TURNOVER OF PROLYL HYDROXYLASE AND AN IMMUNOLOGICALLY RELATED PROTEIN IN RABBIT-TISSUE

The turnover rates of prolyl hydroxylase and immunologically related (cross reacting) protein were examined using labeled leucine as precursor or by measuring the decay of elevated prolyl hydroxylase and immunologically cross-reacting protein back to basal levels. Prolyl hydroxylase and immunologically cross-reacting protein were purified from neonatal rabbit skin at various times following the administration of [3H]leucine. Prolyl hydroxylase was purified by affinity chromatography. Immunologically cross-reacting protein was purified by antibody precipitation from the dialyzed 70% (NH4)SO4 supernatants and subsequent electrophoresis on 10% sodium dodecyl sulfate-polyacrylamide slab gels. The radioactivity of the species isolated, which corresponded to the two major subunits of prolyl hydroxylase, was used in the turnover studies of immunologically cross-reacting protein. The peak incorporation of label into prolyl hydroxylase was found to be 12 h while for immunologically cross-reacting protein this occured within 2 h. The loss of radioactivity from these protein pools denotes an apparent t 1 2 for prolyl hydroxylase of 73 h and a 1 2 for immunologically cross-reacting protein of 53 h. From the specific activity of free skin leucine pools, the effect of reutilization could be corrected and a true t 1 2 for prolyl hydroxylase of 45 h was determined. The t 1 2 values of these proteins were determined by a second method in which prolyl hydroxylase and immunologically cross-reacting protein in the aorta and liver of adult male rabbits were elevated by daily epinephrine-thyroxine treatment for 12 days. The decline of prolyl hydroxylase and immunologically cross-reacting protein with termination of treatment in the aorta denotes values of 42 h for enzyme and 53 h for immunologically cross-reacting protein. Calculated enzyme κd values, by both methods, indicate that breakdown of enzyme does not account for tissue immunologically cross-reacting protein. © 1979.