Developmental changes in prolyl hydroxylase activity and protein in chick embryo.

Changes in prolyl hydroxylase activity and immunoreactive protein were studied in various chick embryo tissues during the embryonic development. Both the enzyme activity and the amoung of immunoreactive protein increased till the 16th day of development and declined thereafter in all tissues studied. Comparison of the enzyme activity to the content of the total immuno-reactive 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 60% of the enzyme was active on the 16th day of development and only 20-30% was active on the 20th day of development; the lowest values were observed in spleen and large vessels, in which below 10% of the enzyme protein was in the active form on the 20th day of development Gel filtration studies demonstrated that in cartilage of 16-day-old chick embryos about 60% of the total immunoreactive enzyme in the tissue was present in the form of active prolylhydroxylase tetramer, whereas on the 20th day of development only 30% of the enzyme protein in cartilage was in the tetramer form. By contrast, in large vessels of the 16-day-old chick embryos, essentially all the enzyme was in the form of prolyl hydroxylase monomers.     

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.     

Research on subcellular localization of procollagen proline hydroxylase from chick embryo liver (author's transl)]

Subcellular fractionation by differential centrifugation was used to study protocollagen proline hydroxylase (EC 1.14.11.2) localization from chick embryo liver. The fractions have been characterized by marker enzymes and electron microscopy. By these methods, it was observed that procollagen-proline-hydroxylase is concentrated in the microsomal fraction which is sedimented at 145 000 X g in 250 mM sucrose.     

Evidence for the association of prolyl hydroxylase from chick embryos with membranes through membrane-bound procollagen.

Proline hydroxylase of membrane pellets from tissues of chick embryo was solubilized by several extraction mediums. Three kinds of enzyme linkage with membranes were pointed out: a labile and non specific adsorption, a specific linkage on endogenous procollagen which is linked with the membranes, and a part of membranes themselves. Our results suggest that, in the liver, these three kinds of linkage exist. Whereas, in tibia bones only labile linkages appear by non specific adsorption or through the link with membrane procollagen.     

Lysyl hydroxylase. Further purification and characterization of the enzyme from chick embryos and chick embryo cartilage.

A purification of up to 4000-fold is reported for lysyl hydroxylase (EC 1.14.11.4) from extract of chick-embryo homogenate and one of about 300-fold from extract of chick-embryo cartilage. Multiple forms of the enzyme were observed during purification from whole chick embryos. In gel filtration the elution positions of the two main forms corresponded to average molecular weights of about 580000 and 220000. These two forms could also be clearly separated in hydroxyapatite chromatography. In addition, some enzyme activity was always eluted between the two main peaks both in gel filtration and in hydroxyapatite chromatography. The presence of the two main forms was also observed when purifying enzyme from chick embryo cartilage. Both forms of the enzyme hydroxylated lysine in arginine-rich histone, which does not contain any -X-Lys-Gly- sequence. No difference was found between the enzyme from whole chick embryos and from chick embryo cartilage in this respect. Lysyl hydroxylase was found to have affinity for concanavalin A, indicating the presence of some carbohydrate residues in the enzyme molecule. Lysyl and prolyl hydroxylase activities increased when the chick embryo homogenate was assayed in the presence of lysolecithin. Preincubation of the homogenate either with lysolecithin or with Triton X-100 increased lysyl hydroxylase activity in homogenate, and in the 1500 x g and 150000 x g supernatants, suggesting that the increase in the enzyme activity was due to liberation of the enzyme from the membranes. Divalent cations were found to inhibit the activity of lysyl and prolyl hydroxylases in vitro. An inhibition of about 50% was achieved with 15 mM calcium 60 muM copper and 3 muM zinc concentrations. The mode of inhibition was tested with Cu2+, and was found to be competitive with Fe2+.     

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.     

Use of soluble, collagenous peptides from medium of chick calvaria cultures as substrate for prolyl hydroxylase

Underhydroxylated collagenous proteins accumulate in the media of embryonic chick calvaria cultured in the presence of α,α′-dipyridyl for 24 h. These soluble collagenous proteins, when labeled with radioactive proline, were shown to be a specific, stable, and highly efficient substrate for in vitro measurement of prolyl hydroxylase. The ability of the media proteins to serve as a substrate for prolyl hydroxylase was abolished by digestion with purified bacterial collagenase. This method of substrate preparation provides a soluble, efficient, economical substrate for routine prolyl hydroxylase assays, and permits the accumulation of sufficient quantities of substrate of one specific activity to serve for extended periods of time.     

Hyaluronate-cell interactions during differentiation of chick embryo limb mesoderm

The mechanism of interaction of hyaluronate with the surface of cells from embryonic chick limbs was studied using cell cultures of mesoderm from various developmental stages. The mode of interaction of hyaluronate with the cell surface changed at the onset of mesodermal cell condensation prior to differentiation of cartilage and muscle. At this time hyaluronate binding sites appeared on the cells and continued to be present on differentiated chondrocytes but not on myotubes. Direct measurement of hyaluronate binding was made using stage 24 mesodermal cells and membranes isolated from cells derived from various limb stages. The stage 24 cells and membranes from stage 22, 24, and 26 cells exhibited hyaluronate binding, but not membranes from stage 19 mesoderm cultures. At stage 38, membranes from chondrocyte cultures exhibited the highest hyaluronate binding, and membranes from myoblasts and fibroblasts intermediate binding, whereas membranes from myotube-enriched cultures lacked binding activity. No significant competition of hyaluronate binding by chondroitin sulfate was observed. Occupied hyaluronate binding sites were measured by the displacement of radiolabeled cell surface hyaluronate with exogenous, unlabeled hyaluronate. Very little hyaluronate was displaced from mesodermal cells derived from the youngest embryos, namely, stage 19 or stage 20-21. However, greater than 50% of cell surface hyaluronate was displaced from stage 22 and 24 mesodermal cells. The addition of exogenous hyaluronate to stage 26 mesoderm, the stage of onset of cartilage differentiation, and to stage 38 chondrocytes resulted in displacement of large proportions of both hyaluronate and chondroitin sulfate. Addition of exogenous chondroitin sulfate did not cause displacement of significant amounts of cell surface hyaluronate or chondroitin sulfate. These results indicate the presence and developmental modulation of specific binding sites for hyaluronate on limb cells during their differentiation.     

Structural studies on prolyl hydroxylase. Conformation from circular dichroism spectroscopy

The circular dichroism spectra of purified prolyl hydroxylase [EC 1.14.11.12] in the native and heat-denatured states were obtained at pH 7.8. Analysis of the far-uv spectrum of the native enzyme gave an estimate of 40% alpha-helix, 40% beta-structure and 20% random coil. The near-uv spectrum contained several negative dichroic bands that can be attributed to phenylalanyl, tyrosyl and tryptophyl residues situated in an asymmetric environment in the protein. These bands were not seen in the enzyme denatured by heat. The denaturation was monitored by changes in the alpha-helical content as well as the activity of the enzyme as a function of temperature. The normalized transition profiles with respect to the change in helical content and the loss of enzyme activity were coincidental, indicating the involvement of the alpha-helical segments in maintaining the enzyme activity.     

Glutamine synthetase localization in cortisol-induced chick embryo retinas

We report here for the first time, in chick retina, Muller cell localization of glutamine synthetase (GS) activity by an immunohistochemical technique, in agreement with previous reports of glial localization of this enzyme in rat brain and retina. Age-dependent changes in the endogenous enzyme activity as well as cortisol-induced changes in GS activity, both in ovo and in vitro, measured biochemically, reflect the changes observed by staining.     

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.     

Isolation and characterization of prolyl hydroxylase from Chlamydomonas reinhardii

Prolyl hydroxylase, which is responsible for the hydroxylation of peptidyl proline residues, has been isolated and purified from the green alga Chlamydomonas reinhardii. The enzyme, which appears to be loosely associated with microsomal membranes, was released into solution by sonication in the presence of detergent. Purification was achieved by ion-exchange chromatography followed by affinity chromatography using the immobilized substrate poly-L-proline. Apart from its differing substrate specificity the enzyme appears to possess similar molecular characteristics to prolyl hydroxylase isolated from animal tissues: the active enzyme is a tetramer of about 240–250 kDa and nonidentical monomers of 65 and 60 kDa. The monomers are capsule shaped having a dimension of 12×7 nm.Abbreviations Da dalton - DEAE diethylaminoethyl - DTT dithiothreitol - Hepes 4-(2-hydroxymethyl)-1-piperazine ethanesulfonic acid - -KGA -ketoglutarate - PAGE polyacrylamide gel electrophoresis - SDS sodium dodecyl sulfate     

Subcellular localization of nerve growth factor receptors. Thirteen-day chick embryo brain.

The subcellular location of the specific binding sites of nerve growth factor (NGF) as judged by binding of the 125I-labeled protein in 13-day chick embryo brain, has been examined. The homogenized tissue was separated into four fractions, P1, P2, P3, and S, by differential centrifugation. Fraction P2, which contained the majority of the specific 125I-NGF binding, was further separated by discontinuous sucrose density gradient centrifugation into three fractions. Fraction B contains many synaptosome-like structures, which, when derived from adult brain, result from the shearing off and resealing of synaptic terminals. This fraction contained 65% of the specific 125I-NGF binding of P2. Following hypoosmotic lysis by water, Fraction B was separated into seven fractions, O, D, E, F, G, H, and I, by discontinuous sucrose density gradient centrifugation. The specific 125I-NGF binding was localized with the denser fractions, G, H, and I, with about a 10-fold purification as compared to the original homogenate. However, only 65% of the binding of Fraction B was found in the sum of the tertiary fractions, indicating that some loss of specific binding accompanied the lysis. By means of marker enzymes and macromolecules, as well as electron microscopy, it was determined that the distribution of cellular components of embryonic tissue in this fractionation technique is very similar to that observed for adult brain tissue. Thus, the properties of the NGF receptors determined in whole brain, which are remarkably similar to those found in peripheral neurons, are the properties of the receptors that appear to be located in the developing synaptosomal structures.     

Stimulation of prolyl hydroxylase activity by chelating agents.

The activity of purified prolyl hydroxylase was enhanced several fold by addition of some chelating agents to the assay medium. Chelating agents could be classified into three groups. The chelating agents of Group I such as α, α′-dipyridyl were inactive until they reached equimolar concentration with ferrous ion in the assay mixture. The Group II agents, EDTA, diethylenetriaminepentaacetic acid, etc., stimulated the enzymatic activity 1.5- to 3-fold at equimolar concentration with ferrous ion. But the agents of both groups precipitously inhibited the enzymatic activity at concentrations greater than ferrous ion. On the other hand, Group III chelating agents, such as nitrilotriacetic acid, enhanced the enzymatic activity 5- to 10-fold at concentrations greater than ferrous ion. Nucleoside triphosphates, which also stimulate the enzymatic activity several fold and whose optimal concentrations are 1–3 × 10^?m, may be analogous to nitrilotriacetic acid of Group III.