Iron-dependent uptake of ascorbate into isolated microsomes

A preliminary study (J.M. Mata, R. Assad, and B. Peterkofsky (1981) Arch. Biochem. Biophys. 206, 93-104) suggested that chick embryo limb bone microsomes took up and concentrated [14C]ascorbate in the presence of cofactors for prolyl hydroxylase. In the present study, we found that the apparent Km for ascorbate in the hydroxylation of intracisternal unhydroxylated procollagen by endogenous prolyl hydroxylase was approximately an order of magnitude less than the value obtained when enzyme solubilized from microsomes was used with an exogenous substrate. These results are compatible with a concentrative uptake of ascorbate into microsomes. The uptake of [14C]ascorbate into microsomes was confirmed and it required only iron, in either the ferrous or ferric form, and was time and temperature dependent, proportional to microsome concentration, and substrate saturable at 2-3 mM ascorbate. Iron-dependent ascorbate uptake also was observed with L-929 cell microsomes. [14C]Ascorbate seemed to be taken up without prior oxidation, since only unlabeled ascorbate, and not dehydroascorbate, competed for uptake into limb bone microsomes. A functional requirement for Fe2+ in ascorbate transport was demonstrated using the intracisternal proline hydroxylating system. L-929 cell microsomes were preincubated with ascorbate with or without the metal and then external ascorbate was oxidized to inactive dehydroascorbate using ascorbic acid oxidase, which cannot penetrate the microsomal membrane. Samples which did not receive iron during the preincubation received it, along with other requirements for prolyl hydroxylase, in a final incubation to measure hydroxylation. Significant hydroxylation was obtained only in samples incubated with iron prior to oxidase treatment, consistent with the conclusion that an iron-dependent process was required to translocate ascorbate and protect it from the oxidase.     

A substance in L-929 cell extracts which replaces the ascorbate requirement for prolyl hydroxylase in a tritium release assay for reducing cofactor; correlation of its concentration with the extent of ascorbate-independent proline hydroxylation and the level of prolyl hydroxylase activity in these cells

Reductant used as cofactor for the prolyl hydroxylase reaction, was measured by a tritium release assay modified from an enzyme assay by making all components of the assay system saturating except for the reductant, but including prolyl hydroxylase. Reduced glutathione (6 mm), which had little activity as a cofactor, and thymol (0.1 mm), an antioxidant which exhibited no cofactor activity at all, were required for optimal proline hydroxylation dependent on reducing cofactor, with thymol fulfilling the previously described requirement for catalase. Ascorbate, cysteine and 6,7-dimethyltetrahydropterin were active as cofactors, in descending order of activity at equimolar concentrations, and activity was concentration dependent for all of these compounds. Sonicates of stationary phase L-929 cells which exhibit ascorbate-independent proline hydroxylation in culture contained reducing cofactor which could replace ascorbate in the cofactor assay, while sonicates of log phase cells which exhibit an ascorbate requirement in culture contained about one-third or less of that amount. NADH and NADPH, which themselves have little or no activity as cofactor, increased the cofactor activity of log phase cell sonicates but had relatively little effect on the activity of stationary cell sonicates suggesting that the cofactor is in a more reduced state in stationary phase. Within 24 h after replating dense, stationary phase cell cultures at low density, conditions where cells return to ascorbate dependence, prolyl hydroxylase activity had decreased to one-fifth the original activity while the concentration of functional reducing cofactor had decreased to less than 1% of its original concentration, largely as a result of oxidation. Ascorbate was not present in L-929 cells sonicates and the levels of tetrahydropterin and cysteine in sonicates could not account for the amount of cofactor activity exhibited by the sonicates in the assay system. Treatment of L-929 cultures with aminopterin did not decrease ascorbate independence, suggesting that tetrahydrofolate did not contribute significantly to cellular proline hydroxylation. These results suggest that an unidentified reductant present in L-929 cells can account for ascorbate-independent proline hydroxylation and also regulate prolyl hydroxylase activity in these cells and that cellular levels of reduced pyridine nucleotides may regulate the reduction state of this substance.     

The effectiveness of inhibitors of soluble prolyl hydroxylase against the enzyme in the cisternae of isolated bone microsomes

Inhibitors of purified, soluble prolyl hydroxylase (K. Majamaa et al. (1984) Eur. J. Biochem. 138, 239-245; K. Majamaa et al. (1986) J. Biol. Chem. 261, 7819-7823) were tested against isolated chick embryo bone microsomes containing intracisternal prolyl hydroxylase and its radiolabeled, unhydroxylated procollagen substrate. Two groups of inhibitors were used which consisted of pyridine-2-carboxylate and 1,2-dihydroxybenzene (catechol) derivatives. The 2,4- and 2,5-pyridine dicarboxylic acids, which are potent inhibitors of the soluble enzyme (Ki values 2 and 0.8 microM, respectively), were effective in the same concentration range against intracisternal prolyl hydroxylase, although their relative affinities were reversed. Inhibition by pyridine-2,4-dicarboxylate in the microsomal system was reversed by increasing the concentration of 2-oxoglutarate. Pyridine-2,4-dicarboxylic acid did not inhibit the uptake of 2-[14C]oxoglutarate into microsomes, so it appears likely that the inhibitor must traverse the microsomal membrane and act directly at the enzyme level. Pyridine-2-carboxylic acid was ineffective in the microsomal system at 1 mM whereas it is a relatively potent inhibitor of the soluble enzyme with a Ki of 25 microM. This finding suggests that the second carboxyl group of the pyridine carboxylate derivatives may be required for their transport into the microsomal lumen. In the soluble system, 3,4-dihydroxybenzoic acid and 1,2-dihydroxybenzene had been found to be competitive inhibitors with relatively low Ki values of 5 and 25 microM, respectively. In the microsomal system, half-maximal inhibition was obtained at approximately 50-100 microM and inhibition was not reversed by increasing the concentrations of either 2-oxoglutarate or ascorbate, alone or together. These results imply that in situ these compounds do not inhibit prolyl hydroxylase directly. Thus, the microsomal system can assess the accessibility of the intracisternal enzyme to potential inhibitors and offers an insight into the in cellulo potential of such compounds.     

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.     

The formation of hydroxyproline in collagen by cells grown in the absence of serum

L-929 and 3T6 cells were conditioned to grow in a chemically defined medium lacking serum and ascorbate. Serum, when added, had a small stimulatory effect on the growth rate of the cells, but ascorbate had no effect either on the growth rate or on the rate of protein synthesis. These cells were also shown to lack gulonolactone oxidase activity and therefore could not synthesize their own ascorbate. Nevertheless, in the absence of serum and ascorbate both cell types were able to hydroxylate peptidyl proline to an appreciable extent. This suggest that reductant other than ascorbate can at least partially satisfy the requirement for a reductant in the prolyl hydroxylase reaction in vivo.     

Proteolytic activity in human skin fibroblasts harvested with trypsin and its effect on measurements of collagen hydroxylase activities.

Lysates of human skin fibroblasts harvested without the use of trypsin do not contain detectable proteolytic activity, but when trypsin is used, lysates may contain activity equal to 10 ng of trypsin/10⁷ cells. The amount of cell lysate ordinarily examined for collagen prolyl and lysyl hydroxylase activity is sufficiently small that such amounts of trypsin have no observable effect on the unhydroxylated collagen substrate. Larger amounts of trypsin cause proteolysis of the unhydroxylated collagen substrate and a reduction of both prolyl and lysyl hydroxylation with lysyl hydroxylation more affected at low trypsin concentration than prolyl hydroxylation.     

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.     

The formation of hydroxyproline in collagen by cells grown in the absence of serum.

L-929 and 3T6 cells were conditioned to grow in a chemically defined medium lacking serum and ascorbate. Serum, when added, had a small stimulatory effect on the growth rate of the cells, but ascorbate had no effect either on the growth rate or on the rate of protein synthesis. These cells were also shown to lack gulonolactone oxidase activity and therefore could not synthesize their own ascorbate. Nevertheless, in the absence of serum and ascorbate both cell types were able to hydroxylate peptidyl proline to an appreciable extent. This suggests that reductants other than ascorbate can at least partially satisfy the requirement for a reductant in the prolyl hydroxylase reaction in vivo.     

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

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 mug of enzyme protein per 10(8) 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 but 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 incultured tendon cells had the same apparent size and the same activity per mug 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 mug per ml of ascorbate, there was a slight increase in the rate of hydroxyproline synthesis. In cultured cells, ascorbate at a concentration of 40 mug per ml caused a 2-fold increase in the rate of hydroxyproline synthesis within 30 min. However, ascorbate did not icrease 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.     

Characterization of collagen hydroxylysyl glycosyltransferases as mainly intramembranous microsomal enzymes

The localization of collagen hydroxylysine galactosyl- and galactosyl-hydroxylysine glucosyltransferases in purified chick embryo bone microsomes was studied by differential solubilization with nonionic detergents. Brij-35 (polyoxyethylene 25-lauryl ether) which selectively releases intracisternal proteins, and Triton X-100, whose specificity varies with its concentration, were used in the presence or absence of high ionic strength NaCl. These methods were used previously to characterize prolyl hydroxylase as intracisternal and lysyl hydroxylase as mainly intramembranous. The distribution of both glycosyltransferases within microsomes was similar to that of lysyl hydroxylase; approximately 70-80% of their activities are intramembranous with the remainder intracisternal. Collagen hydroxylysine glucosyltransferase differed from prolyl and lysyl hydroxylase and the galactosyltransferase in that its activity in vitro was apparently inhibited by membrane vesicles, even in the presence of detergents at concentrations which permeabilize the membrane. Accurate measurement of its activity could be achieved only by its separation from vesicles after detergent treatment. The common location of the major portion of lysyl hydroxylase and the glycosyltransferase activities suggests that they may act as a multienzyme complex to preferentially modify certain lysyl residues in nascent procollagen chains as they traverse the membrane of the endoplasmic reticulum. Since these enzymes do not act on helical collagen, their physical separation from prolyl hydroxylase may ensure that modifications of lysine residues occur prior to formation of hydroxyproline, which stabilizes the helical form.     

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.     

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.     

The stimulation of collagen secretion by ascorbate as a result of increased proline hydroxylation in chick embryo fibroblasts.

Collagen secretion by chick embryo fibroblasts was measured by incorporating [¹⁴C]proline into proteins and then analyzing the amount of collagen in the cell and medium separately by using purified bacterial collagenase. In order to produce varying levels of hydroxylation, cells were incubated with varying concentrations of ascorbate or with varying concentrations of α,α′-dipyridyl in the presence of saturating ascorbate. Ascorbate stimulated both the hydroxylation of proline in collagen and the secretion of collagen; the concentration of ascorbate required for half-maximal stimulation of both proesses was approximately 4.5 × 10^?7, m. Since the cells could concentrate ascorbate 10-fold, this K_M for proline hydroxylation is 100-fold lower than values reported for purified prolyl hydroxylase (Abbot, M. T., and Udenfriend, S. (1974) in Molecular Mechanisms of Oxygen Activation (Hayaishi, O., ed.), p. 173, Academic Press New York; Kivirikko K. I., et al. (1968) Biochim. Biophys. Acta, 151, 558–567). Conversely, α,ga′-dipyridyl inhibited both proline hydroxylation and collagen secretion; half-maximal inhibition of both processes was observed at 7 × 10^?5, m. The results of the two types of experiments show that the secretion of collagen becomes directly proportional to proline hydroxylation when approximately 30% of the proline residues in collagen have been hydroxylated compared to maximal hydroxylation of 50%. Since the stability of triple-helical collagen at 37 °C has been shown to be dependent on the hydroxyproline content of the molecule (Rosenbloom, J., et al. (1973) Arch. Biochem. Biophys., 158, 478–484), we suggest that the observed proportionality between secretion and hydroxylation is a reflection of the increased amount of stable triple helical collagen at 37 °C. When the cells were incubated with a concentration of ascorbate that was saturating for secretion and hydroxylation, there was no significant activation of prolyl hydroxylase as measured in a cell-free extract. These experiments suggest that ascorbate effects collagen secretion by acting at the site of proline hydroxylation but not by increasing the activity of prolyl hydroxylase.     

The effects of lead upon collagen synthesis and proline hydroxylation in the Swiss mouse 3T6 fibroblast.

The effects of lead upon collagen synthesis and proline hydroxylation were examined in the Swiss mouse 3T6 fibroblast. The results indicate that lead reduces proline hydroxylation in stationary phase cultures of 3T6 cells, resulting in increased cellular retention of unhydroxylated procollagen. Inhibition of proline hydroxylation by lead was prevented by increasing the extracellular $\text{Fe}{}^{\mathrm{2+}}\text{Pb}{}^{\mathrm{2+}}$ molar ratio. Interference by lead in the hydroxylation of proline in logarithmic phase cultures of 3T6 cells resulted in increases in the 0.5 n HClO₄ soluble/insoluble hydroxyproline ratio. This was attributed to an increase in the rate of breakdown of lead-induced unhydroxylated procollagen. Kinetic analysis of the lead-iron interaction with proline hydroxylase suggests that the mechanism is competitive.     

Further characterization of embryonic tendon fibroblasts and the use of immunoferritin techniques to study collagen biosynthesis

Morphological studies were carried out on fibroblasts from chick embryo tendons, cells which have been used in a number of recent studies on collagen biosynthesis. The cells were relatively rich in endoplasmic reticulum and contained a well-developed Golgi complex comprised of small vesicles, stacked membranes, and large vacuoles. Techniques were then devised for preparing cell fragments which were penetrated by ferritin-antibody conjuates but which retained the essential morphological features of the cells. Finally, the new procedures were employed to develop further information as to how collagen is synthesized. As reported elsewhere, preliminary studies with ferritin-labeled antibodies showed that prolyl hydroxylase was found in the endoplasmic reticulum of freshly isolated fibroblasts and that procollagen is found in both the cisternae of the endoplasmic reticulum and the large Golgi vacuoles. In the experiments described here, the cells were manipulated so that amino acids continued to be incorporated into polypeptide chains but assembly of the molecule was not completed because hydroxylation of prolyl and lysyl residues was prevented. The results indicated that these manipulations produced no change in the distribution of prolyl hydroxylase. Examination of the cells with ferritin conjugated to antibodies which reacted with protocollagen, the unhydroxylated form of procollagen, demonstrated that protocollagen was retained in the cisternae of the endoplasmic reticulum during inhibition of the prolyl and lysyl hydroxylases. Assays for prolyl hydroxylase with an immunologic technique demonstrated that although the enzyme is found within the endoplasmic reticulum, it is not secreted along with procollagen. The observations provided further evidence for a special role for prolyl hydroxylase in the control of collagen biosynthesis.     

Secretion of unhydroxylated chick tendon procollagen.

The secretion of unhydroxylated procollagen at 37° by isolated chick tendon fibroblasts independent of protein synthesis was examined. The data showed that intact molecules were secreted and that their degradation was an extracellular event. The kinetics of secretion indicated that most of the secreted procollagen appeared in the medium during the initial 30 min following inhibition of protein synthesis and only an additional 35% reached the extracellular space in the subsequent 90 min. The pattern of secretion suggested the existence of an intracellular binding site for the unhydroxylated molecules which was saturated during the early period of secretion. It is speculated that such a binding site could be the enzyme prolyl hydroxylase which has a high affinity for unhydroxylated procollagen at 37°.     

Inhibition of a viral prolyl hydroxylase

The hydroxylation of prolyl-residues in eukaryotes is important in collagen biosynthesis and in hypoxic signalling. The hypoxia inducible factor (HIF) prolyl hydroxylases (PHDs) are drug targets for the treatment of anaemia, while the procollagen prolyl hydroxylases and other 2-oxoglutarate dependent oxygenases are potential therapeutic targets for treatment of cancer, fibrotic disease, and infection. We describe assay development and inhibition studies for a procollagen prolyl hydroxylase from Paramecium bursaria chlorella virus 1 (vCPH). The results reveal HIF PHD inhibitors in clinical trials also inhibit vCPH. Implications for the targeting of the human PHDs and microbial prolyl hydroxylases are discussed.     

The role of reductants in the tyrosine hydroxylase reaction.

The role of several reducing systems in the tyrosine hydroxylase reaction has been studied. A significant dependence upon the reducing systems beyond that required to regenerate the oxidized cofactor has been observed. 2-Mercaptoethanol, NADPH, and ascorbate are each effective at reducing the cofactor, but their abilities to stimulate tyrosine hydroxylase vary over a threefold range. NADPH is a suitable reductant for the tyrosine hydroxylase reaction, even in the absence of pteridine reductase. A reducing system containing ascorbate, ferrous ion, and catalase gives unusually high enzyme activity and low blanks. This ascorbate system, in addition to being useful for in vitro enzyme assays, may serve as a model for the in vivo reaction. Ascorbate may play an important role in the hydroxylation of tyrosine in catecholaminergic tissues. This study demonstrates that an efficient reductant for the tyrosine hydroxylase reaction must, in addition to reducing the pterin cofactor, also interact effectively with the enzyme itself.     

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.     

An activatable form of prolyl hydroxylase in fibrobalast extracts.

An $\text{in vitro}$ increase in prolyl hydroxylase activity has been effected in sonicates of early log phase L 929 mouse skin fibroblasts from either monolayer or suspension cultures. The requirements for activation are identical to those needed for the hydroxylation reaction itself, i.e., ferrous ion, ascorbate and α-ketoglutarate. Catalase, which is not an absolute requirement for the hydroxylation, is also necessary for activation. The activation is time dependent and, under the conditions used, is complete in 3 hr at 30°. Since ferrous ion also appears necessary for the activation in intact cells and since the same level of activation is achieved in intact cells as in sonicates, it appears that the $\text{in vitro}$ activation proceeds in the same manner as that seen in cultured cells.