as heavy polyribosomes in the high S-value range. Microsomal RNA sedimentated in three distinct peaks corresponding to 4 S (tRNA), 18 S (rRNA) and 28 S (rRNA). The mRNA showed a heterogeneous sedimentation behaviour with a broad peak at about 20 S. Evidence for the functional intactness of the isolated polyribosomes and mRNA was obtained by their excellent capacities for in vitro protein synthesis in cell-free systems. The cell-free synthesized proteins were identified by analysis on SDS-polyacrylamide-slab gels and subsequent fluorography. Major radioactivity bands are found in the region of actin, troponin-T, tropomyosin, L 1 and L 2-myosin and myoglobin.
Published Version
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