STUDIES ON THE REGULATION OF PROLYL HYDROXYLASE

Abstract

The hydroxylation of proline is thought to be one of the critical cellular events necessary for the synthesis and secretion of structural collagen. Using antibody directed against prolyl hydroxylase it has been shown that there is an enzymatically inactive protein related to prolyl hydroxylase in mammalian tissue. This cross-reacting protein is always present in excess relative to active hydroxylase and it is not kriown whether it is a precursor or a degradation product 0£ prolyl hydroxylase. The turnover rates of prolyl hydroxylase and immunologically related protein, CRP, were examined using labeled leucine as precursor or by measuring the decay of elevated prolyl hydroxylase and CRP back to basal levels. Prolyl hydroxylase and CRP were purified from neonatal rabbit skin at various times following the administration of 3 H-leucine. Prolyl hydroxylase was purified by affinity chromatography. CRP was purified by antibody precipitation from the dialyzed 70% (NH4)2S04 supernatants and subsequent electrophoresis on 10% SDS polyacrylamide slab gels. CRP was shown to migrate similarly to the two prolyl hydroxylase monomers which had molecular weights of 65,000 and 60,000. A smaller antigenic component (45,000) of CRP was also observed. However, only the higher molecular weight components were used in the turnover studies of CRP. The peak incorporation of label into prolyl hydroxylase was found to be 12 hours while for CRP this occurred within 2 hours. The loss of radioactivity from these protein pools denotes an apparent T1/2 for prolyl hydroxylase of 73 hours and a T ~ for CRP of 53 hours. 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 hours was determined. Prolyl hydroxylase and CRP in the aorta and liver of adult male rabbits were elevated by daily epinephrinethyroxine treatment for 12 days. The decline of prolyl hydroxylase and CRP with termination of treatment in the aorta denotes T ½ values of 42 hours for both. Calculated enzyme Kd values, by both methods, indicate that breakdown of enzyme does not account for tissue CRP.