The hydroxyproline-containing proteins (hyproproteins) synthesized by cultured human fibroblasts have been partially characterized. The hyproprotein extracted from the cell layer was found to be similar to the collagen extracted from skin in the ratio of hydroxyproline to proline, chain composition, solubility, and resistance to proteolytic digestion. In earlier studies, the synthesis of collagen by fibroblasts and other cells in vitro was followed by measuring nondialyzable hydroxyproline, a relatively unique marker for collagen, and by observing extracellular collagen fibrils with the electron microscope (1-6). It has been reported that only about half of the nondialyzable hydroxyproline formed by human fibroblasts was deposited in the cell layer, while the remainder was found in the medium (4). We have observed that most of the hydroxyproline-containing protein (hyproprotein) associated with the cell layer was insoluble in cold neutral salt solution. However, when hyproproteins were synthesized in the presence of 13-aminopropionitrile, a specific inhibitor of collagen cross-linking, a hyproprotein could be readily extracted and characterized. Its properties have been compared with the hyproproteins secreted into the medium.
MATERIALS AND METHODSFibroblasts were obtained from biopsies of normal human skin by standard techniques and grown in plastic culture flasks containing 20 ml of Dulbecco-Vogt medium supplemented with 300 Mig/ml of glutamine, 50 ug/ml of ascorbic acid, 50 units/ml of penicillin, 50 /Ag/ml of streptomycin, and 10% fetal calf serum. Cultures of confluent fibroblasts were labeled with [U-14C ]iproline (2 /ACi/ml) or both [U-14C ]irproline (1 0Ci/ml) and [U-14C ]glycine (2,4Ci/ml) in 15 ml of Eagle's basal medium. In addition, certain flasks received 50 ,ug/ml of f3-aminopropionitrile-HCl (BAPN) to block the cross-linking of collagen (7). After 24 hr, the medium was decanted from the cell layer and dialyzed exhaustively against the appropriate buffers used for chromatography. The cell layer was rinsed with unlabeled medium, removed from the flask surface in 20 ml of 1 M NaCl-0.05 M Tris (pH 7.4), and extracted for 2 days at 40C. The soluble proteins were separated from the insoluble material by centrifugation at 10,000 rpm for 10 min, then dialyzed against the appropriate buffers used for chromatography. Carrier collagen, (20 mg) prepared from the skin of lathyritic rats (8), was added to the medium or cell-extract samples to serve as an internal standard for measuring the recoveries of the radioactive al and a2 chains, as well as to mark their position on elution. In certain experiments, medium or cell extract samples were brought to 0.5 M acetic acid and incubated with pepsin (9) (100 ,ug/ml) for 6 hr at 15'C. The pH of the samples was adjusted to 8.0 with 1 M NaOH and allowed to stand for at least 20 min to irreversibly inactivate pepsin (10). CM-cellulose chromatography was performed on the heat-denatured proteins as described by Piez et al. (8). Molecular-sieve chromatography was p...