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.     

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.     

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.     

Radiommunoassay for human and chick prolyl hydroxylases.

A radioimmunoassay is reported for measuring prolyl hydroxylase. The assay is based on the displacement of radioactively-labelled prolyl hydroxylase from its antibody by the non-labelled enzyme, and on the subsequent precipitation of the enzyme-antibody complex by a cellulose-bound second antibody. Pure prolyl hydroxylase was isolated from foetal human or chick embryo tissues by an affinity column procedure usingpoly(L-proline). The enzyme was labelled with tritium using a technique of reductive alkylation with formaldehyde and sodium [3H]borohydride. No conversion of the enzyme tetramer to its monomers was found to take place during the tritiation reaction. Experiments on the dissociation of the non-labelled enzyme indicated that the degree of displacement of the labelled enzyme was similar regardless of whether the non-labelled enzyme was in the tetramer form or in that of the subunit monomers. The sensitivity of the radioimmunoassay is of the order of 5 -- 10 ng immunoreactive prolyl hydroxylase. The concentrations of the immunoreactive prolyl hydroxylase assayed with the present method in human serum and skin and in several chick embryo tissues are reported.     

Regulation of prolyl and lysyl hydroxylase activities in cultured human skin fibroblasts by ascorbic acid

Studies with confluent human skin fibroblasts maintained in 0.5% serum supplemented medium have given new insight into the regulatory influences of ascorbate. These include a reduction of prolyl hydroxylase activity, a stimulation of lysyl hydroxylase activity, and an acceleration of collagen production. The lack of parallel between prolyl hydroxylase activity and collagen production indicates that the rate of collagen synthesis is not controlled by the level of prolyl hydroxylase.     

Turnover of prolyl hydroxylase and an immunologically related protein in rabbit tissue

The turnover rates of prolyl hydroxylase and immunologically related (cross reacting) protein were examined using labeled leucine as precursor or by measuring the decay of elevated prolyl hydroxylase and immunologically cross-reacting protein back to basal levels. Prolyl hydroxylase and immunologically cross-reacting protein were purified from neonatal rabbit skin at various times following the administration of [³H]leucine. Prolyl hydroxylase was purified by affinity chromatography. Immunologically cross-reacting protein was purified by antibody precipitation from the dialyzed 70% (NH₄)SO₄ supernatants and subsequent electrophoresis on 10% sodium dodecyl sulfate-polyacrylamide slab gels. The radioactivity of the species isolated, which corresponded to the two major subunits of prolyl hydroxylase, was used in the turnover studies of immunologically cross-reacting protein. The peak incorporation of label into prolyl hydroxylase was found to be 12 h while for immunologically cross-reacting protein this occured within 2 h. The loss of radioactivity from these protein pools denotes an apparent $\text{t}{}_{\mathrm{<mtext>1</mtext><mtext>2</mtext>}}$ for prolyl hydroxylase of 73 h and a $\text{1}\text{2}$ for immunologically cross-reacting protein of 53 h. From the specific activity of free skin leucine pools, the effect of reutilization could be corrected and a true $\text{t}\text{1}\text{2}$ for prolyl hydroxylase of 45 h was determined. The $\text{t}\text{1}\text{2}$ values of these proteins were determined by a second method in which prolyl hydroxylase and immunologically cross-reacting protein in the aorta and liver of adult male rabbits were elevated by daily epinephrine-thyroxine treatment for 12 days. The decline of prolyl hydroxylase and immunologically cross-reacting protein with termination of treatment in the aorta denotes values of 42 h for enzyme and 53 h for immunologically cross-reacting protein. Calculated enzyme κ_d values, by both methods, indicate that breakdown of enzyme does not account for tissue immunologically cross-reacting protein.     

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 the mechanism of reduction of prolyl hydroxylase activity by D,L-3,4 dehydroproline.

When chick frontal bone cells in culture were exposed to d,l-3,4 dehydroproline, the specific activity of prolyl hydroxylase was markedly reduced, but the concentration of the protein antigenically related to prolyl hydroxylase was not decreased. The specific activity of purified prolyl hydroxylase from cells grown in d,l-3,4 dehydroproline was significantly lower than that of control cells. Preincubation of a homogeneous preparation of chick embryo prolyl hydroxylase with collagenous peptides containing [¹⁴C]d,l-3,4 dehydroproline resulted in a time-dependent decrease in the enzymatic activity. These observations suggest that the in vivo reduction in prolyl hydroxylase activity by dehydroproline could be either due to an interaction of the enzyme with collagenous peptides containing dehydroproline and/or the synthesis of an aberrant form of prolyl hydroxylase with decreased enzymatic activity.     

In vivo labeling and turnover of prolyl hydroxylase and a related immunoreactive protein

Prolyl hydroxylase and an immunologically related protein (CRP) were purified from neonatal rabbit skin at various time periods following administration of 3H-leucine. The peak incorporation of label into prolyl hydroxylase was found to be 12 hours, while peak incorporation into CRP occurred within 2 hours. Semi-log plots of the loss of radioactivity from these protein pools against time indicated an apparent T 1/2 for prolyl hydroxylase of 78 hours, and a T 1/2 of CRP of 44 hours. Calculated Kd values indicate that that breakdown of active enzyme does not account for the amount of CRP found in tissues.     

Intracellular enzymes of collagen biosynthesis in rat liver as a function of age and in hepatic injury induced by dimethylnitrosamine. Purification of rat prolyl hydroxylase and comparison of changes in prolyl hydroxylase activity with changes in immunoreactive prolyl hydroxylase.

Prolyl hydroxylase was purified from newborn rats by affinity chromatography using poly(L-proline), and antiserum to the enzyme was prepared in rabbits. The rat prolyl hydroxylase was similar to the chick and human enzymes with respect to specific activity, molecular weight and molecular weights of the polypeptide chains. The activity of prolyl hydroxylase and the content of immunoreactive enzyme were measured in rat liver as a function of age in experimental hepatic injury. Active prolyl hydroxylase comprised about 13.2% of the total immunoreactive protein in the liver of newborn rats and the value decreased to about 3.6% at the age of 420 days. This decrease was due to a decrease in the enzyme activity, whereas only minor changes were found in the content of the immunoreactive protein. In hepatic injury, a significant increase was found in the ratio of active enzyme to total immunoreactive protein, owing to an increase in the enzyme activity. The data indicate that prolyl hydroxylase activity in rat liver is controlled in part by a mechanism which does not involve changes in the content of the total immunoreactive protein.     

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.     

Prolyl hydroxylase activity as an index of liver damage induced by ethanol

It was found that chronic intoxication of rats with ethanol results in an increase of prolyl hydroxylase activity in liver and serum of the experimental animals. The increase of enzyme activity precedes the morphological symptoms of liver damage. The possibility arises that the assay of prolyl hydroxylase in serum or in liver biopsy samples could be useful for the diagnosis of the tendency of some individuals to develop liver cirrhosis induced by ethanol.     

Concanavalin A binds of purified prolyl hydroxylase and partially inhibits its enzymic activity.

Prolyl hydroxylase [(EC 1.14.11.2; prolyl-glycyl peptide, 2-oxoglutarate dioxygenase (4-hydroxylating)] was electrophoresed on polyacrylamide gels and the enzyme in the gels was shown to bind [acetyl-³H]concanavalin A. The enzyme-lectin complex was dissociated by treating the gel with methyl α-D-mannopyranoside, a sugar known to inhibit binding of concanavalin A to glycoproteins. Furthermore, prolyl hydroxylase activity was partially inhibited by concanavalin A when the enzyme was assayed in the absence of bovine serum albumin, a protein which enhances enzymic activity. The inhibition of enzyme activity was prevented by sugars known to react with concanavalin A.     

Inhibitory effects of glucocorticoids on collagen synthesis by mouse sponge granulomas and granuloma fibroblasts in culture.

The basis for the glucocorticoid-mediated decrease in tissue collagen was studied in mouse granulomas and in primary granuloma fibroblast cultures. Injection of mice for 12 days with dexamethasone (0.35 mg/kg body weight) resulted in a 50--70% inhibition of collagen synthesis and accumulation in polyvinyl sponge-induced granulomas whereas total protein synthesis was inhibited by only about 25%. The decreased collagen content of the granuloma was accounted for by both a reduced fibroblast number and diminished synthesis per cell. Growth rates, total protein synthesis and collagen synthesis were the same in granuloma fibroblast cultures derived from control or steroid-treated mice. However, addition of 3.10(-7) M hydrocortisone to the culture medium caused a 30--50% inhibition of both collagen and non-collagen protein synthesis in firbroblasts from either source. These inhibitory effects were dose- and time-dependent with a lag time of 12--24 h. Prolyl hydroxylase activity was reduced both in sponge granulomas from glucocorticoid-treated mice and in hydrocortisone-treated fibroblast cultures. However, protein synthesis was inhibited to the same extent as the inhibition of prolyl hydroxylase activity and there was no effect on peptidyl prolyl hydroxylation. These results indicate that the glucocorticoid-induced reduction of collagen synthesis and accumulation observed in mouse granulomas and primary granuloma fibroblast cultures is not specific for this protein. Furthermore, glucocorticoid-induced inhibition of collagen synthesis cannot be attributed to underhydroxylation of collagen prolyl residues.     

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 μM, respectively. Incorporation of proline into total liver protein was unaffected at 30 and 50 μM, but was inhibited 20% at 100μM 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.     

Effect of L-3,4-dehydroproline on collagen synthesis and prolyl hydroxylase activity in mammalian cell cultures.

The incorporation of DL-3,4-dehydro[14C]proline into collagen and total protein of 3T3 cells occurred at approximately one-fifth the rate observed for L-[14C]proline. Addition of L-3,4-dehydroproline to the culture medium inhibited markedly the incorporation of [14C]glycine and L-[3H]lysine into the collagen of 3T3 cells, but there was only slight inhibition of the incorporation of the radiolabeled amino acids into total cellular proteins, indicating that the action of L-3,4-dehydroproline is specific for collagen. When 1 mM L-3,4-dehydroproline was added to the culture medium the [14C]hydroxyproline content was reduced 40% in the cell layer and 70% in the medium. The D isomer of 3,4-dehydroproline did not inhibit [14C]hydroxyproline formation. These findings indicate that L-3,4-dehydroline reduced the hydroxylation of the susceptible prolyl residues in the collagen molecule and the secretion of collagen from the cell. The reduction in the hydroxyproline content is probably related in part to a reduction in the activity of prolyl hydroxylase; when various mammalian cell cultures were exposed to 0.2 mM L-3,4-dehydroproline, the specific activity of prolyl hydroxylase was reduced markedly, while that of lysyl hydroproline, the specific activity of prolyl hydroxylase was reduced markedly, while that of lysyl hydroxylase was not affected. Under these conditions, cell growth and lactic dehydrogenase required protein synthesis. Removal of L-3,4-dehydroproline from the growth medium resulted in a time-dependent increase in the specific activity of prolyl hydroxylase.     

Submicrosomal localization of prolyl hydroxylase from chick embryo limb bone.

A fraction greatly enriched in microsomes was prepared from chick embryo limb bone tissue homogenates by differential centrifugation in a high density solution of Metrizamide. This fraction was used to determine the submicrosomal localization of prolyl hydroxylase. At a low concentration (0.05%) of the non-ionic detergents Triton X-100 and Brij-35, 90 to 93% of prolyl hydroxylase activity was released from microsomes. Concentrations of Triton X-100 greater than 0.1% were required to solubilize the intrinsic membrane enzyme NADH-ferricyanide reductase and to release membrane-bound ribosomes, while Brij-35 did not extensively solubilize membrane components even at concentrations up to 0.4%. In addition, prolyl hydroxylase activity which could subsequently be released from microsomes by Brij-35 was relatively resistant to trypsin proteolysis at concentrations which removed more than 50% of the ribosomes and approximately 40% of the protein from microsomes. These results suggest that 90 to 93% of prolyl hydroxylase activity in connective tissue is located within the cisternae of the endoplasmic reticulum. Gel filtration of prolyl hydroxylase released from microsomes or found in the soluble fraction of limb bone homogenates revealed two peaks of activity corresponding to molecular weights of 230,000 and 450,000 to 500,000. The latter is twice the value reported for purified chick embryo prolyl hydroxylase. A fraction of the total prolyl hydroxylase activity (generally 20 to 35%) in microsome preparations could be measured in the absence of detergent, although the microsomal membrane should be impermeable to the large unhydroxylated collagen chains used as substrate. On the basis of experimental data, it was concluded that detergent-independent activity was most likely due to damaged microsomal membranes and that this damage was sufficient to allow substrate and trypsin to enter the cisternae but not to allow prolyl hydroxylase to be released.     

Stimulation of the activity of prolyl hydroxylase in 3T3 fibroblasts by 1,10-phenanthroline.

In confluent cultures of 3T3 fibroblasts, incubated for 24 h with 1,10-phenanthroline at 10(-5)--10(-9) M, the activity of prolyl hydroxylase was significantly increased. 1,10-Phenanthroline was inhibitory at concentrations greater than 10(-4) M. The stimulatory effect of 1,10-phenanthroline manifests itself after 6 h incubation and increased with time up to 48 h. 2,2'-dipyridyl and 5,6-dimethyl-1,10-phenanthroline were also stimulatory; a nonchelating analog, 1,7-phenanthroline had no effect. Cycloheximide did not modify the 1,10-phenanthroline effect. The stimulatory effect does not seem to depend on the shift of an inactive precursor of prolyl hydroxylase to an active form because 1,10-phenanthroline was shown to be ineffective in logarithmically growing cells. While dialysis of washed and homogenized cells significantly increased prolyl hydroxylase activity in cell extracts, undialyzed 1,10-phenanthroline treated samples exhibited higher prolyl hydroxylase activity than dialyzed controls. These data suggested to us that 1,10-phenanthroline and other chelating agents may be forming complexes with certain metal ions or protein-metal ions which are inhibitory towards prolyl hydroxylase.     

Thyroid hormone binding protein contains glycosylation site binding protein activity

Several lines of evidence provided by other workers indicate that within the same species thyroid hormone binding protein, the beta-subunit of prolyl hydroxylase, and protein disulfide isomerase are the same protein. We sought to determine if glycosylation site binding protein, a lumenal protein of the endoplasmic reticulum, also has the same primary structure. To accomplish this the level of glycosylation site binding protein (GSBP) activity, measured by photolabeling with a glycosylation site peptide probe, was carried out in preparations of 3T3 cells and in E. coli transformed with human thyroid hormone binding protein cDNA. The results strongly support the idea that GSBP is identical to these other lumenal proteins of the endoplasmic reticulum.