Characterization of cell free synthesis of collagen by lung polysomes in a heterologous system
In normal lung growth, post-pneumonectomy lung growth, and in possibly several lung disorders, there are marked alterations in the density of collagen and changes in the rate of synthesis of collagen relative to the synthesis of other lung proteins. To provide a technology to begin to understand these changes at the molecular level, polysomes were prepared from rabbit lung and translated in a heterologous cell-free system including rabbit reticulocyte 0.5 M KCl ribosomal wash fraction and liver tRNA. Collagen was shown in the cell-free product by collagenase sensitivity, hydroxylation of incorporated proline by peptidyl prolyl hydroxylase, agarose gel chromatography, and sodium dodecyl sulfate acrylamide gel electrophoresis. The cell-free system was optimized with respect to K+, Mg2+, amino acids, and ribosomal wash fraction and used under conditions where total protein synthesis and collagen synthesis are linear with respect to time and amount of polysomes. Under these conditions, collagen synthesis was directed almost entirely by polysomes derived from the endoplasmic reticulum. Polysomes isolated from late fetal lung directed collagen synthesis at twice the rate (per polysome) as those polysomes isolated from adult lung. Similar changes were seen if lung tRNA replaced liver tRNA and if lung ribosomal wash fraction replaced reticulocyte wash fraction. Although these changes in cell-free lung collagen synthesis with tissue explants, further studies will have to be carried out to determine whether, in fact, age-related alterations in control of lung collagen synthesis are truly explained by these findings.