The effect of ascorbic acid on collagen polypeptide synthesis and proline hydroxylation during the growth of cultured fibroblasts

Abstract

The rate of collagen synthesis relative to the rate of synthesis of noncollagen protein was determined in several lines of cultured fibroblasts using an assay which measures [ 14C]proline incorporation into the polypeptide chains of collagen. In this assay procedure, collagen is degraded by protease-free collagenase regardless of whether proline and lysine residues are hydroxylated, thus separating the process of polypeptide synthesis from hydroxylation. It was found that the relative rate of collagen synthesis in L-929 cells was approximately 0.8–1% at all stages of growth. There was no significant increase in the relative rate of collagen synthesis in stationary phase compared to log phase cells in the lines Balb 3T3, 3T6, 3T12, and Swiss mouse 3T6. In all cases, the absolute incorporation of [ 14C]proline into both collagen and noncollagen proteins expressed as radioactivity incorporated per milligram of cellular protein, was 2–10 times higher in log phase cells, depending on the line examined. Ascorbic acid did not affect the growth nor the rate of collagen synthesis of L-929 cells. In the presence of ascorbate, the ratio of proline:hydroxyproline in collagenase digests of protein from cells and medium indicated that proline hydroxylation was complete at all stages of growth. In the absence of ascorbate, however, hydroxylation of proline in collagen of the cell layer was about 20% of maximum in early log phase cells and the extent of hydroxylation increased to about 60% in late stationary phase, suggesting that a cofactor which can substitute for ascorbate is synthesized in these cells and accumulates during stationary phase. About 25–30% of the collagen synthesized during log phase was found in the medium and the extent of proline hydroxylation in the absence of ascorbate was approximately the same as that of the collagen in the cell layer. It was also found that ascorbic acid is extremely unstable in tissue culture media incubated at 37 °C. After 24 hr, 88–98% of the compound had disappeared or been altered so that it no longer reacted in a standard ascorbic acid assay.