Prolyl hydroxylase and other enzymes of collagen synthesis have been found in cloned cells, including epithelium, melanoma, liver, HeLa, pituitary, kidney, and neuroblastoma. The significance of collagen biosynthesis in nonfibroblastic cells lines is discussed.

A specific antibody to rat collagen proline hydroxylase has been used to measure the amount of "enzyme" protein in cultured mouse fibroblasts (L-929 cells) during normal growth, and under other conditions that cause an increase in enzyme activity. Collagen proline hydroxylase activity per mg of cell protein increased 24-fold as the cells progressed through the logarithmic to the stationary phase of growth, while the cellular concentration of immunologically reactive protein changed only slightly. Similar results were obtained with cells in early log phase in which enzyme activity was stimulated severalfold by cell concentration or lactate treatment without a corresponding change in cellular antigen. It has also been shown that the enzymatically inactive antigen in these fibroblasts competes effectively for antibody-binding sites with partially purified enzyme. It is concluded that early-log-phase fibroblasts contain an inactive form of collagen proline hydroxylase which may be a precursor of the functional enzyme.Green and Goldberg noted that collagen, measured as protein-bound hydroxyproline, first appeared in cultured fibroblasts in late log phase (1), and that it appeared earlier when lactate was added to the medium (2). Subsequently, Gribble et al. showed (3) that primary collagen chains were, in fact, synthesized in early-log-phase fibroblasts but that collagen proline hydroxylase activity, and hydroxyproline, were present at low levels in these cultures. The activity of the enzyme was found to increase several-fold in late log phase and was paralleled by an increase in peptidyl hydroxyproline. Further studies (4, 5) revealed that the activity of collagen proline hydroxylase in early-log-phase cells could be stimulated several-fold if these cells are concentrated to higher density or if lactate is added to the medium. The increase in enzyme activity produced by both procedures was not blocked by cycloheximide, puromycin, or actinomycin D, which indicated that it was independent of protein and nucleic acid synthesis. The latter investigations pointed to the existence of an inactive form of collagen proline hydroxylase in early-log-phase fibroblasts which was activated during normal cell growth and by the experimental manipulations cited. The object of the present investigation was to examine this hypothesis more directly. An antibody specific for collagen proline hydroxylase was prepared and used to quantify the amount of enzyme protein present during normal cell growth and under various experimental conditions. These data are compared to changes in enzyme activity under the same conditions. MATERIALS AND METHODSThe sources of all reagents and equipment used in the purification and assay of collagen proline hydroxylase and in the Collagen proline hydroxylase was purified from rat and mouse skin according to the method of Rhoads and Udenfriend (6). Mouse skin enzyme was obtained after the first ammonium sulfate fractionation step. The rat enzyme, which was used for antibody production, was further pur...

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L 929 mouse fibroblasts were cultured as monolayer using MEM Eagle medium after addition of bovine serum albumin, antibiotics, antimycotica, ferrous nitrate and ascorbic acid. In 24 hours distances cell number was counted. At the same time peptidyl prolyl hydroxylase activity was determined and related to the protein concentration in the cells. The proliferation of the cells followed a typical curve. The cell number decreased after trypsination, then increased logarithmically, finally reached a stationary phase. Enzyme activity showed a correspondent curve. Active enzyme did not appear until near the end of the log phase. After addition of the mesenchyme active substances in therapeutical concentrations proliferation and synthesis of active peptidyl prolyl hydroxylase in the cells were significantly decreased. The results are discussed in relation to the characteristics of collagen synthesis and proliferation of connective tissue cells under physiological, pathophysiological and therapeutical conditions.