Ascorbate increases the synthesis of procollagen hydroxyproline by cultured fibroblasts from chick embryo tendons without activation or prolyl hydroxylase

An improved procedure was developed to extract prolyl hydroxylase from tendon cells of chick embryos with detergent, and improved assays were developed for both the activity of the enzyme and the amount of enzyme protein. Freshly isolated tendon cells were found to contain approx. 100 μg of enzyme protein per 10⁸ cells and 40–50% of the enzyme protein was active. When the cells were cultured, they were found to contain the same amount of enzyme protein by only 15–20% of the enzyme protein was active. Gel filtration of cell extracts indicated that the active form of prolyl hydroxylase in freshly isolated tendon cells and in cultured tendon cells had the same apparent size and the same activity per μg of immunoreactive protein as enzyme which was shown to be a tetramer. The inactive form was found to have about the same apparent size as subunits of the enzyme.When freshly isolated cells were incubated for 2 h in the presence of 40 μg per ml of ascorbate, there was a slight increase in the rate of hydroxyproline synthesis. In cultured cells, ascorbate at a concentration of 40 μg per ml caused a 2-fold increase in the rate of hydroxyproline synthesis within 30 min. However, ascorbate did not increase the activity of prolyl hydroxylase in extracts from either cell system. Therefore it appears that the influence of ascorbate on synthesis of procollagen hydroxyproline by the cells studied here must be ascribed to a cofactor effect on the hydroxylation reaction similar to that observed with purified enzyme, and it does not involve “activation” of inactive enzyme protein to active enzyme as has been observed in cultures of L-929 and 3T6 mouse fibroblasts.     

Prolyl hydroxylase activity in L-929 fibroblasts incubated with and without ascorbate

An improved procedure was used to assay prolyl hydroxylase activity in both early-log and late-log L-929 fibroblasts grown on plastic surfaces. When 40 μg/ml of ascorbate was added to early-log phase cultures, the rate of hydroxy-[¹⁴C] proline synthesis increased 2-fold within 4 h, but there was no change in prolyl hydroxylase activity per cell. The results indicated therefore that ascorbate did not “activate” prolyl hydroxylase in the sense of converting inactive enzyme protein to active enzyme protein. Instead ascorbate appeared to increase hydroxyproline synthesis in early-log L-929 fibroblasts because the prolyl hydroxylase reaction in such cells was limited by the availability of ascorbate or a similar cofactor. When 40 μg/ml of ascorbate was added to late-log phase cultures, there was essentially no effect on the rate of hydroxyl[¹⁴C]-proline synthesis or prolyl hydroxylase activity. The late-log phase cells, however, contained three times more enzyme activity and about two times more immuno-reactive enzyme protein than early-log phase cells. In addition, the rate of protein synthesis per cell in late-log phase cells was only one-tenth the rate in early-log phase cells. The results suggested that as the cells grew to confluency, collagen polypeptides were more completely hydroxylated in part because the rate of polypeptide synthesis decreased and at the same time prolyl hydroxylase activity per cell increased. The results appear to provide an alternate explanation for previous observations on the effects of ascorbate and “crowding” on hydroxy[roline synthesis in cultures of L-929 fibroblasts.     

Studies on enzymes of collagen biosynthesis and the synthesis of hydroxyproline in macrophages and mast cells

The activities of four intracellular enzymes of collagen biosynthesis were assayed in freshly isolated rat peritoneal macrophages and mast cells and compared with the same enzymes in freshly isolated chick-embryo tendon cells. The macrophages were found to contain activities of all four enzymes, those of prolyl and lysyl hydroxylase being 7 and 12% respectively of those in the tendon cells when expressed per cell or 3 and 4% when expressed per unit of soluble cell protein. The corresponding values for hydroxylysyl galactosyltransferase and galactosylhydroxylysyl glucosyltransferase activities were about 82 and 68% or 32 and 24% respectively. When the macrophages were incubated in suspension with [(14)C]proline, they synthesized a small but significant amount of non-diffusible hydroxy[(14)C]proline. The synthesis per cell was only about 0.1% of that formed by the tendon cells, and its distribution between the cells and the medium also differed from that in the tendon cells. The hydroxy[(14)C]proline synthesized by the macrophages may be present in the Clq subcomponent of the complement, but its amount was too small to allow any characterization of the protein. All four enzyme activities, and in particular the two hydroxylysyl glycosyltransferase activities, seem to be present in macrophages in a large excess compared with the very low rate of synthesis of hydroxy-proline-containing polypeptide chains. The mast cell extract was found to inhibit all four enzyme activities, but even when corrected for this inhibition, prolyl and lysyl hydroxylase activities in the mast cells were less than 0.08% and the two hydroxylysyl glycosyltransferase activities less than 1% of those in the tendon cells. The intracellular enzyme pattern of collagen biosynthesis in the mast cells is thus completely or virtually completely repressed.     

Prolyl hydroxylase production can be uncoupled from the regulation of procollagen synthesis

Primary avian tendon (PAT) cells increase the production of procollagen from 10-12% to 40-50% of total protein synthesis in response to the addition of ascorbate and an increasing cell density. We now show that prolyl hydroxylase (PH) also increases its activity by greater than five-fold in response to increasing cell density; but unlike procollagen production, this is independent of the presence of ascorbate. The increased activity is a result of greater enzyme production and not a shift in the ratio of inactive to active forms which remains constant at about 10% of the total enzyme proteins. We present the possibility that at low cell density the levels of PH activity could limit production of collagen.     

Phosphorylation of pig brain microtubule proteins. General properties and partial characterization of endogenous substrate and cyclic AMP-dependent protein kinase.

Collagen synthesis and the activities of prolyl hydroxylase, lysyl hydroxylase, collagen galactosyltransferase and collagen glucosyltransferase were studied in isolated chick-embryo tendon cells after the administration of cortisol acetate to the chick embryos. When the steroid was injected 1 day before isolation of the tendon cells, collagen synthesis was decreased, even though the enzyme activities were not changed. When cortisol acetate was given as repeated injections over a period of 4 days, both collagen synthesis and the enzyme activities decreased. The hydroxylase activities decreased even more than the two collagen glycosyltransferase activities, both in isolated cells and in whole chick embryos. The amount of prolyl hydroxylase protein diminished to the same extent as the enzyme activity, indicating that cortisol acetate inhibits enzyme synthesis. The inhibitory effect of cortisol acetate on collagen synthesis and on the enzyme activities was partially reversible in 3 days. Total protein synthesis was completely restored within this time. Only massive doses of cortisol acetate inhibited collagen synthesis in vitro. Additional experiments indicated that cortisol acetate did not decrease the rate of the enzyme reactions when added directly to the enzyme incubation mixtures. The results suggest that cortisol acetate decreases collagen synthesis both by its direct effect on collagen polypeptide-chain synthesis and by decreasing the activities of enzymes involved in post-translational modifications.     

Localization of collagen prolyl hydroxylase to the hepatocyte : Studies in primary monolayer cultures of parenchymal cells from adult rat liver

Significant levels of prolyl hydroxylase activity (prolyl-glycyl-peptide, 2-oxoglutarate: oxygen oxidoreductase; EC 1.14.11.2) have been found in freshly isolated hepatocytes prepared from normal or regenerated adult rat liver and primary non-proliferating monolayer cultures of these cells. Four days after partial hepatectomy, the intact regenerated liver contained two times the normal level of prolyl hydroxylase activity. Freshly isolated hepatocytes contained 24% of the total prolyl hydroxylase activity in normal liver and 47% of that in regenerated liver. Upon incubation of hepatocytes for 24 h in a chemically defined culture medium containing insulin, prolyl hydroxylase activity rose 2- to 3-fold, and gradually declined during the next 48 h. The rise in prolyl hydroxylase activity was blocked by addition of cycloheximide to the culture medium. The presence of prolyl hydroxylase activity in hepatocyte cultures was not likely due to contamination with non-parenchymal liver cells. The latter cells contained less than 20% of the total enzyme activity recovered in all cells isolated from the liver. Furthermore, prolyl hydroxylase was localized by immunofluorescence uniformly to the hepatocytes in culture. Cultured hepatocytes converted [¹⁴C]proline to [¹⁴C]hydroxyproline at rates comparable to those reported for whole liver. However, only a small portion of the hydroxyproline containing product was present as collagen protein, suggesting its rapid degradation in culture. We conclude that the liver parenchymal cell may actively participate in collagen synthesis and possibly in collagen degradation.     

The assembly of tetrameric prolyl hydroxylase in tendon fibroblasts from newly synthesized α-subunits and from preformed cross-reacting protein

Embryonic-chick tendon cells were incubated in suspension for 4h with (14)C-labelled amino acids, cell extracts were subjected to gel filtration, and the effluent was examined by rocket immunoelectrophoresis by using antibodies specific for the beta-subunit of chick prolyl hydroxylase. Two peaks of immunoreactive protein were found. The first peak contained 40% of the immunoreactive protein eluted from the column and 100% of the enzyme activity. Polyacrylamide-slab-gel electrophoresis in sodium dodecyl sulphate of an immunoprecipitate of this peak demonstrated that it consisted of the tetrameric form of prolyl hydroxylase, subunit composition alpha(2)beta(2) where alpha and beta are non-identical subunits. Only the alpha-subunits were labelled, indicating that they were synthesized during the 4h labelling period. The beta-subunits were unlabelled, indicating that they had been synthesized before the labelling period. The second peak eluted from the gel-filtration column contained 60% of the immunoreactive protein eluted from the column and was enzymically inactive. Polyacrylamide-slab-gel electrophoresis of an immunoprecipitate of this peak indicated that it consisted of a single labelled polypeptide chain, identified as cross-reacting protein, which was related to, but not identical with, the beta-subunit of prolyl hydroxylase. Pulse-chase experiments were performed on cultured chick tendon cells to demonstrate that alpha-subunits and cross-reacting protein had half-lives of about 60h. The half-life of beta-subunits was considerably longer, and the kinetic pattern was consistent with their being derived from a labelled precursor such as cross-reacting protein. The data presented here indicate that the active tetrameric form of prolyl hydroxylase in cells is assembled from alpha-subunits which are newly synthesized, and from beta-subunits which are derived from cross-reacting protein.     

Induction of prolyl hydroxylase activity in a nonadherent population of human leukocytes

A nonadherent population of human monocytes has been shown to express the collagen hydroxylating enzyme prolyl hydroxylase in vitro. Enzyme levels present in freshly isolated nonadherent cells were induced 300% during the first 72 hours of culturing, which could be suppressed by cycloheximide. Maximum induction required both a feeder layer of adherent leukocytes, and 10-15% autologous plasma. Biosynthesis of Clq, a protein which also is hydroxylated by prolyl hydroxylase, by the nonadherent cells was significantly less than the adherent monocytes. Therefore, this collagen biosynthetic marker enzyme was not associated with Clq synthesis, which suggests that the enzyme is present for collagen biosynthesis.     

Regulation of collagen post-translational modification in transformed human and chick-embryo cells.

Changes in the regulation of collagen post-translational modification in transformed cells were studied in three established human sarcoma cell lines and in chick-embryo fibroblasts freshly transformed by Rous sarcoma virus. The collagens synthesized by all but one of these and by all the control human and chick-embryo cell lines were almost exclusively of types I and/or III. The relative rate of collagen synthesis and the amounts of prolyl hydroxylase activity and immunoreactive protein were markedly low in all the transformed human cell lines. The other enzymes studied, lysyl hydroxylase, hydroxylysyl galactosyltransferase and galactosylhydroxylysyl glucosyltransferase, never showed as large a decrease in activity as did prolyl hydroxylase, suggesting a more efficient regulation of the last enzyme than of the three others. The chick-embryo fibroblasts freshly transformed by Rous sarcoma virus differed from the human sarcoma cells in that prolyl hydroxylase activity was distinctly increased, whereas the decreases in immunoreactive prolyl hydroxylase protein and the three other enzyme activities were very similar to those in the simian-virus-40-transformed human fibroblasts. It seems possible that this increased prolyl hydroxylase activity is only a temporary phenomenon occurring shortly after the transformation, and may be followed by a decrease in activity later. The newly synthesized collagens of all the transformed cells that produced almost exclusively collagen types I and/or III had high extents of lysyl hydroxylation, and there was also an increase in the ratio of glycosylated to non-glycosylated hydroxylysine. The data suggest that one critical factor affecting modification is the rate of collagen synthesis, which affects the ratio of enzyme to substrate in the cell.     

Collagen biosynthesis. Characterization of subcellular fractions from embryonic chick fibroblasts and the intracellular localization of protocollagen prolyl and protocollagen lysyl hydroxylases

1. Subcellular fractions of freshly isolated matrix-free embryonic chick tendon and sternal cartilage cells have been characterized by chemical analysis, electron microscopy and the location of specific marker enzymes. These data indicate the fractions to be of a high degree of purity comparable with those obtained from other tissues, e.g. liver and kidney. 2. When homogenates were assayed for protocollagen prolyl hydroxylase and protocollagen lysyl hydroxylase activities, addition of Triton X-100 (0.1%, w/v) was found to stimulate enzyme activities by up to 60% suggesting that the enzymes were probably membrane-bound. 3. Assay of subcellular fractions obtained by differential centrifugation for protocollagen prolyl hydroxylase activity indicated the specific activity to be highest in the microsomal fraction. Similar results were obtained for protocollagen lysyl hydroxylase activity. 4. Submicrosomal fractions obtained by discontinuous sucrose-gradient centrifugation were assayed for the two enzymes and protocollagen prolyl hydroxylase and protocollagen lysyl hydroxylase were found to be associated almost exclusively with the rough endoplasmic reticulum fraction in both tendon and cartilage cells.     

CRP,immunologically cross-reacting protein of prolyl hydroxylase. Its role in assembly of active prolyl hydroxylase and cellular localization in L-929 fibroblasts

There are two forms of prolyl hydroxylase in L-929 flbroblasts. One is the enzymatically active tetramer having two α- and two β-subunits. The other is monomeric cross-reacting protein which is enzymatically inactive but is structurally related to β-subunit of the enzyme. Cultured L-929 fibroblasts at mid-log phase were labeled by ³H-labeled amino acid mixture and the radioactivity was chased for 24 h while cells were harvested and plated at higher cell densities in cultures. The results indicated that both α-subunit of the tetrameric prolyl hydroxylase and cross-reacting protein were labeled, but the β-subunit of the tetrameric active prolyl hydroxylase was not labeled until the cells were crowded for 24 h. Using immunofluorescent techniques with antibodies directed against pure tetrameric prolyl hydroxylase, capping or patching was observed when the cells were incubated at 37 °C. Also, it was found that phagosomes prepared from L-929 flbroblasts contained about 30% of total enzyme protein as determined immunologically but contained no significant prolyl hydroxylase activities. Labeling cells with ¹²⁵I by lactoperoxidase, cross-reacting protein was labeled but both α- and β-subunits of tetrameric active prolyl hydroxylase were not labeled. The results indicate that cross-reacting protein can be utilized as the precursor of β-subunit by the cells to form tetrameric active prolyl hydroxylase and that cross-reacting protein is found associated with cytoplasmic membranes.     

In vitro hydroxylation of proline in the collagen of the cysticercus of Taenia solium

1. In vitro hydroxylation of proline in cysticercus collagen was carried out using two different vertebrate enzymes. 2. Chick embryo enzyme is more active on cysticercus collagen than new-born rabbit enzyme. 3. Hydroxylation of cysticercus collagen is more efficient at 40 than at 37 degrees C. 4. No prolyl hydroxylase activity was detected in cysticercus extracts. 5. Collagen from the adult tapeworms Taenia solium and T. saginata lack hydroxyproline. 6. SLS crystallites from T. solium and T. saginata collagen show the same band pattern as cysticercus collagen.     

Hydroxylation of prolyl residues in type II procollagen in vitro and in cellulo. Lack of preferential hydroxylation of specific regions of the protein

[14C]Proline-labeled protocollagen, the unhydroxylated form of procollagen, was isolated from cartilage cells incubated with alpha, alpha'-dipyridyl. For examination of the initial steps in the hydroxylation of the protein, it was incubated in vitro with prolyl hydroxylase so that an average of 1.3-2.7 prolyl residues per chain was hydroxylated. The partially hydroxylated alpha chain were cleaved with cyanogen bromide, and the fragments were separated by polyacrylamide gel electrophoresis or column chromatography. The cyanogen bromide fragments were hydroxylated to the same degree. The results indicated, therefore, that in the initial hydroxylation of alpha chains in vitro, there was no preferential hydroxylation of any specific regions of the protein. In a second series of experiments, cartilage cells were incubated with [14C]proline and alpha, alpha'-dipyridyl so that prolyl hydroxylase in the cells was extensively, but not completely, inhibited. Partially hydroxylated alpha chains were isolated, and cyanogen bromide fragments of the alpha chains from the cells were assayed for hydroxy[14C]proline. The alpha chains contained an average of two residues of hydroxyproline per chain, and the cyanogen bromide fragments were hydroxylated to about the same degree. The results indicated, therefore, that when prolyl hydroxylase activity in cells is low relative to the rate at which pro alpha chains are synthesized, hydroxylation of prolyl residues occurs as it does in vitro, and there is no preferential hydroxylation of a specific region of the protein.     

Inhibition of hydroxyproline synthesis by palladium ions.

Palladium ions, administered as PdSO4, markedly affect the incorporation of L-[3,4-3H2] proline into non-dialyzable fractions in 10-day chick embryo cartilage explants with a 55-65% reduction in the concentration range 0.06-0.6 mM. Under these conditions the synthesis of [3H]hydroxyproline was nearly completely inhibited. Experiments with prolyl hydroxylase (EC 1.14.11.2) indicated a strong irreversible inhibition of the enzyme with a competition between Fe2+ and Pd2+. The Ki for the inhibition was 0.02 mM. Pd2+-treated enzyme remained inactive after extensive dialysis. These studies suggest that Pd2+ may inhibit collagen synthesis by replacing Fe2+ in the active site of prolyl hydroxylase and forming strong complexes with the enzyme. These studies also point to a potential mechanism of Pd2+ toxicity.     

Cell density-dependent increase in prolyl hydroxylase activity in cultured L-929 cells requires de novo protein synthesis.

Prolyl hydroxylase activity in cultured L-929 cells was found to increase when cells grew from log phase to stationary phase and when cells were harvested at the mid-log phase and replated at higher cell densities. Cycloheximide and actinomycin D inhibited the cell density-dependent increase in prolyl hydroxylase activity indicating that the increase in prolyl hydroxylase activity required de novo synthesis of protein and RNA. Prolyl hydroxylase was purified from cultured L-929 cells and antibodies against the protein were raised in rabbits. The antibodies were used to demonstrate that L-929 cells contained two forms of prolyl hydroxylase: an enzymatically active, tetrameric form consisting of two alpha and two beta polypeptide chains and an enzymatically inactive form containing immunologically cross-reacting protein. The polypeptide chains alpha, beta and cross-reacting protein were obtained by immunoadsorption. Peptide map analysis indicated that cross-reacting protein was similar if not identical to beta in primary structure, and alpha was different from both beta and cross-reacting protein. The results suggested that the prolyl hydroxylase levels in cells or tissues may be regulated by new protein and/or RNA synthesis.     

The association between prolyl hydroxylase metabolism and cell growth in cultured L-929 fibroblasts

Prolyl 4-hydroxylase (EC 1.14.11.2) is a key enzyme in collagen biosynthesis, its active form is a tetramer (alpha 2 beta 2). In L-929 fibroblasts in the log phase of culture there is a low level of active enzyme. When the cell culture reaches confluency, prolyl hydroxylase activity in cells increases by a process that requires de novo RNA and protein synthesis. The same result may be achieved by crowding the cells (replating log phase cells at the density of stationary phase cells). In the work reported here we further examined induction of the enzyme. RNA synthesis necessary for enzyme induction is complete 6 h after "crowding" while protein synthesis requires 12 h. Thymidine (0.2-0.5 mM) added to log phase cells will also cause enzyme induction to the level found in "crowded" or resting cells. We also looked at the decay of the enzyme activity after subculture. This occurs rapidly (enzyme half-life is 1-2 h) and is concurrent with the re-entry of resting cells into cell cycle; however, thymidine added at the time of subculture to block DNA synthesis does not prevent the loss of prolyl hydroxylase activity. These results suggest that when cells are not engaged in propagation, they begin to synthesize luxury proteins such as prolyl hydroxylase. However, the loss of prolyl hydroxylase during subculture is probably not a direct consequence of DNA synthesis.     

Formation of trans-caffeoyl-CoA from trans-4-coumaroyl-CoA by Zn2+-dependent enzymes in cultured plant cells and its activation by an elicitor-induced pH shift

A novel hydroxylase activity catalyzing the formation of trans-caffeoyl-CoA from trans-4-coumaroyl-CoA was identified in crude extracts from cultured parsley cells. The extracts were less active (Vmax/Km) in converting trans-4-coumaric to trans-caffeic acid. Optimal hydroxylase activity was found at pH 6.5 with a steep decline toward both pH 7.4 and pH 5.0. The enzyme activity requires ascorbate and Zn2+ at optimal concentrations of 50 and 0.5 mM, respectively. No other reductant could replace ascorbate, whereas high concentrations of Ca2+ partially substituted for Zn2+. The enzyme is soluble and appears to be located in the cytoplasm. The unusual pH optimum suggests that the hydroxylase is inactive at the normal cytoplasmic pH. Upon treatment of parsley cells with an elicitor derived from Phytophthora megasperma f. sp. glycinea, the cytoplasmic pH dropped by approximately 0.25 pH unit within 55 min as determined by 31P NMR spectroscopy. Our results suggest that this shift in the cytoplasmic pH is sufficient for the activation of the hydroxylase, eventually leading to the formation of caffeoyl and feruloyl esters. Such esters may be a part of a very rapid resistance response of the plant cells, which would leave no time for de novo enzyme synthesis.     

Ascorbate-independent proline hydroxylation resulting from viral transformation of Balb 3T3 cells and unaffected by dibutyryl cAMP treatment.

Collagen synthesis, hydroxylation of proline in collagen, and collagen secretion were studied in the contact-inhibited mouse fibroblast line, Balb 3T3; the Kirsten virus transformed line, Ki-3T3; and dibutyryl cAMP (dbcAMP)-treated Ki-3T3 cells, during the various phases of the growth cycle. Transformed cells in both logarithmic and stationary phase produced lower levels of collagen than the parent line but 85-90% of the theoretically possible hydroxyproline residues of the collagen were formed even when ascorbic acid was not added to the culture medium. Moreover, the transformed cells showed only about a 20% increase of collagen secretion upon addition of ascorbate. This was in contrast to the ascorbate requirement for maximal proline hydroxylation and the 2-3 fold stimulation of collagen secretion by ascorbate in the parent Balb 3T3 cells. Although dbcAMP treatment caused Ki-3T3 cells to assume a more normal morphology and increased the relative rate of collagen synthesis to levels similar to that of 3T3, such treatment did not restore an ascorbate requirement for proline hydroxylation or collagen secretion. The specific activity of the enzyme prolyl hydroxylase also was not affected by dbcAMP treatment although collagen synthesis was increased by such treatment. In addition, it was found that ascorbic acid was not effective in activating prolyl hydroxylase derived from Ki-3T3 or dbcAMP-treated Ki-3T3 cell cultures either in logarithmic phase or stationary phase. Ki-3T3 cultures did not accumulate ascorbic acid in cells or medium nor was ascorbic acid synthesized from the precursor 14C-glucuronate in cell homogenates. The results suggest that virally transformed Balb 3T3 cells acquire the capacity to synthesize a reducing cofactor for prolyl hydroxylase and that this function may be related to the increased glycolytic metabolism of these cells since neither cellular metabolism nor ascrobate-independent hydroxylation was altered by treatment with dbcAMP.     

Liver collagen hydroxylation in murine schistosomiasis.

The activity of procollagen prolyl hydroxylase was measured in fibrotic liver obtained from mice with hepatosplenic schistosomiasis, an animal model of the most prevalent form of human liver fibrosis. Measurable activity of prolyl hydroxylase in fibrotic liver supernatants was 47-fold higher than that of normal liver. The effect of prolyl hydroxylase inhibition on collagen synthesis in fibrotic liver slices was studied, using 8,9-dihydroxy-7-methyl benzo[b]quinolizinium bromide (GPA 1734). This compound was shown in other systems to inhibit prolyl and lysyl hydroxylations by iron chelation at concentrations which did not affect total protein synthesis. The formation of nondialyzable labelled hydroxyproline was inhibited by GPA 1734, 40, 70 and 95% at 30, 50 and 100 micrometer, respectively. Incorporation of proline into total liver protein was unaffected at 30 and 50 micrometer, but was inhibited 20% at 100 micrometer GPA 1734. Underhydroxylated collagen synthesized by liver slices with GPA 1734 was extracted with neutral salt solution and was subsequently hydroxylated with partially-purified prolyl hydroxylase to the same extent as control material synthesized in the absence of GPA 1734.     

Primary and secondary effects of ascorbate on procollagen synthesis and protein synthesis by primary cultures of tendon fibroblasts.

The effects of ascorbic acid on collagen biosynthesis were studied in primary cultures of fibroblasts from chick embryo tendons. Addition of ascorbate to the cultures increased the rate of synthesis of procollagen hydroxyproline, but the effect was not explained by activation of prolyl hydroxylase as has been seen in other cell cultures. Instead the increase in the rate of hydroxyproline synthesis appeared to be the result of some direct cofactor effect of the vitamin. In the presence of ascorbate, most of the newly synthesized procollagen was hydroxylated and became triple helical. In the absence of ascorbate, the overall degree of hydroxylation in newly synthesized procollagen was reduced, but a small fraction of newly synthesized procollagen was near-maximally hydroxylated and became triple helical. When cultures were exposed to ascorbic acid for more than 6 h, there was an increase in rate of protein synthesis, rate of procollagen synthesis, and fraction of membrane-bound ribosomes. The increases in these parameters in the presence of ascorbate appeared to be a secondary effect produced by the accumulation of stable triple-helical procollagen in the culture system.