Incorporation of 3,4-Dehydroproline into Protocollagen and Collagen: LIMITED HYDROXYLATION OF PROLINE AND LYSINE IN THE SAME POLYPEPTIDE

Abstract

Abstract Chick embryo cartilage incubated with dl-3,4-dehydroproline (3,4-dehydroproline) synthesized collagen containing the analogue in place of proline. It was not possible to determine the degree to which 3,4-dehydroproline replaced proline, but under comparable conditions 14C-dl-3,4-dehydroproline was incorporated at about one-fifth the rate of 14C-l-proline. The hydroxyproline content of the collagen was decreased because the analogue replaced prolyl residues which were normally hydroxylated, and because the proline still incorporated was not hydroxylated to the same extent as in control tissues. With tissues incubated with 14C-proline and 40 to 100 µg per ml of dl-3,4-dehydroproline the hydroxylation of 14C-proline in collagen was decreased to one-third to one-half of the control value. The hydroxylation of 14C-lysine in collagen was depressed to about the same degree. The analogue did not inhibit protocollagen proline hydroxylase, and gel filtration indicated the proteins synthesized in the presence of the analogue were of the same size as proteins from control tissues. The 14C-hydroxyproline content of collagen containing 3,4-dehydroproline could not be increased by incubating it with excess protocollagen proline hydroxylase. Collagenous polypeptides prepared by incubating cartilage with puromycin and 3,4-dehydroproline formed stable enzyme-substrate complexes similar to complexes formed with polypeptides which did not contain 3,4-dehydroproline. However, the polypeptides containing 3,4-dehydroproline differed from polypeptides which did not contain the analogue in that they remained attached to the enzyme after incubation with excess enzyme and cofactors. This observation may explain the decreased hydroxylation of collagen containing the analogue in that tight binding of the enzyme to a 3,4-dehydroproline residue at one position in a substrate polypeptide may sterically hinder the hydroxylation of adjacent prolyl or lysyl residues in the same molecule. Autoradiographic experiments indicated that collagen containing 3,4-dehydroproline was not extruded from cartilage cells as rapidly as normal collagen.