In vitro synthesis of collagen prolyl hydroxylase. The newly synthesized enzyme contains two newly synthesized subunits of the alpha and beta size.

Neonatal rat lung prolyl hydroxylase was synthesized in a cell-free system. Total polysomes were isolated from neonatal rat lung homogenates after Triton X-100 and deoxycholate solubilization. The polysomes were then translated in a wheat germ lysate in the presence of tritiated leucine. The radioactive product was precipitated with antibody to rat prolyl hydroxylase and co-precipitated with protein A (Pansorbin). The synthesis of rat lung prolyl hydroxylase was optimal at 4.6 mM magnesium and 135 mM potassium chloride. Spermine (16.6 mM) was also present in the cell-free system. On nondenaturing gels, 61% of the labeled protein which was precipitated with antibody alone comigrated with tetrameric prolyl hydroxylase. The radioactive precipitated product was also analyzed on sodium dodecyl sulfate-polyacrylamide gels after denaturation with urea and reduction with mercaptoethanol. On denaturing gels, 64% of the radioactive product migrated coincidentally with the subunits of the mature rat skin enzyme. Total neonatal rat lung polysomes contain. prolyl hydroxylase activity which was inhibited by antibody. The prolyl hydroxylase-containing polysomes were isolated from total lung polysomes by immunoprecipitation. The antibody-precipitated polysomes did not synthesize collagen. However, 44% of the total radioactive leucine-labeled product migrated coincidentally with the purified enzyme subunits on denaturing gels. Therefore, neonatal rat lung polysomes are able to direct the synthesis of prolyl hydroxylase in the wheat germ lysate system. These data indicate that newly synthesized lung prolyl hydroxylase i composed of two newly synthesized and distinct subunits which are of the same apparent size on sodium dodecyl sulfate gels as the a and f i subunits of the mature enzyme.