Basement membrane biosynthesis secretion without deposition of underhydroxylated basement membrane collagen by parietal yolk sacs

Abstract

1. 1. In contrast to published results with other collagen-synthesizing systems the rate of secretion of basement membrane collagen synthesized by the rat parietal yolk sac was essentially the same in the presence or absence of either dipyridyl or GPA 1734 (8,9-dihydroxy-7-methyl-benzo( b)quinolizinium bromide), with no evidence for intracellular accumulation of underhydroxylated basement membrane collagen. This was shown by measuring total radioactivity and [ 14C]hydroxyproline in collagenase digests of tissue and medium proteins after incubation for 3 and 6 h with [ 14C]proline or [ 14C]proline plus [ 3H]glycine. The secreted underhydroxylated basement membrane collagen contained 2–10% of the hydroxyproline found in controls without hydroxylation inhibitors. The identity of the non-dialyzable radioactivity in the medium after incubation with dipyridyl or GPA 1734 with underhydroxylated basement membrane collagen was established (a) by tripeptide analyses of the collagenase digests, which showed about 20% of the radioactivity to be Gly-Pro-Pro and (b) by incubation with partially purified prolyl hydroxylase from rat skin, which converted the material to hydroxyproline-containing protein with a [ 14C]hydroxyproline to total [ 14C]protein ratio close to that of control basement membrane collagen. 2. 2. Normal 14C-labelled basement membrane collagen, whether found intracellularly, secreted into the medium or deposited on Reichert's membrane, after reduction and denaturation appears to be the same molecular size on SDS-agarose chromatography. Similarly, 14-labeled basement membrane collagen from the medium synthesized in the presence of hydroxylation inhibitors and chromatographed under conditions which prevent proteolytic digestion appears as a single peak with the same molecular size as control basement membrane collagen. 3. 3. Underhydroxylated basement membrane collagen was not deposited on Reichert's membrane as evidenced by measurements of total [ 14C]proline and [ 14C]hydroxyproline in collagenase-digests of membranes free of trophoblast and epithelial cells. 4. 4. The inhibition of hydroxylation did not affect the synthesis of the glucosamine-containing moiety of basement membrane collagen. This was shown by pulsing with [ 14C]glucosamine in the presence of dipyridyl and chasing with 0.5 mM glucosamine in fresh medium containing Fe 2+ and cycloheximide. Glucosamine incorporated intor basement membrane collagen during the pulse stage was found to deposited on Reichert's membrane after hydroxylation of proline and lysine and secretion at the chase stage.