Developmental Changes in Prolyl Hydroxylase Activity and Protein in Chick Embryo

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1 Eur. J. Biochem. 66, (976) Developmental Changes in Prolyl Hydroxylase Activity and Protein in Chick Embryo Leena TUDERMAN Department of Medical Biochemistry, University of Oulu (Received March 3, 976) Changes in prolyl hydroxylase activity and iminunoreactive protein were studied in various chick embryo tissues during the embryonic development. Both the enzyme activity and the amount of immunoreactive protein increased till the 6th day of development and declined thereafter in all tissues studied. Comparison of the enzyme activity to the content of the total immunoreactive protein indicated that there are distinct differences in the degree of enzyme activity between different chick embryo tissues, and in the same tissue between different stages of embryonic development. The highest relative enzyme activities were found in cartilage and skin, in which about 6% of the enzyme was active on the 6th day of development and only 2-3% was active on the 2th day of development; the lowest values were observed in spleen and large vessels, in which below % of the enzyme protein was in the active form on the 2th day of development. Gel filtration studies demonstrated that in cartilage of 6-day-old chick embryos about 6% of the total immunoreactive enzyme in the tissue was present in the form of active prolyl hydroxylase tetramer, whereas on the 2th day of development only 3% of the enzyme protein in cartilage was in the tetramer form. By contrast, in large vessels of the 6-day-old chick embryos, essentially all the enzyme was in the form of prolyl hydroxylase monomers. Prolyl hydroxylase catalyzes the synthesis of hydroxyproline in collagen by hydroxylation of certain prolyl residues in peptide linkages (for recent reviews, see [l-4). The enzyme was initially purified to near homogeneity by conventional protein purification procedures from chick embryos [5,6] and newborn rat skin [7]. More recently two affinity column procedures were developed for purifying prolyl hydroxylase, and the enzyme was isolated as a pure protein from chick embryos [8,9] and from foetal human tissues [lo]. The molecular weight of prolyl hydroxylase from chick embryos [6,8,9] and human tissues [lo] is about 24, and it is a tetramer consisting of two different types of subunits with molecular weights of about 6 and 64 [7-lo]. The enzymes purified from chick embryos, rat skin and human tissues have also been characterized by preparing antisera to them [lo-3. Studies of prolyl hydroxylase in cultured fibroblasts have demonstrated that the enzyme is partly present as an inactive precursor, which can be activated by the addition of micromolar concentrations of ascorbate to the medium [,4-5. The activation of prolyl G7z>mc. Prolyl hydroxylase or prolyl-glycyl-peptide,2-oxoglutarate : oxygen oxidoreductase (4-hydroxylating) (EC.4. I.2). hydroxylase was also demonstrated in vitvo in sonicates of early log phase L-929 fibroblasts, but the activation in vitro required, in addition to ascorbate, ferrous iron and 2-oxoglutarate [6]. The presence of inactive prolyl hydroxylase was demonstrated in other fibroblasts too [7], but the enzyme activity in cultured fibroblasts from chick embryo leg tendon [7] or frontal bones [8] is not subjected to an ascorbaterequiring control mechanism. It has also been further demonstrated that mammalian tissues contain large amounts of inactive immunologically reacting protein [9,28], suggesting that prolyl hydroxylase may be present partly as an inactive pro-enzyme in mammalian cells too, and that the activation of the enzyme may be of physiological significance. Changes in prolyl hydroxylase activity in animal and human tissues have been reported in a number of experimental and clinical states (see [3,4]). However, it is not known whether such changes in any state are due to activation of the enzyme protein, or whether they are due to induction of enzyme synthesis. The presence of hydroxyproline in chick embryos has been detected as early as after 6 h incubation [2]. Prolyl hydroxylase activity has been shown to increase until the 4th day of embryonic development

2 66 Developmental Changes in Prolyl Hydroxylase and to decline thereafter [2]. Distinct differences were reported in the enzymic activity between various chick embryo tissues [2]. No attempt, however, has previously been made to find out whether the changes in the enzymic activity are associated with similar changes in the immunologically reacting protein. In the present work changes in prolyl hydroxylase activity and in the immunologically reacting protein were studied in several tissues during chick embryo developmen t. MATERIALS AND METHODS Preparation of the Chick Embryo Extracts Fertilized eggs of white Leghorn chickens were purchased from Siipikarjanhoitajien liitto ry. (Hameenlinna, Finland) and were incubated at 37 "C in humidified incubators. Embryos were dissected free of surrounding membranes, and various tissues were carefully dissected at "C. After weighing they were immediately frozen in liquid nitrogen and stored at -7 "C. The chick embryo tissues were thawed and homogenized in a cold ( "C) solution consisting of. M NaC,. M glycine,. Triton X- and. M Tris-HCI buffer, adjusted to ph 7.4 at 4 "C (4 ml solution/g tissue). Soft tissues of all embryos, and bone and whole embryos up to the age of 4 days were homogenized with a Teflon-and-glass homogenizer. In other instances the homogenization was carried out with an Ultra-Turrax homogenizer 4 times for 5 s each time. The homogenate was allowed to stand with occasional stirring for h, after which it was centrifuged at 5 x g for 3 min. The supernatant was stored frozen until assayed for prolyl hydroxylase activity, for immunoreactive protein and for protein concentration [22]. Assay qf Prolyl Hydroxy/ase Activity The assay of prolyl hydroxylase activity was carried out using ['4C]proline-labelled protocollagen as a substrate. The reaction was carried out in a final volume of 2. ml, which contained.5-3 p of the chick embryo extract, 5 dis./min of [4C]prolinelabelled protocollagen,.5 mm FeS4,. mm 2-oxoglutarate, 2 mm ascorbic acid,. mg catalase [23],. mm dithiothreitol [24], 2 mg bovine serum albumin [24] and 5 mm Tris-HCI buffer, adjusted to ph 7.8 at 25 'C (see [5,7,25]). The samples were incubated for 3 min at 37 'C and then hydrolyzed in 6 M HCI at 2 'C for 8 h. The samples were evaporated to dryness over a steam bath, dissolved in 4. ml of distilled water and used in 3.5-ml aliquots for the assay of ['4C]hydroxyproline [26]. The [4C]proline-labelled protocollagen substrate was prepared in isolated chick embryo tendon cells as described previously [27] with minor modifications [27aJ and it was stored frozen. The experiments were designed so that embryos of different ages were analyzed on the same day, and the levels of separate experiments were adjusted by using a crude ammonium sulphate enzyme [9] (3-65 X saturation) prepared from chick embryos and stored frozen in aliquots. In order to compare the enzyme activity to the immunoreactive protein it was necessary to convert the synthesis of hydroxyproline to units of enzyme activity. Because the units of prolyl hydroxylase activity have been defined with a saturating concentration of a synthetic peptide substrate [9] and because the assays of prolyl hydroxylase activity in the chick embryo tissue homogenates were carried out with [4C]proline-labelled protocollagen substrate, it was not possible to convert the synthesis of [4C]hydroxyproline directly to units of enzyme activity. Therefore, a conversion factor had to be experimentally determined for each new [4C]protocollagen substrate by incubation with purified enzyme of known specific activity. The units of enzyme activity were further converted to ng active enzyme protein by assuming a specific activity of 8. U/mg for the pure enzyme, as reported previously [9]. It should be noted that the levels of different hydroxylation experiments, even with the same protocollagen substrate preparation, vary by about *lo%. Thus the level of absolute values of percentage of active enzyme compared to total immunoreactive protein is subject to some errors. However, all samples were analyzed together with standard samples in the same incubations, and thus the magnitudes of the changes are not subject to the errors described above. Measurement of the Concentration oj the Inzmunoreac'tive Prolyl Hydroxylase The amount of immunologically reactive prolyl hydroxylase was measured with a specific radioimmunoassay based on the displacement of radioactively labelled prolyl hydroxylase from its antibody by the non-labelled enzyme and on the precipitation of the enzyme. antibody complex with a cellulosebound second antibody [28]. Gel Filtration of Prolyl Hydroxyluse in Clzick Embryo E-xtracts To study further the relationship between the enzyme activity and the immunoreactive protein, the enzyme was extracted as described above. About ml of the 5 xg supernatant from each tissue was applied on a.5 x 9-cm Agarose A-.5 m (Bio-

3 L. Tuderman Table. Prolyl hydroxylase activity and immunorructive protein in 6 g 3.- a, diffrrent tissues on the 6th day of embryonic deveiopment.- 7 g Tissue Enzyme activity Immunoreactive 2 ' 225. r protein. 2 g 3 75 w/mg PU/% ng/mg nglpg I 8$ 5 5 wet extract wet extract._ c 2 25 tissue protein tissuc protein ";.-- Whole embryos % N E Cartilage $ Skin E Heart ' Liver Development time (days) Lung R Fig.. Prolyl hydroxyu.w activirj and immunoreactive protein Kidney during chick embryo dtvelopment in whole chick embryos. The Spleen enzyme activity () is expressed as pu/mg wet tissue weight and Large vessels the immunoreactive enzyme () as ng/mg wet tissue weight Gel, 2-4 mesh) gel filtration column. The column was equilibrated and eluted with a solution containing. M NaCI,. M glycine, pm dithiothreitol and. M Tris-HCl buffer adjusted to ph 7.8 at 4 "C. Fractions of 3.5 ml were collected and enzyme activity and immunoreactive prolyl hydroxylase in the fractions were measured. RESULTS Changes in Prolyl Hydroxyluse Activity and Protein during Chick Embryo Dcvelopmenf In order to study the changes in prolyl hydroxylase activity and protein during chick embryo development, the enzyme activity and immunoreactive protein were measured in the 5Oxg supernatants of chick embryo homogenates between the 4th and 2th days of development. Both the enzyme activity and the immunoreactive protein in extract from whole chick embryos, calculated per unit wet weight, increased until the 6th day of development and declined thereafter (Fig.). Similar changes were seen in all tissues studied (Fig. 2 A- H). The changes in the amount of immunoreactive prolyl hydroxylase usually corresponded to those in the enzyme activity, except in cartilage, skin and whole chick embryos, in which the activity increased in proportion more than the immunoreactive enzyme until the 36th day of development and subsequently declined to a proportionally smaller value than the immunologically reacting protein. The maximal prolyl hydroxylase activity was observed on the 6th day of chick embryo development when the enzyme units were expressed per unit wet weight of the tissues. However, if the values are calculated per unit dry weight of the tissue, the highest activities will be found on the 3th day of development because the content of dry substance in growing chick embryo increases by about 6% during this 3-day period [2]. When the values were expressed per unit wet weight, the highest values of enzyme activity and immunoreactive protein were present in cartilage and skin. The lowest values were found in spleen and large vessels. Expression of the values per pg of extract protein gave a slightly different pattern. The enzyme activity was again highest in cartilage and skin and the immunoreactive protein was also slightly higher than in other tissues. The lowest values were now found in heart and liver. Comparison of the values for enzyme activity and immunoreactive protein in various tissues on the 6th day of development expressed per mg wet weight and per pg extract protein is shown in Table. Ratio qf Active Enzyme to Total Immunoreactive Protein in Dlfrerent Tissues In order to determine the ratio of active prolyl hydroxylase to the total amount of immunologically cross-reacting protein, the units of enzyme activity were converted to ng as described in Methods. Marked differences were found in the percentages of active enzyme between different tissues (Fig. 3). The highest relative activities were observed in cartilage and skin, in which 66 and 59% respectively of the prolyl hydroxylase protein was found in the active form at the time of maximal enzyme activity. The lowest relative activities were found in spleen and large vessels, the amount of active prolyl hydroxylase being maximally 5'%; and 3% respectively of the total immunoreactive enzyme protein, and at the time of minimal relative activity, 8 and 6 "/, respectively. In spleen, large vessels, kidney and lung only minor changes were found in the relative activity during the development of chick embryos (Fig. 3). In heart an initial increase of from 2 % to 4 ';d in the ratio of active prolyl hydroxylase to the immunoreactive enzyme

4 68 Developmental Changes in Prolyl Hydroxylase >,z 3 - B lu 2- :I/ ', lb ; 2 3 lk 5 lk 7 t'8 9 2b Development time (days) 2 i l_i 8-6 E f E. ; 3." 4 : 2 ; 9 5 ; ; '3 '4 ; k b : Development time (days) t -6 2 mo G jy 8 8 h ' q ;,., 3,,,,,,,,, 2,.g2 6 f -6 L m.- (u ! 2 F A )..%._ 2 + m a, 5. c w OA 9 ' ; '3 '4 ; 6 ; 8 9 -lo ;lo Develepment time (days) ' Development time (days) protein in diiferent fissurs during chick emhry dewlopment. The enzyme activity () is Fig. 2. Prolyl hydroxyluse actrvirj, ond ~t~~inut~or~~u(~fil'e expressed as puimg wet tissue weight and the immunoreactive enzyme () as ng/mg wet tissue weight. Tissue studied are cartilage (A), skin (B), heart (C), lung (D), kidney (E), liver (F), spleen (C) and large vessels (H)

5 L. Tuderman 69 7 I I x) 2 Development time (days) Fig. 3. Ratio qf active prolyl hydroxylase to the immunoreactive enzyme in different tissues during chick embryo development. The ratio is expressed as percentages of active enzyme of the total immunoreactive protein. The conversion of enzyme units to ng is calculated as described in the text. () Cartilage, (m) skin, () heart, (A) liver, (3) kidney, (A) lung, ( x) spleen, () large vessels, (+) whole chick embryos was noticed between the 9th and the 3th day, after which small changes in the relative activity were found. A similar increase of activity took place in liver, where 8% of the enzyme was active on the 9th day and 3% on the 2th day. A small decline was noticed in this tissue after the 6th day of development. The largest developmental changes were found in cartilage and skin. The relative amounts of active enzyme increased from 38% in cartilage and from 39% in skin on the 9th day to the maximal values of 66% and 59% on the 6th day, but declined rapidly thereafter to 3 % and 22% respectively on the 2th day of development. The pattern of changes in the relative enzyme activity in whole chick embryos resembled that in cartilage and skin (Fig. 3). Gel Filtration Studies of Prolyl Hydroxyluse in Chick Embryo Tissues To examine further the active and inactive prolyl hydroxylase, the enzymes extracted from cartilage and large vessels of 6-day-old and from cartilage of 2-day-old chick embryos were applied on a gel flltration column as described in Methods. Earlier experiments with the same column had demonstrated that active, tetrametric chick embryo prolyl hydroxylase eluted as a peak in fractions no. 8-2 (not shown). Inactive monomers, prepared by reducing tetramers with mm dithiothreitol [28], eluted as a peak in fractions no (not shown). When the enzyme from cartilage of 6-day-old chick embryos was studied, about 6% of the total immunoreactive enzyme protein was recovered in fractions corresponding to the elution position of the enzyme tetramer and the remainder in fractions. - Fraction numb Fig. 4. Gel Jiltration ojprolyl hydroxylase rxtractedjiom cartilage and large vessels. The enzyme was extracted from the tissues and applied on the gel filtration column as described in Methods. Immunoreactive enzyme measured in the fractions () is expressed as pg/ml and enzyme activity () as mu/ml. Prolyl hydroxylase for gel filtration was extracted from cartilage of 6-day-old (A) and 2-day-old (B) chick embryos and from large vessels of 6-day-old (C) chick embryos corresponding to the elution position of the prolyl hydroxylase monomers (Fig. 4A, ). The enzymic activity was found only in the first peak (Fig.4A, ). In the gel filtration of cartilage extract from 2-day-old embryos only about 3% of the enzyme protein was found in the position of enzyme tetramer and about 7 % was eluted in the monomer peak (Fig. 4B). As an example of a tissue with low prolyl hydroxylase activity, an extract from large vessels was subjected to gel filtration studies (Fig.4C). Only a tiny peak of immunoreactive enzyme was found in the first peak and practically all the enzyme protein was eluted in the second peak. DISCUSSION Prolyl hydroxylase activity has earlier been measured in a number of mammalian tissues (for reviews, see [l -4) and in the developing chick embryo [2]. Remarkable differences have been noticed between the enzyme activities in different tissues and at different stages of chick embryo development [2]. The maximal values of enzyme activity were found in whole chick embryos, cartilage and skin on the 4th day of development, whereas no peak activities were noticed in other tissues [2]. In the present work a peak activity

6 62 Developmental Changes in Prolyl Hydroxylase was noticed on the 6th day of development in all the tissues studied. The 2-day discrepancy in the times of maximal enzymic activity between these studies may be due to differences in developmental stages of the embryos as a result of minor differences in the incubation of the eggs. The presence of protein immunologically crossreacting with prolyl hydroxylase has been reported in several rat [9], mouse [9], chick embryo [28] and human tissues [28], as well as in isolated L-929 cells [29] and in isolated and cultured tendon fibroblasts [7]. In the present study comparison of the enzyme activity to the content of the total immunoreactive protein clearly indicated that there are differences in the relative enzyme activities between different chick embryo tissues and in the same tissue between different stages of chick embryo development. The highest relative activities were observed in cartilage and skin, where about 6 % of prolyl hydroxylase was active at the time of maximal enzyme activity. The lowest relative enzyme activities were found in spleen and large vessels, in which the relative activities at certain stages were below %. In other tissues studied, about 2-4 % of the enzyme was in the active form. When the relative activities in the same tissue were compared during chick embryo development, the largest changes were found in cartilage and skin, in which percentage activity on the 9th day was only about one-half of that on the 6th day, and on the 2th day the value was again one-half to one-third of the maximal value. The nature of the inactive immunologically crossreacting enzyme remains unsolved. Gel filtration studies of the inactive immunoreactive protein from L-929 fibroblasts [29] and mouse skin [9] have indicated a molecular weight corresponding to that of the subunit monomer of the enzyme. The inactive protein from isolated tendon cells was observed to be of about same size [7], and gel filtration studies conducted during the present work also confirm these values. Interestingly, changes in the relative enzyme activity corresponded to changes in the proportions of the relative amounts of enzyme tetramer to total immunoreactive enzyme protein by gel filtration. In cartilage from 6-day-old chick embryos, in which about 6% of prolyl hydroxylase was active, about 6% of enzyme applied to the gel filtration column eluted in the position of the enzyme tetramers and about 4% in the position of the enzyme monomers. By contrast, on the 2th day, when the relative enzyme activity had declined to about 3%, the tetramer peak consisted of about 3% and the monomer peak of about 7% of the enzyme protein. The gel filtration pattern of enzyme protein from large vessels, the tissue with the lowest relative enzyme activity, showed the presence of little enzyme tetramer, and essentially all the enzyme protein eluted in the posi- tion of enzyme monomers. Thus it seems possible that the proportion of the active enzyme to the total immunoreactive protein reflects changes in the relative amounts of the enzyme tetramer and monomers in the tissue. The role of this inactive small-molecular-weight protein in the regulation of prolyl hydroxylase activity is not known. No definite data exist showing that this protein is identical with an intact enzyme subunit. Small structural differences and small changes in the molecular weight, such as the possible presence of short peptide extensions ( pro-subunit ) or partial degradation, cannot be detected by gel filtration. Studies in cultured L-929 fibroblasts have suggested control of prolyl hydroxylase activity by subunit association after addition of micromolar concentrations of ascorbate to the culture medium [4]. However, this ascorbate activation has not been observed in cultured fibroblasts from chick embryo frontal bones [8] or leg tendon [7]. The activation of prolyl hydroxylase in vitvo in sonicates of L-929 cells did not involve subunit association and the activatable form of the enzyme was as large or larger than the active prolyl hydroxylase 6. Attempts to associate and activate the purified enzyme subunits by incubation with the enzyme cofactors have not been successful [27]. A recent work demonstrated a decrease in the relative amount of prolyl hydroxylase tetramer in isolated tendon cells when these are cultured [7]. In freshly isolated cells 4-5 % of the enzyme was in the tetramer form, but in cultured cells only about 5 % was in this form. This result may be interpreted as showing that at the time of high enzyme activity a larger part of the enzyme is in the tetramer form than at the time of low enzyme activity. The present data on developmental changes in the same tissue and on comparison of relative enzyme activities between different tissues are consistent with these findings. It is further of interest that during the first three days after experimental hepatic injury, prolyl hydroxylase activity in the liver increases by about 6 without any change in the amount of immunoreactive enzyme protein [27 a]. Thus one could propose that the inactive small-molecular-weight immunoreactive protein in the tissues either forms a pool of inactive enzyme precursor, which associates to active enzyme tetramer when more rapid collagen synthesis is needed, or alternatively that it represents a pool of degradation products into which the enzyme dissociates more slowly at the time of high enzyme activity. This work was supported in part by a grant from the Medical Research Council of the Academy of Finland. The author is grateful to Professor Kari I. Kivirikko, M. D., for helpful advice and to Mrs Raija Harju and Mrs Lea Torvela for expert technical assistance.

7 L. Tuderman 62 REFERENCES. Bornstein, P. (974) Annu. Rev. Biochem. 43, Cardinale, G. J. & Udenfriend, S. (974) Adv. Enzymol. 4, Kivirikko, K. I. & Risteli, L. (976) Med. Biol. 54, Prockop, D. J., Berg, R. A., Kivirikko, K.. & Uitto, J. (976) in Biochemistry of Collagen (Ramachandran, G. N. & Reddi, A. H., eds) in press, Plenum Publishing Corp. 5. Halme, J. Kivirikko, K. I. & Simons, K. (97) Biochim. Biophys. Acta, 98, PankClainen, M., Aro, H., Simons, K. & Kivirikko, K. I. (97) Biochim. Biophys. Acta, 22, Rhoads, R. E. & Udenfriend, S. (97) Arch. Biochem. Biophys. 3, Berg, R. A. & Prockop, D. J. (973) J. Bid. Chem. 248, Tuderman, L., Kuutti, E.-R. & Kivirikko, K. I. (975) Eur. J. Bioclzem. 52, Kuutti, E.-R., Tuderman, L. & Kivirikko, K. I. (975) Eur. J. Biochem. 57, McGee, J. O D., Langness, U. & Udenfriend, S. (97) Proc. Nut Acad. Sci. U.S.A. 68, Roberts, N. E., McGee,.I. O D. & Udenfriend, S. (973) Connective Ti.rsue Res. 2, Berg, R. A,, Olsen, B. R. & Prockop, D. J. (972) Biochim. Biophys. Acta, 285, Stassen, F. L. H., Cardinale, G. J. & Udenfriend, S. (973) Proc. Not Acad. Sci. U.S. A. 7, Levene, C. J., Aleo, J. J., Prynne, C. J. & Bates, C. J. (974) Biochim. Biophys. Acta, 338, Kuttan, R., Cardinale, G. J. & Udenfriend, S. (975) Biochcm. Biophys. Res. Commun. 64, Kao, W. W.-Y., Berg, R. A. & Prockop, D. J. (975) Biochim. Biophys. Acta, 4, Blanck, T. J. J. & Peterkofsky, B. (975) Arch. Biochem. Biophys. 7, Stassen, F. L. H., Cardinale, G. J., McGee, J. O D. & Udenfriend, S. (974) Arch. Biochem. Biophys. 6, Kivirikko, K. I. (963) Acta Physiol. Scand. Suppl. 29, Halme, J. (969) Biochim. Biophys. Acta, 92, Lowry,. H., Rosebrough, N. J., Farr, A. L. & Randall, R. J. (95) J. Biol. Chem. 93, Kivirikko, K. I. & Prockop, D. J. (967) Proc. Nut Acud. Sci. U.S.A. 57, Rhoads, R. E., Hutton, J. J. & Udenfriend, S. (967) Arch. Biochem. Biophys. 22, Kivirikko, K. I., Kishida, Y., Sakakibara, S. & Prockop, D. J. (972) Biochim. Biophys. Acta, 27, Juva, K. &Prockop, D. J. (966) Anal. Biochem. 5, Berg, R. A. & Prockop, D. J. (973) Biochemistry, 2, a. Risteli, J., Tuderman, L. & Kivirikko, K. I. (976) Biochem. J., in press. 28. Tuderman, L., Kuutti, E.-R. & Kivirikko, K. I. (475) Eur. J. Bioc,hem. 6, McCee, J. O D. & Udenfriend, S. (972) Arch. Biochem. Biophys. 52, L. Tuderman, Oulun Yliopiston Liiaketieteellisen Kemian Laitos, Kajadnintie 52A, SF-922 Oulu 22, Finland

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