The size of the polypeptide precursor of collagen hydroxyproline.

Studies with both cell-free' and intact cell systems2 from chick embryos indicated that the hydroxyproline present in collagen is synthesized by hydroxylation of pro- line residues already incorporated into polypeptide linkage.This finding raised the question of the nature of this polypeptide material.In this paper we report studies on (1) the size of this substrate, (2) the possibility that it terminates in an sRNA molecule, and (3) some of its properties, in particular its stability.Materials.-Uniformlylabeled C'l-proline, approximately 186 Asc/iimole, was obtained from New England Nuclear Corporation in 0.01 N HCl and was added as such to incubations.Puro- mycin dihydrochloride was purchased from Sigma Chemical Co., and a,a'-dipyridyl from the Matheson Co., as was the prepurified nitrogen used in anaerobic experiments.The crystalline RNase and the purified collagenase (no.CLSP-A) were products of the Worthington Biochemical Corp. White Leghorn 7-day embryonated eggs were purchased from Hall Bros. Hatchery, Wal- lingford, Conn.Ichthyocol was generously provided by Drs. Blumenfeld and Gallop.The conditions of gelatinization of ichthyocol and of treatment with NH20H (for 2.5 hr) were as described by Blumenfeld, Rojkind, and Gallop.3The fragments obtained after this treatment are referred to in the present paper as subunits, in accordance with the usage of these authors.Methods.-Preparation of acetic acid extracts of minces of chick embryos: The embryos were minced in Krebs-Ringer phosphate,4 in ratios of embryos to medium indicated below, with 56 complete strokes of a loosely fitting Potter-Elvehjem homogenizer, turning at low speed.After addition of C'4-proline and other materials where indicated, the minces were incubated at 370 with shaking.At the end of the incubation, the entire incubation was chilled to near 00 and centrifuged at 30 for 2 min at about 10,000 X g in a Servall SS-1 centrifuge.The sediment was dispersed in'ice-cold 0.5 N acetic acid, 6 ml per 10 ml of the original incubation mixture, with a Dounce homogenizer and was shaken for 1 hr at 30 on a Burrell wrist-action shaker, prior to centrifugation at about 15,000 X g for 5 min.The sediment was discarded and the supernatant was dialyzed overnight against at least two batches of distilled H20.Gel filtration on Sephadex G-200: One column of Sephadex G-200, bead form (3 X 74 cm), was used without repacking for all experiments.The dialyzed acetic acid extracts were concentrated before gel filtration either by means of a flash evaporator or by treatment with dry Sephadex G-200.The sample in 0.25 N acetic acid was applied in a final volume of 4.7 ml.The column was developed at room temperature with 0.25 N acetic acid, and 5-ml fractions were collected.The extracts from minces contained material absorbing at 280 mu, and the fraction containing the maximum concentration of this material (32-35 in different runs) was used as an internal marker to bring all elution profiles into register with that of the ichthyocol subunits shown in Figure 1.Disc electrophoresis: The conditions of Nagai et al.5 were used.Conditions of the incubation to prepare the substrate for the hydroxylation reaction: The incubation mixtures contained, per ml, 0.36 embryos (7-day, 1.1 gm wet weight), 0.6 ml of Krebs-Ringer phosphate, 0.7 JAc of C'4-proline (186 uc/Mmole), and 1 jsmole of a,a'-dipyridyl.After incubation