Isolation and Characterization of Polyribosomes From The Epidermis of The Newborn Rat

Elucidation of the steps in the biosynthesis of unusual “histidine-rich” protein in the epidermis of the newborn rat has been hampered by an inability to reconstruct an efficient epidermal system for synthesis of any protein in vitro. Inhibitory activities (e. g. RNase and proteolytic enzymes) associated with epidermal catalytic components and failure to obtain active epidermal polyribosomal fractions have provided the main difficulties. Epidermal polyribosomes which actively incorporate labeled amino acids in the presence of a “pH 5 enzyme” preparation from liver, have now been obtained from the newborn rat. The epidermis, free of dermis, is homogenized in 0.25 M sucrose containing 1% sodium deoxycholate, 25 mM KCI, 5 mM magnesium acetate, 0.4 mM dithiothreitol and 50 mM Tris buffer, pH 7.5. The homogenate is submitted to differential centrifugation and the postmitochondrial fraction is further purified by ultracentrifugation in sucrose density gradients to obtain the polyribosomal fraction. Electron microscopy and analytical ultracentrifugation confirm the identity and composition of the RNase-sensitive polyribosomal fraction.