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PubMed2000

The regulatory expression of procollagen COOH-terminal proteinase enhancer in the proliferation of vascular smooth muscle cells.

Intimal hyperplasia following arterial endothelial denudation results in large part from the proliferation of vascular smooth muscle cells (SMCs) and matrix accumulation. Procollagen COOH-terminal proteinase enhancer (PCPE) binds procollagen COOH-propeptides and potentiates procollagen COOH-proteinase activity to cleave COOH-propeptides of procollagens I-III. Here we report the enhanced expression of PCPE in cultured SMCs and in intimal thickening induced by arterial injury. The levels of PCPE mRNA in parallel with the level of p21(Cip1) mRNA, as a negative regulator of cellular proliferation, increased under serum deprivation or reduced cellular proliferation in cultured SMCs. In contrast, rapidly proliferating cells show the decreased levels of PCPE mRNA. In vivo, the marked induction of PCPE in injured rat arteries occurred at 14 days after endothelial denudation. The induced expression levels of PCPE as well as p21(Cip1) were maintained until 42 days, although cyclin E expression declined. Furthermore, transforming growth factor beta1 (TGF-beta1), an important regulator of cellular proliferation in atheroma, increased the levels of the PCPE mRNA in cultured SMCs. Thus, the regulatory expression of PCPE dependent on cellular proliferation, and particularly contact inhibition, may play a key role in the proliferation of SMCs and matrix production during the process of atheroma formation.

Animals↗
PubMed2002

Site-specific interaction of bone morphogenetic protein 2 with procollagen II.

Bone morphogenetic proteins (BMPs) play a critical role in embryo development, organogenesis, and regeneration of damaged tissues. Biological activity of BMPs depends on their local concentration, which is regulated by intracellular enzymatic processing of pro-BMPs, and then the binding of secreted BMPs to antagonizing extracellular proteins. It has been suggested that BMPs interact with structural proteins of the extracellular matrix, but this process is poorly understood. To study interactions of BMPs with fibrillar collagens in detail we expressed recombinant procollagen II variants in which specific domains that correspond to the D-periods were deleted. Subsequently, the procollagen II variants were used in biosensor and immuno-precipitation binding assays to map the regions of procollagen II with a high affinity for the BMP-2. Our data suggest that interaction of BMP-2 with procollagen II is site-specific, and that the high-affinity binding site is located in the D4-period of the collagen triple helix. We hypothesize that the binding of BMP-2 to collagen II reflects a general mechanism of interaction between the fibrillar collagens and morphogens that belong to the transforming growth factor (TGF)-beta superfamily.

Binding Sites↗
PubMed1973

Secretion and extracellular processing of procollagen by cultured human fibroblasts.

Cultures of human diploid fibroblasts were labeled with radioactive proline and glycine, and the precursor of collagen (procollagen) in cells and medium was characterized by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. A covalently assembled molecule with the composition (pro alpha1)(2).pro alpha2 (approximate molecular weight, 360,000) appeared intracellularly soon after synthesis of the constituent chains, and could be detected in the medium after 60 min of labeling. The molecule was stabilized by disulfide bonds between cysteine residues in the amino-terminal procollagen peptide sequences of the three chains. Collagenase digested the molecule to peptides of 30,000 molecular weight or less. Limited digestion with pepsin excised nonhelical procollagen peptides, yielding native, triple-helical tropocollagen. Pulse-chase experiments indicated that a peptidase in the medium sequentially excised the nonhelical peptides from the molecule, generating tropocollagen molecules that aggregated as fibers in the cell layer. The excised, nonhelical procollagen peptides contained little or no proline or glycine. Intramolecular bonds of the lysyl aldehyde type were not detected in the secreted molecule, as reduction of the medium always resulted in quantitative recovery of free pro alpha chains in dodecyl sulfate-urea. Lysyl-derived, covalent bonds appeared to form between tropocollagen molecules aggregating in the cell layer. We suggest the term "pro-tropocollagen" for the assembled, secreted precursor of collagen.

Carbon Isotopes↗
PubMed1974

Ferritin-conjugated antibodies used for labeling of organelles involved in the cellular synthesis and transport of procollagen.

An improved procedure was used to conjugate ferritin to antibodies specific for the NH(2)-terminal extensions on the precursor form of collagen known as procollagen. The ferritin-antibody conjugates were then used to determine the localization of procollagen in fibroblasts isolated from chick-embryo tendons. Procollagen was found in the cisternae of the endoplasmic reticulum, indicating that the protein passes into this compartment early in its biosynthesis. Specific labeling of large Golgi vacuoles was also observed, suggesting that this compartment is also involved in the secretory process. When cells were incubated with colchicine so that the secretion of procollagen was delayed, there was an increase of large, smooth-surfaced vacuoles in the cells and these vacuoles were labeled with the ferritin-antibody conjugate.

Animals↗
PubMed1975

Interchain disulfide bonds in procollagen are located in a large nontriple-helical COOH-terminal domain.

Tadpole collagenase (EC 3.4.24.3) cleaved chick cranial bone procollagen into two triple-stranded fragments, PCA and PCB. Only PCB, with an estimated molecular weight of about 60,000 for each component chain after reduction, was found to contain interchain disulfide bonds. The analogous cleavage of collagen is known to produce a large NH2-terminal fragment with a molecular weight of 70,000 for each chain and a small COOH-terminal fragment containing chains of about 25,000 molecular weight. Since PCB was too small to represent the product NH2-terminal to the site of collagenase cleavage, localization of interchain disulfide bonds to a COOH-terminal domain in procollagen was indicated. This assignment was conformed by Dintzis-type short-term labeling experiments. Procollagen obtained by acid extraction of bone lacked the COOH-terminal disulfide-bonded domain. The findings support a model for procollagen consisting of three proalpha chains each containing nonhelical NH2-terminal extensions of 20,000 molecular weight and COOH-terminal extensions of about 35,000 molecular weight, the latter linked by interchani disulfide bonds.

Animals↗
PubMed2005

Procollagen type I gene expression and cell proliferation are increased in lipodermatosclerosis.

BACKGROUND: Lipodermatosclerosis (LDS) is characterized by a hardening and hyperpigmentation of lower leg skin as a consequence of chronic venous insufficiency. The degree of skin hardening or fibrosis associated with LDS is proposed to relate directly to skin breakdown and venous ulcer formation as well as to a subsequent delay in ulcer healing. OBJECTIVES: To determine whether elevated procollagen type I gene expression and increased cell proliferation are responsible for the fibrotic changes associated with LDS. METHODS: Skin biopsies were obtained from the legs of patients with varying degrees of chronic venous disease and were assessed for procollagen gene expression by in-situ hybridization and for cell proliferation by immunolocalization of proliferating cell nuclear antigen. RESULTS: The number of cells expressing procollagen type I mRNA (COL1A1) was significantly higher in the dermis of LDS-affected skin compared with samples from the other patient groups. In addition, there was a significant increase in the number of dermal fibroblasts undergoing proliferation in both LDS samples and skin samples prior to LDS changes compared with control samples. However, there was no significant difference in level of inflammation in biopsy samples between patient classes. CONCLUSIONS: These results suggest that enhanced cell proliferation and procollagen gene expression are both involved in LDS development. Furthermore, fibrotic changes may occur in the absence of, or subsequent to, any significant inflammatory response, indicating that additional profibrotic factors produced in the skin as a consequence of chronic venous insufficiency may play a role in LDS formation.

Aged↗
PubMed2006

Angiotensin II-mediated oxidative stress and procollagen-1 expression in cardiac fibroblasts: blockade by pravastatin and pioglitazone.

Angiotensin II (ANG II), a product of renin-angiotensin system activation, enhances collagen synthesis, which is a key event in cardiac remodeling after myocardial infarction. Inhibition of cardiac remodeling is now a target of multiple therapies, including 3-hydroxy-3-methylglutaryl coenzyme A reductase inhibitors, commonly known as statins, and peroxisome proliferator-activated receptor-gamma (PPAR-gamma) ligands. We examined the potential antifibrotic effect of the combination of a statin (pravastatin) and a PPAR-gamma ligand (pioglitazone) in ANG II-treated mouse cardiac fibroblasts. ANG II treatment induced procollagen-1 expression, which was inhibited by pravastatin and pioglitazone in a dose-dependent fashion. Pretreatment of fibroblasts with low therapeutic concentrations of either pravastatin (0.1 microM) or pioglitazone (5 microM) only slightly decreased ANG II-induced NADPH oxidase expression, superoxide anion production, and procollagen-1 expression; however, the combination of pravastatin and pioglitazone markedly modulated these effects of ANG II. The combination also blocked ANG II-mediated p38 MAPK and p44/42 MAPK activation. Electrophoretic mobility shift assay showed that ANG II activated transcription factors NF-kappaB and activator protein-1 (AP-1). Although pravastatin and pioglitazone alone had a variable effect on NF-kappaB and AP-1 activation, their combination exerted a potent inhibitory effect on the activation of both NF-kappaB and AP-1. The effects of pravastatin and pioglitazone in combination on superoxide generation and procollagen-1 expression mimicked those of alpha-tocopherol and gamma-tocopherol, two potent antioxidants. Thus it appears that there is a positive interaction between pravastatin and pioglitazone in modulating ANG II-mediated oxidative stress, inhibiting MAPK activation, and procollagen-1 expression.

Angiotensin II↗
PubMed2004

Transcriptional regulation of human alpha1(I) procollagen gene in dermal fibroblasts.

AIM: To clarify the fractional activity of promoters from human alpha1(I) procollagen gene, the interaction between cis-elements and consensus DNA-binding proteins responsible for high promoter activity, and the potential application of promoter competitors as well as cytokines for antifibrogenesis. METHODS: Sequence between 2483 bp upstream of the start of transcription and 42 bp downstream of this site was investigated with serial 5'-deletion. The 5'-deleted promoters recombined with chloramphenicol acetyltransferase (CAT) as reporter gene were transiently transfected to human dermal fibroblasts. Electrophoretic mobility shift assay was performed to show the DNA-protein binding capacity of the promoter sequence. Cytokines including tumor necrosis factor alpha (TNFalpha) and interferons (INFs) were added to the culture medium of transiently transfected fibroblasts. Competitor DNA for the binding sites of Sp-1, Ap-1 and NF-1 was individually cotransfected transiently in order to block the promoter-driven CAT expression. RESULTS: Sequences of -2483 to +42 bp and -268 to +42 bp of human alpha1(I) procollagen gene had high activity as promoters. Binding sites for Ap-1 and Sp-1 were among the cis-regulatory elements recognizing consensus transcription factors responsible for basal promoter activity of sequence -268 to +42 bp. TNFalpha, IFNalpha, IFNbeta showed inhibitory effects on sequence -2 483 to +42 bp as promoter with activities 43%, 62% and 60% of control respectively. Transfection of the promoter competitors could reverse the promoter activity of -268 to +42 bp 40-60%. CONCLUSION: Sequences of -2 483 to +42 bp recombined with reporter gene provide an ideal construction for transcriptional study of alpha1(I) procollagen gene. The anti-collagen capacity of TNFalpha and IFNs is associated with their transcriptional regulation. Ap-1 and Sp-1 mediate the basal transcriptional activation of human alpha1(I) procollagen gene in dermal fibroblasts. Competitors for highly active promoters might be a novel potential candidate in fibrotic blockade.

Cells, Cultured↗
PubMed2002

Alternative splicing of type II procollagen gene in the dedifferentiation of rat epiphyseal chondrocytes serially cultured in monolayer.

Type II procollagen is synthesized in two forms (IIA and IIB), generated by the alternative splicing of its precursor mRNA. The purpose of this study was to determine whether alternative splicing of type II procollagen pre-mRNA occurs during the dedifferentiation of chondrocytes in monolayer culture. We serially cultured rat epiphyseal chondrocytes for eight weeks in monolayer, and investigated the expression patterns of the two forms of type II procollagen transcripts according to the duration of the culture period. Reverse transcription-polymerase chain reaction and northern analysis revealed that both transcripts peaked on the day of cell isolation, but the expression level of IIB form was much higher than that of IIA form. The expression of IIA form decreased gradually, whereas the expression of IIB form decreased so rapidly that the ratio of type IIB and type IIA transcripts was reversed in the second week. In situ hybridization showed that all cells expressed type IIA mRNA throughout the culture period. The signal intensity for type IIA transcript decreased gradually in accordance with the results of RT-PCR and northern analysis. In situ hybridization of type IIB mRNA showed different results from in situ hybridization of type IIA mRNA. Like type IIA transcript, the freshly isolated chondrocytes and the cells in the first week showed positive signals for type IIB transcript. However, in the second week, positive signal was detected neither in polygonal cells nor large fibroblastic cells. Only a few cells of small size were weakly positive. From the third to the eighth week, no positive signal was detected. These findings indicate that alternative splicing of type II procollagen gene does occur and is regulated during the dedifferentiation of chondrocytes in monolayer culture.

Alternative Splicing↗
PubMed1975

Inhibition of the assembly and secretion of procollagen by incorporation of a threonine analogue, hydroxynorvaline.

Hydroxynorvaline (DL-alpha-amino-beta-hydroxyvaleric acid) was shown to competitively inhibit the activation of threonine and valine when tested with tRNA and synthetases prepared from whole chick embryos. However, the hydroxynorvaline was transferred only to threonyl-tRNA and not valyl-tRNA. The hydroxynorvaline had no effect when tested with other amino acids. The Km for threonine was 25 muM and the Ki for hydroxynorvaline was 181 muM. When fibroblasts from embryonic chick tendons were incubated with [3H]threonine and increasing concentrations of hydroxynorvaline, there was a progressive decrease in the incorporation of [3H]threonine so that 1 mM hydroxynorvaline the incorporation into nondialyzable protein was 26% of the control value. A much smaller decrease in the incorporation of other radioactive amino acids was observed. When the cells were incubated hith [14C]proline and 1 mM hydroxynorvaline, the labeled procollagen containing hydroxynorvaline accumulated intracellularly and very little was secreted. Control experiments demonstrated that free hydroxynorvaline did not inhibit the secretion of unsubstituted procollagen. Although the individual pro alpha chains containing hydroxynorvaline were of normal molecular weight (125,000) and hydroxyproline content, only about 50% of this intracellularly retained procollagen was triple helical within the cell as 37 degrees as measured by sensitivity to pepsin digestion. Also only approximately 50% of the pro alpha chains were disulfide-linked to form triple stranded molecules as compared to greater than 85% linkage in unsubstituted procollagen. We postulate that incorporation of hydroxynorvaline alters the conformation of the propeptide extension sufficiently so that: (a) normal assembly of disulfide-linked, triple helical molecules is reduced and (b) assembled triple helical molecules are not properly recognized by the secretory mechanism.

Amino Acyl-tRNA Synthetases↗
PubMed2002

[Basic fibroblast growth factor inhibits promoter activities of human alpha 1(I) procollagen gene induced by transforming growth factor-beta 1].

OBJECTIVE: To investigate the effects of basic fibroblast growth factor (bFGF) on the promoter activities of human alpha 1(I) procollagen gene and the interaction between bFGF and transforming growth factor-beta 1 (TGF-beta 1). METHODS: Fibroblasts of the hypertrophic scar and normal skin from a 3-year-old patient were primarily cultured and subcultured in vitro. Both of the fibroblasts were transient transfected with phCOL 2.5, containing -2.5 kb of 5'f lank sequence of human alpha 1(I) procollagen gene and CAT reporter gene by FuGENE transfection reagent; and treated thereafter by 16 ng/ml bFGF, 2 ng/ml TGF-beta 1 and 16 ng/ml bFGF + 2 ng/ml TGF beta 1 for 24 hours. The relative CAT expression values were determined by CAT-ELISA. RESULTS: TGF-beta 1 strongly induced the CAT expression level, however, bFGF not only inhibited the basal CAT expression but also reduced the CAT expression up-regulated by TGF-beta 1 in normal skin and hypertrophic scar fibroblasts (P < 0.05). CONCLUSION: bFGF can reduce the promoter activities of human alpha 1(I) procollagen gene and antagonize the role of TGF-beta 1 in up-regulating the promoter activities of human alpha 1(I) procollagen gene in normal skin and hyertrophic scar fibroblasts.

Cells, Cultured↗
PubMed2006

Inhibitory effects of polymyxin B on NF-kappaB activation and expression of procollagen I, III in pre-eclamptic umbilical artery smooth muscle cells.

BACKGROUND: It has been identified that in patients with pre-eclampsia, several factors were released into the serum, which can regulate the proliferation of vascular smooth muscle cells and stimulate the synthesis of extracellular matrix (ECM). However, the signal transduction pathway of the growth factors and cytokines synthesized by endothelial cells leading to the proliferation and ECM synthesis of human umbilical arterial smooth muscle cell (HUASMC) is unknown. The aim of this study was to research the effects of protein kinase C (PKC) inhibitor, polymyxin B (PMB), on the proliferation, nuclear factor-kappa B (NF-kappaB) activation, and expression of procollagen I and III mRNA in HUASMC cultured with pre-eclamptic umbilical sera. METHODS: Normal HUASMCs were treated with pre-eclamptic umbilical serum (pre-eclamptic group), pre- eclamptic umbilical serum plus PMB (PMB group), or normal umbilical serum (normal group). The expression of I-kappaB, NF-kappaB was detected by Western blotting after the HUASMC was incubated for 2 hours. The proliferation of HUASMC was evaluated by MTT, the cell cycle was analyzed by flow cytomerty, and the expression of procollagen I, III mRNA was measured by RT-PCR assay after HUASMC was incubated for 48 hours. ANOVA was used for statistical analysis. RESULTS: Umbilical sera in pre-eclampsia could stimulate the proliferation, the DNA synthesis, the transition of G(0) + G(1) phase or G(2)/M phase to S and phase, the activation of NF-kappaB, and the expression of procollagen I mRNA of HUASMC as compared with normal umbilical sera (P < 0.01). PMB could inhibit the proliferation, the DNA synthesis, the transition of G(0) + G(1) or G(2)/M phase phase to S phase, the activation of NF-kappaB, and the expressions of procollagen I mRNA of HUASMC stimulated by umbilical sera in pre-eclampsia (P < 0.01). CONCLUSION: PKC-NF-kappaB signal transduction pathway may play a key role in SMC phenotype modulation, which is more important in the pathogenesis of placental blood vessel in pre-eclampsia.

Cell Cycle↗
PubMed2006

[Effect of methylprednisolone on procollagen type III of rats with acute lung injury].

OBJECTIVE: To observe the effect of methylprednisolone on procollagen type III of rats with acute lung injury (ALI) induced by endotoxin. METHODS: Forty-five rats were randomly divided into three groups: endotoxin group, endotoxin plus methylprednisolone group, and control group, with 15 rats in each group. The model of ALI in rat was reproduced by intravenous injection of endotoxin in the dose of 6 mg/kg. On 1, 3, 14 days after the onset of ALI in rats, total cell number, classification of polymorphonuclear neutrophil (PMN), macrophages and procollagen type III in serum and bronchoalveolar lavage fluid (BALF) were determined by radioimmunology. RESULTS: Total cell number, classification of PMN and macrophages in BALF as well as procollagen type III level in serum and BALF were obviously increased in endotoxin group on the first day, then decreased gradually (all P<0.01). Methylprednisolone inhibited the increase of the above-mentioned parameters (P<0.05 or P<0.01). CONCLUSION: Methylprednisolone can inhibit the increase of the procollagen type III levels in serum and BALF in rats with acute lung injury induced by endotoxin.

Acute Lung Injury↗
PubMed1999

Procollagen folding and assembly: the role of endoplasmic reticulum enzymes and molecular chaperones.

Procollagen assembly occurs within the endoplasmic reticulum, where the C-propeptide domains of three polypeptide alpha-chains fold individually, and then interact and trimerise to initiate folding of the triple helical region. This highly complex folding and assembly pathway requires the co-ordinated action of a large number of endoplasmic reticulum-resident enzymes and molecular chaperones. Disease-causing mutations in the procollagens disturb folding and assembly and lead to prolonged interactions with molecular chaperones, retention in the endoplasmic reticulum, and intracellular degradation. This review focuses predominantly on prolyl 1-hydroxylase, an essential collagen modifying enzyme, and HSP47, a collagen-specific binding protein, and their proposed roles as molecular chaperones involved in fibrillar procollagen folding and assembly, quality control, and secretion.

Collagen↗
PubMed1981

Collagen biosynthesis by human skin fibroblasts. III. The effects of ascorbic acid on procollagen production and prolyl hydroxylase activity.

Human skin fibroblasts were cultured under conditions optimized for collagen synthesis, and the effects of ascorbic acid on procollagen production, proline hydroxylation and the activity of prolyl hydroxylase were examined in cultures. the results indicated that addition of ascorbic acid to confluent monolayer cultures of adult human skin fibroblasts markedly increased the amount of [3H]hydroxyproline synthesized. Ascorbic acid, however, did not increase the synthesis of 3H-labeled collagenous polypeptides assayed independently of hydroxylation of proline residues, nor did it affect the amount of prolyl hydroxylase detectable by an in vitro enzyme assay. Also long-term cultures of the cells or initiation of fibroblast cultures in the presence of ascorbic acid did not lead to an apparent selection of a cell population which might be abnormally responsive to ascorbic acid. Thus, ascorbic acid appears to have one primary action on the synthesis of procollagen by cultured human skin fibroblasts: it is necessary for synthesis of hydroxyproline, and consequently for proper triple helix formation and secretion of procollagen.

Adult↗
PubMed1986

Iron-containing metallocenes as active site directed inhibitors of the proteinase that cleaves the NH2-terminal propeptides from type I procollagen.

Derivatives of ferrocene (dicyclopentadienyliron) (Fc) were examined as active site directed inhibitors of type I procollagen N-proteinase, the enzyme that cleaves the NH2-terminal propeptides from type I procollagen. The compounds were shown here to be reversible, competitive inhibitors of the enzyme. The effectiveness of the Fc inhibitors varied with modification of the cyclopentadienyl (cp) rings. The monocarboxylic acid (I) and the 1,1'-dicarboxylic acid (II) derivatives of Fc inhibited 50% of the enzymic activity (I50) at concentrations of 1.0 and 0.5 mM, respectively. The Ki values were 0.3 mM for both I and II. Derivatization of the carbonyl alpha to the cp ring of compound I (FcCOCH2CH2COOH, III) increased the inhibitory activity (I50 = 0.100 mM; Ki = 0.065 mM). Removal of the carbonyl alpha to the cp ring of III did not improve inhibitory activity: FcCH2CH2COOH, I50 = 2 mM; FcCH = CHCOOH, I50 = 1.5 mM. The active inhibitory species apparently contained iron in the 3+ valence state since two ferrocenium derivatives were very effective inhibitors: ferrocenium tetrachloroferrate, IV (I50 = 0.030 mM; Ki = 0.004 mM), and carboxyferrocenium hexafluorophosphate, V (I50 less than 0.1 mM; Ki less than 0.05 mM). In addition, reduction of III with ascorbic acid abolished its inhibitory activity. Compounds I and III stabilized the enzyme to heat denaturation in the absence of exogenous calcium; compound IV did not stabilize the enzyme. Further observations indicated that Fc derivatives were specific inhibitors of procollagen N-proteinase.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1988

Processive action of the two peptide binding sites of prolyl 4-hydroxylase in the hydroxylation of procollagen.

The number of peptide binding sites of prolyl 4-hydroxylase was manipulated with the peptide photoaffinity label N-(4-azido-2-nitrophenyl)glycyl-(Pro-Pro-Gly)5, and the effect on hydroxylation of the relatively short peptide substrate (Pro-Pro-Gly)5 and of the long natural substrate procollagen was studied. With (Pro-Pro-Gly)5 as a substrate, a linear relation was found between enzyme activity and the amount of covalently bound photoaffinity label, approximately 50% inactivation being reached at 1 mol of label/mol of enzyme. No difference in Km value for (Pro-Pro-Gly)5 was detected between unlabeled and partially labeled enzyme preparations. These results indicate that enzyme molecules with only one free active site hydroxylated the synthetic substrate (Pro-Pro-Gly)5 with the same Km and at half the rate of native enzyme. In contrast, with procollagen as a substrate a 5-10-fold increase in Km was found with the fraction of enzyme containing only one free active site, as compared to the Km for procollagen with nonlabeled enzyme. This finding is explained by an enzyme-kinetic model based on a processive action of the two peptide substrate binding sites of prolyl 4-hydroxylase, preventing dissociation of the enzyme-substrate complex between successive hydroxylations of a long peptide with multiple substrate sites. Such a mechanism leads to a low Km for a long peptide by overcoming the diffusional constraints on the rate of association between the enzyme and the individual substrate sites.

Affinity Labels↗
PubMed1979

Translation of embryonic-chick tendon procollagen messenger ribonucleic acid in two cell-free protein-synthesizing systems.

Embryonic-chick tendon poly(A)-containing RNA was translated in the wheat-germ and mRNA-dependent rabbit reticulocyte-lysate systems. The ability of each system to synthesize polypeptides similar to pro-alpha chains of collagen was tested on the bases of electrophoretic mobility and susceptibility to highly purified bacterial collagenase. Very small amounts of polypeptides in the size range of pro-alpha chains were synthesized in the wheat-germ system, whereas efficient synthesis of two polypeptides similar to pro-alpha1 and pro-alpha2 chains was achieved in the reticulocyte lysate. The collagenous nature of the major high-molecular-weight products synthesized was demonstrated by their susceptibility to collagenase and ability to act as a substrate for purified collagen proline hydroxylase. Determinations of the relative amounts of these translation products suggest that the 2:1 ratio of pro-alpha1 and pro-alpha2 chains found in type I procollagen is reflected in proportional amounts of translatable mRNA for pro-alpha1 and pro-alpha2 chains. Comparisons of the electrophoretic mobilities of hydroxylated and unhydroxylated reticulocyte-lysate translation products were made with appropriate standards of hydroxylated and unhydroxylated procollagen polypeptides. The results suggest that, in common with a number of secreted proteins, procollagen is synthesized as pre-pro molecules consistent with the ;Signal Hypothesis'.

Animals↗