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

https://doi.org/10.1016/0003-9861(72)90221-4Get rights and content

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.

References (33)

  • R. Miller et al.

    Arch. Biochem. Biophys

    (1970)
  • E.L. Lazarides et al.

    J. Mol. Biol

    (1971)
  • B. Peterkofsky et al.

    J. Biol. Chem

    (1963)
  • G. Manner

    Exp. Cell Res

    (1971)
  • O.H. Lowry et al.

    J. Biol. Chem

    (1951)
  • R.L. Margolis et al.

    Arch. Biochem. Biophys

    (1971)
  • J.J. Jeffrey et al.

    Biochim. Biophys. Acta

    (1966)
  • P.B. Ramaley et al.

    Fed. Eur. Biochem. Soc. Lett

    (1971)
  • S. Udenfriend
  • B. Peterkofsky et al.
  • K. Juva et al.

    Science

    (1966)
  • I.B. Chatterjee et al.

    Ann. N. Y. Acad. Sci

    (1961)
  • R.E. Priest et al.

    Lab. Invest

    (1967)
  • H. Eagle

    Science

    (1965)
  • B. Goldberg et al.

    J. Cell Biol

    (1964)
  • H. Green et al.

    Nature London

    (1966)
  • Cited by (324)

    • Intracellular ascorbate enhances hypoxia-inducible factor (HIF)-hydroxylase activity and preferentially suppresses the HIF-1 transcriptional response

      2014, Free Radical Biology and Medicine
      Citation Excerpt :

      Ascorbate was relatively stable in medium under our experimental conditions, with a half-life of ~5–6 h (Supplementary Fig. 1). This is somewhat longer than reported by others in medium without cells or serum [25], but is similar to the findings of others who also monitored ascorbate in culture medium with serum and cells [26,27] and most likely reflects the presence of protective factors (serum iron chelators) in these conditions. Intracellular uptake peaked at 8 h and remained stable for up to 48 h.

    • Ascorbic Acid and Cancer: Animal and Cell Culture Data

      2012, Natural Antioxidants in Human Health and Disease
    • IGF-I and vitamin C promote myogenic differentiation of mouse and human skeletal muscle cells at low temperatures

      2011, Experimental Cell Research
      Citation Excerpt :

      VC plays various essential roles in vivo, which includes serving as a cofactor for the hydroxylation of proline and lysine during collagen synthesis. Although L-ascorbic acid is unstable and rapidly degraded under the normal culture conditions [13], a phosphate derivative of L-ascorbic acid is considerably stable, with 85% still remaining after a 1-week incubation at 37 °C [14]. L-ascorbic acid phosphate was reported to increase myogenin expression in muscle cells and promote their myogenic differentiation by accelerating collagen synthesis at 37 °C [15,16].

    View all citing articles on Scopus
    View full text