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Peptides containing the unphysiological amino acid 5-oxaproline (Opr) in the sequence R1-Xaa-Opr-Gly-OR2 were found to inactivate prolyl 4-hydroxylase from chick and human origins. Of the substances investigated, compounds with aromatic substituents R1 and R2 were particularly effective when compared with those with an aliphatic group or without a C-terminal blocking group. Both affinity of the individual peptides for the enzyme and partition ratio contributed to the differences in efficiency. Benzylcarbonyl-Phe-Opr-Gly-benzyl ester was the most effective substance tested, its concentration giving 50% inactivation in 1 h being 0.8 microM. Inactivation was only observed in the presence of 2-oxoglutarate and Fe2+. The Opr peptides enhanced the decarboxylation of 2-oxoglutarate by prolyl 4-hydroxylase, the Vmax values obtained with the individual peptides being positively correlated with their inactivating efficiency. Inactivation was prevented by high concentrations of peptide substrate and ascorbate. Lineweaver-Burk kinetics experiments suggested noncompetitive inhibition with respect to peptide substrate and ascorbate. Lysyl hydroxylase was not affected by Opr peptides in concentrations of up to 1.5 mM in either the presence or absence of prolyl 4-hydroxylase. The results suggest that the oxaproline compounds are specific syncatalytic inactivators of prolyl 4-hydroxylase.

Protein disulfide-isomerase was isolated as a homogeneous protein from 15-day-old chick embryos. The enzyme has a molecular weight of 56,000 in SDS-polyacrylamide gel electrophoresis. Its Km value for randomly cross-linked ribonuclease, a protein used as a substrate for the enzyme, was 0.3 microM, and the Km value for DTT was 1.0 microM. Its optimum pH was 7.5 and its optimum temperature, 33 degrees C. The maximal velocity of pure protein disulfide-isomerase from chick embryos under optimal conditions was about 29,000 units/g. Protein disulfide-isomerase was able to activate purified prolyl 4-hydroxylase 2- to 3-fold, the activation being higher for enzyme stored for a longer time. This activation is probably due to the repairing of disulfide exchanges occurring in the prolyl 4-hydroxylase structure during purification and storage. Prolyl 4-hydroxylase activity was very stable in microsomes, however, and protein disulfide-isomerase was unable to increase the microsomal prolyl 4-hydroxylase activity, suggesting that prolyl 4-hydroxylase retains its native conformation in microsomes. Protein disulfide-isomerase was able to reactivate prolyl 4-hydroxylase inactivated by mild H2O2 treatment. The activity obtained after this treatment and protein disulfide-isomerase incubation corresponded to the amount of prolyl 4-hydroxylase tetramer found after H2O2 treatment. The data suggest that protein disulfide-isomerase is able to activate only the tetramer part of the enzyme preparation.(ABSTRACT TRUNCATED AT 250 WORDS)

From the structure-activity relationships of known competitive inhibitors, coumalic acid (2-oxo-1,2H-pyran-5-carboxylic acid) was deduced to be a potential syncatalytic inhibitor for chick-embryo prolyl 4-hydroxylase. The compound caused time-dependent inactivation, the reaction rate being first-order. The inactivation constant was 0.094 min-1, the Ki 17 mM and the bimolecular rate constant 0.09 M-1 X S-1. Human prolyl 4-hydroxylase and chick embryo lysyl hydroxylase were also inactivated, though to a lesser extent. Inactivation could be prevented by adding high concentrations of 2-oxoglutarate or its competitive analogues to the reaction mixture. In Lineweaver-Burk kinetics, coumalic acid displayed S-parabolic competitive inhibition with respect to 2-oxoglutarate. The inactivation reaction had cofactor requirements similar to those for the decarboxylation of 2-oxoglutarate. Enzymic activity was partially preserved in the absence of iron, but the rescue was incomplete, owing to decreased stability of the enzyme under this condition. Coumalic acid also decreased the electrophoretic mobility of the alpha-subunit, but the beta-subunit was not affected. Prolonged incubation of coumalic acid above pH 6.8 led to loss of its inactivating potency, owing to hydrolysis. It is concluded that the inactivation of prolyl 4-hydroxylase by coumalic acid is due to a syncatalytic mechanism. The data also suggest that the 2-oxoglutarate-binding site of the enzyme is located within the alpha-subunit.

The kinetics of the lysyl hydroxylase (peptidyllysine, 2-oxoglutarate:oxygen 5-oxidoreductase, EC 1.14.11.4) reaction were studied using enzyme from chick embryos by varying the concentration of one substrate in the presence of different fixed concentrations of the second substrate, while the concentrations of the other substrates were held constant. Intersecting lines were obtained in double-reciprocal plots for all possible pairs involving Fe2+, alpha-ketoglutarate, O2 and the peptide substrate, whereas parallel lines were obtained for pairs comprising ascorbate and each of the other substrates. The pair composed of Fe2+ and alpha-ketoglutarate gave an asymmetrical initial veolcity pattern, indicating binding of these two reactants in this order, that of Fe2+ being at thermodynamic equilibrium. The initial velocity patterns are identical with those reported for prolyl 4-hydroxylase, and the apparent Km and Kd values calculated from these data are also very similar. The largest difference was fo-nd in Km and Kd for alpha-ketoglutarate, which were about 4 times the corresponding values for prolyl 4-hydroxylase. Ascorbate was found to be a quite specific requirement for lysyl hydroxylase, but the enzyme catalyzed its reaction for a short time at a high rate in the complete absence of this vitamin, suggesting that the reaction with ascorbate does not occur during each catalytic cycle. Lysyl hydroxylase catalyzed an uncoupled decarboxylation of alpha-ketoglutarate in the absence of the peptide substrate, the rate being about 4% of that observed in the presence of a saturating concentration of the peptide substrate. This uncoupled decarboxylation required the same cosubstrates as the complete reaction.

Possible changes in collagen biosynthesis were studied in 50 patients with neuromuscular disorders and 14 controls. Type III procollagen aminoterminal propeptide concentrations and galactosylhydroxylysyl glucosyltransferase (GGT) activities were assayed in serum, and prolyl 4-hydroxylase and GGT activities were assayed in muscle biopsy specimens. All four assays showed significantly elevated values in cases of polymyositis, adult forms of muscular dystrophy, and amyotrophic lateral sclerosis, the concentration of muscular collagen also being significantly increased in the last two conditions. Some abnormalities were also seen in polyneuropathy, myotonia congenita, and undefined myopathy. High correlations were found among the values for the four assays, but no marked correlations with muscular collagen concentration or enzyme activities characteristic of neuromuscular disorders were found. The four assays may reflect changes in actual collagen synthesis in the diseased muscle.

Denatured citrate-soluble collagen was coupled to agarose by the cyanogen bromide activation technique, and columns prepared from this material were studied for affinity chromatography of collagen glycosyltransferases. Both collagen glycosyltransferases became bound to the column, the degree of binding and the capacity of the column being higher with the glucosyltransferase than with the galactosyltransferase than with the galactosyltransferase. The addition of Mn2+ enhanced the binding, especially with the glucosyltransferase. The enzymes were eluted from the column with small peptides prepared from collagen, and they were separated from the peptides by gel filtration. With this procedure a collagen glucosyltransferase purification of about 5000-fold and a collagen galactosyltransferase purification of about 1000-fold was obtained from chick embryo extract by relatively simple steps.

Product inhibition of lysyl hydroxylase (peptidyllysine, 2-oxoglutarate:oxygen 5-oxidoreductase, EC 1.14.11.4) was studied with succinate, CO2, dehydroascorbate and hydroxylysine-rich polypeptide chains. The product inhibition patterns and addition data are consistent with a reaction mechanism involving an ordered binding of Fe2+, alpha-ketoglutarate, O2 and the peptide substrate to the enzyme in this order, and an ordered release of the hydroxylated peptide, CO2, succinate and Fe2+, in which Fe2+ need not leave the enzyme during each catalytic cycle and in which the order of release of the hydroxylated peptide and CO2 is uncertain. Ascorbate probably reacts by a substitution mechanism, either after the release of the hydroxylated peptide, CO2 and succinate or after the release of all products, including Fe2+, and dehydroascorbate is released before the binding of Fe2+. It is suggested that the ascorbate reaction is required to reduce either the enzyme-iron complex or the free enzyme, which may be oxidized by a side-reaction during some catalytic cycles, but not the majority. The mechanisms of the prolyl 4-hydroxylase and lysyl hydroxylase reactions are suggested to be identical. Zn2+, several citric acid cycle intermediates, nitroblue tetrazolium and homogentisic acid inhibited lysyl hydroxylase competitively with regard to Fe2+, alpha-ketoglutarate, O2 and ascorbate respectively, and epinephrine non-competitively with regard to all cosubstrates. Apparent Ki values are given for the product and other inhibitors.

A single polypeptide is shown to act both as the beta subunit of the proline hydroxylase (EC 1.14.11.2) and as a protein disulfide-isomerase (EC 5.3.4.1). When isolated from chick embryos or rat liver, the beta subunit of prolyl 4-hydroxylase and the enzyme protein disulfide-isomerase have identical molecular weights and peptide maps as produced by digestion with Staphylococcus aureus V8 protease. The apparent molecular weights of both proteins isolated from human placental tissue are slightly higher, and the human beta subunit and one of its peptides have molecular weights about Mr 500 higher than the protein disulfide-isomerase and its corresponding peptide. Experiments with polyclonal and monoclonal antibodies also suggest a structural identity between the two proteins. The beta subunit isolated from the prolyl 4-hydroxylase tetramer has protein disulfide-isomerase activity similar to protein disulfide-isomerase itself, and even the beta subunit when present in the prolyl 4-hydroxylase tetramer has one-half of this activity.

Prolyl 4-hydroxylase (EC 1.14.11.2) catalyzes the formation of 4-hydroxyproline in collagens by the hydroxylation of proline residues in X-Pro-Gly sequences. The reaction requires Fe2+, 2-oxoglutarate, O2, and ascorbate and involves an oxidative decarboxylation of 2-oxoglutarate. Ascorbate is not consumed during most catalytic cycles, but the enzyme also catalyzes decarboxylation of 2-oxoglutarate without subsequent hydroxylation, and ascorbate is required as a specific alternative oxygen acceptor in such uncoupled reaction cycles. A number of compounds inhibit prolyl 4-hydroxylase competitively with respect to some of its cosubstrates or the peptide substrate, and recently many suicide inactivators have also been described. Such inhibitors and inactivators are of considerable interest, because the prolyl 4-hydroxylase reaction would seem a particularly suitable target for chemical regulation of the excessive collagen formation found in patients with various fibrotic diseases. The active prolyl 4-hydroxylase is an alpha 2 beta 2 tetramer, consisting of two different types of inactive monomer and probably containing two catalytic sites per tetramer. The large catalytic site may be cooperatively built up of both the alpha and beta subunits, but the alpha subunit appears to contribute the major part. The beta subunit has been found to be identical to the enzyme protein disulfide isomerase and a major cellular thyroid hormone-binding protein and shows partial homology with a phosphoinositide-specific phospholipase C, thioredoxins, and the estrogen-binding domain of the estrogen receptor. The COOH-terminus of this beta subunit has the amino acid sequence Lys-Asp-Glu-Leu, which was recently suggested to be necessary for the retention of a polypeptide within the lumen of the endoplasmic reticulum. The alpha subunit does not have this COOH-terminal sequence, and thus one function of the beta subunit in the prolyl 4-hydroxylase tetramer appears to be to retain the enzyme within this cell organelle.

The activities of prolyl 4-hydroxylase and beta-glucuronidase, the concentration of hydroxyproline as well as reticulin and collagen type III, IV and V stainings were followed in skeletal muscle during a 20-day period after a 9-h treadmill running in untrained and trained male mice, aged 4-6 months. The prolonged 9-h running of untrained mice temporarily increased prolyl 4-hydroxylase activity 2, 5 and 10 days after exercise, more prominently in the red than in the white part of quadriceps femoris-muscle, and in analogical manner as beta-glucuronidase activity in tibialis anterior-muscle. Twenty days after exercise these enzymatic activities were back to the control level. The hydroxyproline content of red muscle was increased for 10 and that of white muscle for 20 days after the exertion. Training for 45 days did not affect hydroxyproline content and prolyl 4-hydroxylase activity was at the control level after the training. A 9-h exercise increased prolyl 4-hydroxylase activity much less in trained muscle than in the untrained muscle and did not affect muscle collagen content. Histological observations showed fiber necrosis 2 days and signs of fiber regeneration 5 days after the exertion in untrained mice. Twenty days afterwards the regeneration was nearly completed. Reticulin staining was increased in injured muscle areas 10-20 days after the exertion. In immunohistochemical staining, antibodies to all studied collagen types (type III, IV and V) showed increased staining 5-20 days after the exertion in the areas of muscle injuries and regeneration.(ABSTRACT TRUNCATED AT 250 WORDS)

A high correlation was found between the activities of protein disulphide isomerase and prolyl 4-hydroxylase when assayed in cells synthesizing various collagen types or the same type at markedly different rates. The highest activities of both enzymes were found in freshly isolated chick-embryo tendon and cartilage cells, intermediate activities in confluent cultures of human skin and lung fibroblasts and mouse 3T6 fibroblasts, and the lowest values in three human sarcoma cell lines, the difference in protein disulphide isomerase activity between the freshly isolated tendon cells and confluent simian-virus-40-transformed human lung fibroblasts being about 25-fold. All these differences are in good agreement with differences reported between the various cells in their rates of collagen synthesis. A great similarity was also found between the changes in the two enzyme activities measured per cell during the growth of 3T6 fibroblast cultures from the early logarithmic phase to the stationary phase. No correlation was found between protein disulphide isomerase activity and the type of collagen synthesized. The data suggest that protein disulphide isomerase may be involved in the formation of intra-chain and inter-chain disulphide bonds in procollagens, but there is no collagen type-related variation in this enzyme activity of a magnitude that would explain the marked differences in the rates of formation of inter-chain disulphide bonds between the various collagen types.

The assembly of reduced pro-alpha chains of type I and type II procollagen into the native triple-helical molecule was examined in vitro in the presence and absence of pure protein disulfide isomerase. The data clearly indicates that protein disulfide isomerase is able to accelerate the formation of native interchain disulfide bonds in these procollagens. It takes about 6 min after disulfide bonding before triple-helical molecules exist, while the time required to produce triple-helical type I procollagen in the presence of protein disulfide isomerase is 9.4 min and that for type II procollagen 17.2 min. These values agree with those obtained for type I and II procollagen in vivo suggesting that protein disulfide isomerase is also an enzyme catalyzing interchain disulfide bond formation in procollagen in vivo. The formation of native disulfide bonds can proceed without any enzyme catalysis but then requires the presence of reduced and oxidized glutathione. Bonding is rather slow in such a case, however, resulting in a delay in the formation of the triple helix.

Endurance training over 3, 10 or 20 days increased the activity of prolyl 4-hydroxylase (PH) in the left ventricle of mice. No increase was observed in the weight of the left ventricle, in galactosylhydroxylysyl glucosyltransferase activity or in hydroxyproline concentration. The increase in PH suggests that the synthesis of collagen increases during physiological adaptation of the heart to endurance exercise without changes in the ventricle weight or its total collagen content.

The effect of angiotensin II-induced hypertension on selected biochemical parameters was studied in Sprague-Dawley rats. Angiotensin II infusion at rates of 41.7 micrograms h-1 kg-1 and 12.5 micrograms h-1 kg-1 for 2, 5, 10 and 15 days elevated the systolic blood pressure from 143 +/- 7 mmHg to 215-230 mmHg (P less than 0.001) and 185-195 mmHg (P less than 0.001), respectively. The left ventricular weight/body weight ratio increased 10-14% (P less than 0.05) and 23-32% (P less than 0.001) after 2-15 days in rats treated at the lower and higher infusion rates, respectively. Prolyl 4-hydroxylase (PH) activity, a marker of collagen synthesis, was evenly distributed in the left ventricle. PH activity increased by about 100% in both subendocardial and subepicardial layers of the left ventricular wall after angiotensin II infusion for 10 days at 41.7 micrograms h-1 kg-1, but remained unaltered at 12.5 micrograms h-1 kg-1. No change was observed in hydroxyproline concentration. Myosin isoenzymes (V1-V3), which reflect myocardial contractility, were unevenly distributed in the left ventricular wall: the proportion of the fast-turnover isoenzyme (V1) was smaller in the subendocardial layer than in the subepicardial layer. The proportion of V1 decreased after treatment in both layers. Alkaline phosphatase activity, a marker of capillary density, was evenly distributed transmurally in the left ventricular wall. Angiotensin II caused a slight decrease in this activity in both myocardial layers. The results suggest that the elevation of blood pressure leads to transmurally evenly distributed changes in biochemical parameters reflecting collagen synthesis, capillary density and contractile properties of the myocardium.

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.

A modified purification procedure, consisting of affinity chromatographies on concanavalin A-agarose, collagen-agarose and UDP-glucose-derivative-agarose and one gel filtration, is reported for galactosylhydroxylysyl glucosyltransferase. The enzyme obtained is entirely pure when studied by sodium dodecyl sulphate/polyacrylamide-gel electrophoresis. The enzyme protein was rich in glutamic acid + glutamine, aspartic acid + asparagine, glycine and alanine. The enzyme catalysed no significant glucose transfer to any of the glycoproteins tested, except for collagens. This included all the glycoproteins that have previously served as glucosyl acceptors for impure enzyme preparations, thus indicating a high degree of specificity of the enzyme for galactosylhydroxylysine. Galactosylsphingosine would act as a glucosyl acceptor, however. This compound has a close structural similarity to galactosylhydroxylysine in that they both have an unsubstituted amino group next to the hydroxy group to which the galactose is attached.

The influence of streptozotocin diabetes, glucose, and insulin on the metabolism of basement membrane collagen and proteoglycan was studied in the murine EHS tumor. No differences were found in the amino acid composition of tumors grown in diabetic and control mice, suggesting that the relative amount of collagen was not increased. Similarly, no significant differences were observed in any of the intracellular enzyme activities of collagen biosynthesis in the tumor tissue, but in diabetic mice, a significant increase was found in all four enzyme activities studied in the kidneys. High glucose concentrations increased total protein synthesis of the tumor tissue in vitro whereas insulin had no effect. Basement membrane collagen synthesis was also increased but to the same extent as protein synthesis in general. No qualitative changes were found in the pro-alpha 1(IV) and pro-alpha 2(IV) chains synthesized in vitro with or without insulin at different glucose concentrations. Hyperglycemia secondary to insulin deficiency may thus be responsible for the basement membrane thickening in diabetes but this cannot explain the relative increase in collagen content of these matrices. The incorporation of 35SO4 into proteoglycan in vitro was likewise increased at high glucose concentrations, indicating that the synthesis of this component may also be altered in diabetes.

The chromosomal location of the human gene coding for both the beta-subunit of prolyl 4-hydroxylase (P4HB) and the enzyme disulfide isomerase (PDI) was determined using mouse x human somatic cell hybrids and three different methods for identifying either the human P4HB/PDI protein or the respective gene: (1) immunoblotting with species-specific monoclonal antibodies; (2) radioimmunoassay with species-specific polyclonal antibodies; and (3) Southern blotting after cleavage of the DNA with EcoRI, HindIII, or BamHI, followed by hybridization with a mixture of two cDNA probes for human P4HB. All three methods gave identical data, demonstrating complete cosegregation of the human protein or its gene in all 17 cell hybrids tested with human chromosome 17. A cell hybrid lacking an intact chromosome 17 localized the gene to 17p11----qter.

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.

A purification of over 2000-fold is reported for collagen glucosyltransferase from Triton X-100 extract of whole chick embryos and one of about 160-fold from similar extract of chick embryo cartilage. The addition of the detergent more than doubled the enzyme activity in the homogenates. The purified enzyme preparations from whole chick embryos showed one major band and two or three minor bands in polyacrylamide gel electrophoresis and were entirely free of collagen galactosyltransferase activity. The molecular weight of collagen glucosyltransferase from both sources was about 52000 -- 54000, as determined by gel filtration. In some enzyme preparations an additional form was observed, with an elution position corresponding to a molecular weight of about 130000. Manganese was the most effective metal co-factor for the purified enzyme, but partial replacement could be obtained with Co2+, Mg2+ and Ca2+, whereas no replacement was found with other metals. The activity of the purified enzyme was stimulated by the addition of dithiothreitol to the incubation system and inhibited by preincubation with p-mercuribenzoate. UDP-glucose or the collagen substrate partially protected the enzyme against p-mercuribenzoate inactivation in the presence of Mn2+ but not in its absence. Some protection was also noted with Mn2+ alone.

Glucosylation of galactosylhydroxylysyl residues in various collagen polypeptide chains and in small peptides prepared from collagen was studied in vitro using collagen glucosyltransferase purified about 200 to 500-fold from extract prepared from chick embryos. When various denatured polypeptide or peptide chains were compared as substrates for the enzyme, no significant differences were found between citrate-soluble collagens from normal or lathyritic rats and isolated alpha1 and alpha2 chains. In contrast, gelatinized insoluble calf skin collagen, and peptides prepared from collagen and having an average molecular weight of about 500 were clearly less effective substrates as judged from their Km and V values. A marked difference was found between native and heat-denatured citrate-soluble collagen in that no synthesis of glucosylgalactosylhydroxylysine was observed with the native collagen when the reaction was studied at 30 degrees C with different times, enzyme concentrations, and substrate concentrations. When the reaction was studied as a function of temperature, little glucosylation of native collagen was observed below 37 degrees C, but there was a sharp transition in the rate of glucosylation of native collagen at temperatures above 37 degrees C, similar to that observable in the melting curve of collagen. The data suggest that triple-helical conformation of collagen prevents that glucosylation of galactosylhydroxylysyl residues.

The anthracyclines doxorubicin and daunorubicin were found to act as irreversible inhibitors of prolyl 4-hydroxylase. The reaction rate for enzyme from both chick and human origin was first order, the concentration of inhibitor giving 50% inhibition being 60 microM for both compounds after 1 h. The effect was dependent on the presence of iron ions in the reaction mixture. Inactivation could be prevented by addition of high concentrations of ascorbate, but not 2-oxoglutarate, before the inactivation period. The same results were obtained with competitive analogues of these cosubstrates. Lysyl hydroxylase from chick embryos was also susceptible to inactivation. Its activity was decreased by 50% after incubation for 1 h with a 150 microM concentration of the inhibitors. When chick-embryo prolyl 4-hydroxylase was incubated with [14-14C]doxorubicin, both enzyme subunits were radioactively labelled, about 70% of the total radioactivity being found in the alpha-subunit. Since the anthracyclines are known to undergo a redox reaction generating semiquinone radicals with Fe3+ only, the results suggest that the enzyme-bound iron ion is oxidized to a tervalent intermediate in uncoupled reaction cycles. The data also suggest that both enzyme subunits contribute to the catalytic site of prolyl 4-hydroxylase.

Factors responsible for the high extent of intracellular posttranslational modifications in type IV collagens were studied in a cultured human tumor cell line, HT-1080. These cells do not synthesize any detectable amounts of interstitial collagens but produce type IV collagen at a high rate, corresponding to about one-third of the production of interstitial collagens by cultured human skin fibroblasts. Prolyl 4-hydroxylase activity was lower in the HT-1080 cells than in human skin fibroblasts, there being a rough correlation between this enzyme activity and the rate of 4-hydroxyproline formation in these two cell types. The differing extents of the respective modifications could largely be explained by differences in the activities of lysyl hydroxylase and the hydroxylysyl glycosyltransferases between the two cell types. No difference ws found in prolyl 3-hydroxylase activity, however, even though the extent of 3-hydroxylation of proline residues was about 6-fold in the type IV collagens. In experiments where the HT-1080 cells were studied in suspension, a lag of about 100 min was found before the secretion of type IV collagen from the cells became linear. Pulse-chase experiments in suspension indicated that all the intracellular enzyme reactions proceeded for about 40 min, presumably due to the slow triple-helix formation in type IV collagens. This slow helix formation apparently contributed to the high extent of all the intracellular modifications but was not a major factor.

Antibodies were prepared against chick-embryo galactosylhydroxylysyl glucosyltransferase and further purified by immunoaffinity chromatography. The antibodies gave a single precipitation line of identity by double immunodiffusion against crude or pure chick-embryo glucosyltransferase. The ability of the antibody to precipitate the transferase was not altered by destroying the secondary structure of the enzyme. The antibody also inhibited the enzyme activity. The degree of inhibition was higher with denatured citrate-soluble rat skin collagen as the substrate than with gelatinized rat skin insoluble collagen or free galactosylhydroxylysine. The cross-reactivity of the glucosyltransferase between different species was low when studied by double immunodiffusion or inhibition kinetics. The antiserum showed no detectable cross-reactivity against other intracellular enzymes of collagen biosynthesis. A line of complete identity was found in double immunodiffusion between the transferases from whole chick embryos and chick embryo tendon, kidney and cartilage. Inhibition by the antiserum of the enzyme from chick embryo tissues synthesizing different collagen types was relatively similar. The data do not support the hypothesis that galactosylhydroxylysyl glucosyltransferase has isoenzymes with markedly different specific activities or immunological properties.

The activities of five intracellular enzymes of collagen biosynthesis were determined during cartilage and bone formation induced in rats by demineralized bone matrix. The five enzymes, prolyl 4-hydroxylase, prolyl 3-hydroxylase, lysyl hydroxylase, hydroxylysyl galactosyltransferase and galactosyl-hydroxylysyl glucosyltransferase, exhibited broadly parallel profiles; the activities rising steeply from day one to reach their highest values on day nine and decreasing gradually thereafter. The maximal enzyme activity correlated with the period of chondrogenesis and hypertrophic cartilage characterized by the synthesis of cartilage-specific type II collagen. Prolyl 4-hydroxylase was also studied in respect of its tissue distribution and cellular location using indirect immunofluorescence. The enzyme was mainly located in the mesenchymal cells on day three, in the chondrocytes and hypertrophic chondrocytes on days seven to nine, and in the osteoblasts on day eleven and thereafter.

The activities of prolyl 4-hydroxylase (PH) and galactosylhydroxylysyl glucosyltransferase (GGT), and the concentration of 4-hydroxyproline were measured in red and white parts of quadriceps femoris muscle of mice after 3, 10, and 20 sessions of daily endurance training. The activities of PH and GGT increased in the red part of the muscle after training for 3 and 10 times and returned to the control level after 20 training sessions. In the white muscle the increase of PH activity was less than in the red muscle. No alteration in GGT activity was observed in the white muscle. The concentration of hydroxyproline was unchanged in the both types of skeletal muscle. The results suggest that collagen turnover in leg muscles may be enhanced during the early phase of adaptation to endurance training. The enhancement is more prominent in red than in white skeletal muscle.

Cultures of human epidermal keratinocytes obtained from adult epidermis were initiated using irradiated BALB/3T3 cells as feeder layers. At different stages of confluence of the epidermal islands, feeder cells were removed and the extracellular matrix proteins of both pure component cells and cocultures were analyzed biochemically and by immunochemical methods and compared to those of skin fibroblasts of the same donors. The keratinocytes synthesized and secreted fibronectin and small amounts of laminin and type IV collagen. In addition, a nondisulfide-linked collagenous polypeptide (Mr = 120,000) was synthesized by the keratinocytes and was confined to the cell layers. Collagenous polypeptides with Mr = 120,000 were also synthesized by organ cultures of epidermal tissue and were detected in its acid or detergent extracts but again no secretion to culture medium was found. The Mr = 120,000 collagen had biochemical and immunological properties distinct from those of types I-V collagens. In immunofluorescence of keratinocyte cultures, fibronectin staining was prominent in the lining marginal cells of the expanding periphery of the epidermal cell islands but was not detected in the terminally differentiating cells in the upper layers of stratified colonies. Very little type IV collagen was found deposited in pericellular matrix form by the keratinocytes. In contrast, the mouse 3T3 feeder cells were found to produce both type IV collagen and laminin in addition to the previously identified connective tissue glycoproteins of fibroblasts, interstitial procollagens, and fibronectin. Basement membrane collagen of the 3T3 cells was found deposited as apparently unprocessed procollagen alpha 1(IV) and alpha 2(IV) chains. The production in culture conditions of basal lamina glycoproteins by the fibroblastic feeder cells may promote the attachment and growth of the cocultured keratinocytes.