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.     

Characterization of procollagen synthesized by matrix-free cells isolated from chick embryo tendons.

The genetic type and molecular structure of the precursor forms of collagen synthesized by matrix-free tendon cells isolated from 17-day old chick embryos were examined by chromatographic and electrophoretic techniques. The [14C]proline-labeled collagenous proteins secreted by the cells resolved on diethylaminoethylcellulose into two peaks, A and B. Both peaks contained type I collagenous proteins since on chromatography on carboxymethylcellulose, after limited pepsin proteolysis, both peaks contained alpha1 and alpha2 chains of collagen in a 2:1 ratio, and cyanogen bromide peptide maps of the 14C-labeled protein in both peaks were similar to cyanogen bromide peptide maps derived from authentic type I collagen. Enzymatic digestion with purified mammalian collagenase demonstrated that the collagen precursor in peak B contained noncollagenous peptide extensions at both the amino- and carboxy-terminal ends of the molecule, while peak A had only carboxy-terminal extension peptides. Although both the amino- and carboxy-terminal extensions incorporated radioactive cystine, only the carboxy-terminal extensions contained interchain disulfide bonds. The carboxy-terminal extensions were also shown to incorporate radioactive tryptophan. Since most of the precursor forms of collagen recovered in the incubation medium chromatographed in peak B, it is concluded that matrix-free tendon cells secrete only type I procollagen with extension peptides at both the amino- and carboxy-terminal ends of the molecule.     

Type I procollagen carboxyl-terminal proteinase from chick embryo tendons. Purification and characterization

Procollagen carboxyl-terminal proteinase, the enzyme which cleaves the carboxyl-terminal propeptides from type I procollagen, was extensively purified in a yield of 25% from pooled culture media of 17-day-old chick embryo tendons using a procedure which involved chromatography on Green A Dye matrix gel, concanavalin A-Sepharose and heparin-Sepharose, and filtration gels of Sephacryl S-300 and S-200. The purified enzyme is a neutral, Ca2+-dependent proteinase which is inhibited by metal chelators, but not by inhibitors for serine and cysteine proteinases. Calcium in a concentration of 5-10 mM is required for optimal activity. The molecular weight of the enzyme was determined to be 97,000-110,000 by gel filtration and by polyacrylamide gel electrophoresis in sodium dodecyl sulfate. Other properties of the carboxyl-terminal proteinase are: 1) the Km for the type I procollagen is 96 nM at pH 7.5 and 35 degrees C; 2) the activation energy for the reaction with type I procollagen is 21,000 cal mol-1; 3) amino acid sequencing of the released carboxyl-terminal propeptide indicated the enzyme specifically cleaves an -Ala-Asp- bond in both the pro-alpha 1(I) and pro-alpha 2(I) chains; 4) the enzyme specifically cleaves the carboxyl-terminal propeptides of a homotrimer of pro-alpha 1(I) chains and type II and III procollagens, but it does not cleave type IV procollagen. The results suggest that the enzyme is involved in the processing of type I procollagen in vivo.     

Declining procollagen mRNA sequences in chick embryo fibroblasts infected with rous sarcoma virus. Correlation with procollagen synthesis.

Chick cells infected with Rous sarcoma virus are characterized by a wide variety of changes known collectively as transformation. Among these are decreases in the level of procollagen biosynthesis and in the level of procollagen mRNA. In this communication, we examine the time course of the decrease in procollagen biosynthesis, as measured by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and collagenase assay, and compare it with the decrease in procollagen mRNA sequences measured by hybridization to a complementary DNA. Procollagen biosynthesis and procollagen mRNA sequences decrease simultaneously after infection. Even the initial decrease in procollagen biosynthesis, therefore, is due to a decline in the level of procollagen mRNA.     

Growth-inhibitory glycopeptides obtained from the cell surface of cultured chick embryo fibroblasts

Cell surface glycopeptides were obtained from cultured chick embryo fibroblasts (CEF) by digestion with Pronase E, and a fraction exerting growth-inhibitory activity on CEF was isolated by high performance gel permeation chromatography. The active fraction, tentatively termed cell surface glycopeptide-2 (CSGP-2), was soluble in 5% trichloroacetic acid (TCA) or 75% ethanol. It inhibited the growth of CEF reversibly at 10-20 micrograms sugar/ml, but did not inhibit BALB/c mouse 3T3, SV40-transformed 3T3, and human diploid cells at similar concentration. The growth-inhibitory activity of CSGP-2 was reduced or lost after digestion with neuraminidase or oxidation with sodium metaperiodate. Cellulose acetate electrophoresis revealed that CSGP-2 was a mixture of sialoglycopeptides. A similar growth inhibitor was also isolated from chicken embryonic tissues.     

Regulation of procollagen synthesis during the development of chick embryo calvaria. Correlation with procollagen mRNA content

During the embryonic development of chick calvaria (membranous cranial bones), the relative rate of procollagen synthesis increased from about 12% of total protein synthesis on Day 10 to about 65% on Day 17. This increase is due to a 1.7-fold increase in the absolute rate of procollagen synthesis and a 3-fold decrease in the synthesis of noncollagenous proteins. The increase in procollagen synthesis is directly proportional to an increase in procollagen mRNA content per cell as measured either by cell-free translation or by hybridization with complementary DNA. The results indicate that translational control of procollagen mRNA does not play a substantial role during calvaria development and that the specialization in the synthesis of this protein is largely due to the loss or inactivation of mRNAs for noncollagenous proteins.     

Effects of spermidine synthase inhibition in cultured chick embryo fibroblasts.

The administration of bis-cyclohexylammonium sulfate (BCHS), a spermidine synthase inhibitor, to in vitro cultures of chick embryo fibroblasts caused a decrease in cellular spermidine levels and an increase in putrescine and spermine. Cell proliferation rate and DNA synthesis were also inhibited. As protein synthesis did not change, it would seem that low levels of cellular spermidine inhibit cell growth depressing DNA synthesis.     

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.     

Procollagen N-proteinase. Properties of the enzyme purified from chick embryo tendons

Procollagen N-proteinase was purified about 3700-fold from chick embryo tendons. Electrophoresis of the protein after iodination and denaturation suggested it was homogeneous. However, the native enzyme could not be examined by gel electrophoresis, and therefore homogeneity of the preparation was not conclusively established. Antibodies to the enzyme completely inhibited activity and gave a single precipitant line by double immuno-diffusion. The Km for a native procollagen substrate was 0.3-0.5 microM. The same protein after denaturation inhibited activity. The enzyme did not cleave type III procollagen from human fibroblasts or a type IV procollagen from a mouse sarcoma. Ca2+ was required for maximal enzymic activity. The data suggested a second metal requirement, but this was not identified. Reducing agents and metal chelators inhibited activity, but there was little if any inhibition from several inhibitors of other neutral metalloproteinases.     

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.     

Depression by hyaluronic acid of glycosaminoglycan synthesis by cultured chick embryo chondrocytes

Proteins synthesized during the preimplantation period of mouse embryogenesis were labeled with radioactive tyrosine and lysine and fractionated by electrophoresis on polyacrylamide disc gels containing sodium dodecyl sulfate. For interstage comparisons and comparisons of the incorporation of different amino acids at the same developmental stages, the embryos were incubated with either ³H- or ¹⁴C-labeled amino acids. The embryos were then combined, and the proteins were isolated and electrophoresed simultaneously. The data were analyzed with a dual isotope computer program and expressed in the form of ${}^{14}\text{C}{}^3\text{H}$ ratios.Approximately 20–25 labeled protein components of apparent molecular weights between 25,000 and 115,000 can be defined, and 5 are most significant quantitatively. Of the latter, there are developmental increases in the rates of synthesis of 3 (with apparent molecular weights of 35,000 to 37,000, 37,000 to 41,000, and 66,000 to 70,000), a decrease in the rate of synthesis of another (53,000 to 57,000), and little change in the last (46,000 to 49,000). Developmental changes in the rates of synthesis of several other components are also demonstrated by the ${}^{14}\text{C}{}^3\text{H}$ incorporation ratios. The relative amounts of the different proteins synthesized by day 3 (early blastocyst) embryos over an 8-hr period remain constant, as does the relative labeling by lysine and tyrosine at each developmental stage examined. Similarly, there is no change in the pattern of the radioactive proteins when day 2 (8–16 cell) embryos are labeled for 2 hr and then incubated for an additional 24 hr. The greatest change in the overall pattern of protein synthesis occurs quite early, between day 1 (2 cell) and day 2, and lesser changes occur at later stages. These findings are in contrast to the major change in the rate of protein synthesis which occurs after day 2.     

Electron microscopic studies of procollagen from cultured human fibroblasts

Tropocollagen was extracted from the extracellular fibers of the cell layers of cultured human fibroblasts and procollagen was isolated from the medium. The tropocollagen precipitated as segment long spacing (SLS) aggregates from an ATP-acetic acid solution, but procollagen formed predominantly fibrous long spacing (FLS) crystallites in the same solution. The procollagen aggregates were distinguished from those of tropocollagen by the presence of globular caps at one (SLS form) or both (FLS form) ends. The globular caps were selectively removed by limited digestion with pepsin. They are thought to be formed from the nonhelical, aminoterminal peptides of the procollagen molecule. These globular peptides coil back 200 Å on the long axis of the molecule and contribute no more than 90 Å to its length. They appear to prevent the molecules from aggregating as native fibers in vitro.     

Studies on the nature of procollagen synthesized by chick embryo polysomes

Analysis of pro-α chains released from chick embryo membrane polysomes indicates that they are not disulfide bonded and have a molecular weight of approximately 120,000. Therefore, disulfide bonding which has been observed between pro-α chains is not essential for their completion and release.