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1.
D F Counts 《Biochimica et biophysica acta》1980,626(1):208-217
Collagen was isolated by acetic acid extraction in the presence of protease inhibitors and also by pepsin digestion from the skins of dogs affected with the Ehlers-Danlos syndrome and the skins on non-affected dogs. The collagen preparations isolated by acetic acid extraction from the Ehlers-Danlos syndrome-affected dog skin contained a greater proportion of alpha-chains than the collagen preparations from the normal dog skin. When the collagen from the Ehlers-Danlos syndrome-affected dog skin was reduced with NaBH4 before heat denaturation, and electrophoresis, there was a greater proportion of beta-chains present. The collagen isolated from the normal dog skin was not affected by the NaBH4 reduction. Collagen preparations isolated by pepsin digestion from both the Ehlers-Danlos syndrome-affected dog skin and the non-affected dog skin contained the same quantity of alpha- and beta-chains. In addition, collagen from both affected and non-affected dog skins isolated by pepsin digestion contained 10-11% type III collagen as determined by the interrupted sodium dodecyl sulfate polyacrylamide gel electrophoresis method. Pepsin digestion of the collagens isolated by acetic acid extraction in the presence of protease inhibitors from the skins of affected and non-affected dogs eliminated the differences between the alpha:beta ratios of the affected and non-affected collagen preparations. 相似文献
2.
海蜇胶原蛋白的提取及纯化(英文) 总被引:1,自引:0,他引:1
通过胃蛋白酶的限制性酶切、选择性盐析及阳离子交换柱的层析作用,低温从海蜇中胶层提取可得到胃蛋白酶促溶且去除端肽(telopeptide)的胶原蛋白(PSC)。紫外-可见扫描测得PSC的最大吸收峰位于235nm。凝胶电泳及氨基酸分析显示这些都符合Ⅰ型胶原的特征,同水母S.nomurai和Rhopilema asamushi来源的胶原蛋白基本一致,作为一个低等动物,其氨基酸分析显示了脊椎动物和非脊椎动物的微小差异。 相似文献
3.
Calf skin collagen was solubilized by incubating acid-extracted calf skin with pepsin at pH 2.0 and 25 degrees C, conditions that did not cause degradation of the triple helical region of collagen. Type III collagen was separated from type I collagen by differential salt precipitation at pH 7.5. The isolated type III collagen contained mainly gamma and higher molecular weight components cross-linked by reducible and/or non-reducible bonds. The isolated alpha1 (III) chains had an amino acid composition characteristic of type III collagen. Denatured but unreduced type III collagen, chromatographed on carboxymethyl-cellulose, eluted in the alpha 2 region, while after reduction and alkylation the alpha1 (III) chains eluted between the positions of alpha1 (I) and alpha2. The mid-point melting temperature temperature (tm) of type III collagen (35.1 degrees C) in a citrate buffer at pH 3.7 was somewhat lower than that of type I collagen (35.9 degrees C). Renaturation experiments at 25 degrees C showed that denatured type III collagen molecules with intact intramolecular disulfide bridges (gamma components) reform the triple helical structure of collagen much faster than reduced and carboxymethylated alpha1 (III) chains. 相似文献
4.
Hee-Seok Jeong Jayachandran Venkatesan Se-Kwon Kim 《Biotechnology and Bioprocess Engineering》2013,18(6):1185-1191
In the present study, we isolated collagen from Thunnus obesus bone, which was physiochemically characterized. Two different kinds of methods were used to isolate the collagen; they are the Acid Soluble Collagen (ASC) and Acid Soluble Enzyme Collagen (ASEC) methods. The isolated collagen was characterized with Fourier Transform Infrared Spectroscopy (FT-IR), SDS-polyacrylamide gel electrophoresis (SDS-PAGE), Optical Microscopy (OM) and Scanning Electron Microscopy (SEM). FT-IR results revealed the presence of collagen. SEM and OM results depicted that collagen was in the form of fiber sponge-like scaffolds. The isolated collagen scaffold was checked with pre-osteoblast (MC3T3-E1) cell line for biocompatibility. The in vitro results revealed that the collagen scaffolds were highly biocompatible and nontoxic in nature. Herewith, we are suggesting that marine fish-derived collagen will be an excellent material for leather, film industry, pharmaceutical, cosmetics, biomedical and food applications. 相似文献
5.
L Bruckner-Tuderman U W Schnyder K H Winterhalter P Bruckner 《European journal of biochemistry》1987,165(3):607-611
The triple-helical domain of type VII collagen was isolated from human placental membranes by mild digestion with pepsin, and polyclonal antibodies were raised in rabbits against this protein. After affinity purification the antibodies specifically recognized type VII collagen in both the triple-helical and the unfolded state. They also reacted with the fragments P1 and P2, derived from the triple-helical domain by further proteolysis with pepsin, but did not crossreact with other biochemical components of the dermal connective tissue. In skin the presence of a fragment of type VII collagen, similar to that isolated from placenta, was demonstrated by SDS-PAGE and immunoblotting. Type VII collagen represented less than 0.001% of the total collagen extracted by pepsin digestion from newborn or adult skin. The tissue form of type VII collagen was obtained from dermis after artificial epidermolysis with strongly denaturing buffers under conditions reducing disulfide bonds. The protein was identified by immunoblotting with the antibodies. The molecule was composed of three polypeptides with an apparent molecular mass of about 250 kDa, each. Similar large-molecular-mass chains could be identified by immunoblotting in extracts of human fibroblasts in culture. 相似文献
6.
The effects of 1-hydroxyethane-1,1-diphosphonate and dichloromethanediphosphonate on collagen synthesis by rabbit articular chondrocytes and rat bone cells.
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Investigations were performed to assess the effects of dichloromethanediphosphonate on the synthesis of collagen by (1) isolated rabbit articular chondrocytes, (2) isolated rat calvaria bone cells and (3) bone explants from rats treated with the diphosphonates. The studies showed that dichloromethanediphosphonate, but not 1-hydroxyethane-1,1-diphosphonate, causes articular chondrocytes to increase net collagen biosynthesis, both when measured as 3H-labelled or as non-radioactive material, in a dose-related fashion. The increment in collagen synthesis was still evident with cells that were exposed continuously to the diphosphonate in primary as well as secondary culture; however, it declined with cells in tertiary culture and was absent after the fourth subculture. The type of collagen was not affected by the diphosphonate. The synthesis of collagen by bone cells was likewise increased with dichloromethanediphosphonate. No effects were detected with 1-hydroxyethane-1,1-diphosphonate was tested. Finally, when calvaria and tibiae from diphosphonate-treated rats were cultured in vitro, the positive effect of dichloromethanediphosphonate on collagen synthesis was also evident. 1-Hydroxyethane-1,1-diphosphonate, on the other hand, decreased the incorporation of [3H]proline into the collagen of calvaria and osseous tibial shafts and showed no effect on the collagen synthesis of the cartilaginous tibial heads. 相似文献
7.
Experiments were carried out to determine whether bone cells isolated from rat calvaria degrade newly synthesized collagen intracellularly prior to secretion and to assess the effect of dichloromethylenebisphosphonate, a compound shown to stimulate collagen synthesis during this event. The findings indicate that isolated bone cells grown in culture degraded a proportion (average 16%) of newly synthesized collagen prior to secretion. This process was markedly reduced by exposure to dichloromethylenebisphosphonate in a dose-related manner. Concomitantly with the observed decrease of degradation, an increase of collagen synthesis was detected as determined by the incorporation of [3H]proline into collagenase-digestible proteins or by the conversion of [3H]proline into [3H]hydroxyproline. No similar enhancement on total non-collagenous protein synthesis was evident. Dichloromethylenebisphosphonate did not influence the extracellular degradation of collagen. Although the reduction in intracellular degradation accounted only for part of the bisphosphonate mediated increase in net collagen synthesis, it is conceivable that the rate of collagen synthesis is regulated, at least in part, by mechanisms that modulate the level of intracellular degradation. 相似文献
8.
Assignment of the human collagen alpha 1 (XIII) chain gene (COL13A1) to the q22 region of chromosome 10 总被引:2,自引:0,他引:2
T B Shows L Tikka M G Byers R L Eddy L L Haley W M Henry D J Prockop K Tryggvason 《Genomics》1989,5(1):128-133
Type XIII collagen is a recently described collagen that resembles in structure the short-chain collagens of types IX, X, and XII. Unlike any other collagen, the type XIII is found in several different forms generated through alternative splicing. A 2.0-kb genomic fragment from the human alpha 1 (XIII) collagen gene was isolated and shown by DNA sequencing to contain exon 12 as counted from the 3' end. This fragment was used as a probe to localize the gene. The gene (COL13A1) was assigned to chromosome 10 by hybridization of the probe to DNA isolated from a panel of human-mouse somatic cell hybrids containing different human chromosomes. Furthermore, the gene was mapped to the q22 region by in situ hybridization to metaphase chromosomes. 相似文献
9.
The glycosylations of hydroxylysine during collagen biosynthesis in isolated chick-embryo tendon cells were studied by using pulse-chase labelling experiments with [14C]-lysine. The hydroxylation of lysine and the glycosylations of hydroxylysine continued after a 5 min pulse label for up to about 10 min during the chase period. These data differ from those obtained previously in isolated chick-embryo cartilage cells, in which, after a similar 5 min pulse label, these reactions continued during the chase period for up to about 20 min. The collagen synthesized by the isolated chick-embryo tendon cells differed markedly from the type I collagen of adult tissues in its degree of hydroxylation of lysine residues and glycosylations of hydroxylysine residues. When the isolated tendon cells were incubated in the presence of L-azetidine-2-carboxylic acid, the degree of glycosylations of hydroxylysine during the first 10 min of the chase period was identical with that in cells incubated without thcarboxylic acid for at least 60 min, whereas no additional glycosylations took place in the control cells after the 10 min time-point. As a consequence, the collagen synthesized in the presence of this compound contained more carbohydrate than did the collagen synthesized by the control cells. Additional experiments indicated that azetidine-2-carboxylic acid did not increase the collagen glycosyltransferase activities in the tendon cells or the rate of glycosylation reactions when added directly to the enzyme incubation mixture. Control experiments with colchicine indicated that the delay in the rate of collagen secretion, which was observed in the presence of azetidine-2-carboxylic acid, did not in itself affect the degree of glycosylations of collagen. The results thus suggest that the increased glycosylations were due to inhibition of the collagen triple-helix formation, which is known to occur in the presence of azetidine-2-carboxylic acid. 相似文献
10.
Type VIII collagen was isolated from bovine Descemet's membranes by pepsin treatment and salt fractionation, as described by Kapoor et al. [(1986) Biochemistry 25, 3930-3937]. Contaminating type IV collagen was removed by ion-exchange chromatography. Purified type VIII collagen consisted of two different polypeptide chains and, compared to the fiber forming collagens, showed a higher thermal stability. Corresponding fractions isolated from pepsinized human Ewing's sarcoma and fetal calf aorta reacted immunologically with a protein of similar molecular mass. After extraction of Descemet's membranes with guanidine hydrochloride, a peptide of about 60 kDa was obtained. This seems to be the tissue form of type VIII collagen. 相似文献
11.
Osteoblast collagenase: collagenase synthesis by clonally derived mouse osteogenic (MC3T3-E1) cells 总被引:1,自引:0,他引:1
Latent collagenase was isolated by heparin-Sepharose affinity chromatography from the culture medium of clonally derived mouse osteogenic (MC3T3-E1) cells. Collagenase synthesis by MC3T3-E1 cells was significantly stimulated by the addition of parathyroid hormone to the serum-containing culture medium. The cellular origin of the isolated collagenase was confirmed by demonstrating the characteristic 3/4 and 1/4 fragments of collagen alpha-chain, as well as inhibition of the enzyme by anti-mouse bone collagenase antibody. 相似文献
12.
M Laurent M O Lonchampt F Regnault J Tassin Y Courtois 《Experimental cell research》1978,115(1):127-142
Lens epithelial cells isolated from adult bovine were maintained in long-term culture. They synthesised important extracellular fibrils which had a similar pattern to capsule-like material in electron microscopic pictures. These fibrils were sensitive to a highly purified collagenase. After addition of labelled proline in the culture medium, the solubilized material obtained from culture preparation contained radioactive hydroxyproline. When the culture was maintained at confluency for several months, extracellular fibres with a pattern similar to fibrous long spacing collagen were observed. The collagen newly synthesised by epithelial cells has been isolated from the cells and from the medium. It has been chromatographed on agarose A5m column and analysed on sodium dodecyl sulphate polyacrylamide gel electrophoresis. Confluent lens cells were stained with anti-serum to lens capsule collagen type IV, the anti-serum stained the fibrils which are shed between the cells. Thus the collagen newly synthesised by epithelial cells has been isolated and tentatively identified as basement membrane collagen. 相似文献
13.
Type VI collagen. Studies on its localization, structure, and biosynthetic form with monoclonal antibodies 总被引:24,自引:0,他引:24
Monoclonal antibodies were prepared by immunization with whole tissue and were selected for their reactivity with extracellular matrices in tissue immunofluorescence. Two such antibodies were used to isolate the corresponding antigen from pepsin extracts of human placental tissue by immunochromatography. In each case, polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate showed that the isolated material was composed of four polypeptides of Mr between 57,000 and 85,000 that were disulfide-bonded into a high molecular weight aggregate. Amino acid analyses showed that the isolated material was partly collagenous. The material was shown to be antigenically related to previously isolated peptic fragments of type VI collagen and it shared their unique structure as revealed by electron microscopy. Based on these findings, it was concluded that the isolated material was a form of type VI collagen. In immunofluorescence, the monoclonal antibodies localized type VI collagen throughout the connective tissue and in the extracellular matrix of cultured fibroblasts. Polypeptides presumably comprising the intact form of this collagen were isolated from cultures of metabolically radiolabeled fibroblast cell cultures using the two monoclonal antibodies. The isolated material consisted of two polypeptides of Mr 240,000 and 140,000 that were extensively disulfide cross-linked. Four additional monoclonal antibodies bound the same radioactive polypeptides from fibroblast cultures, but only one of them reacted with the fragments isolated from pepsin-digested placenta. Since all six antibodies were originally selected based on tissue immunofluorescence, and therefore react with the tissue form of the protein, the tissue form appears to be more similar to the polypeptides detected in fibroblast cultures than to the pepsin-resistant fragments. Since these monoclonal antibodies apparently recognize different parts of the molecule, they will be useful for further study of the structure and function of the intact form of type VI collagen. 相似文献
14.
Two microscopical methods for the evaluation of molecular length--the measurement of SLS aggregates and the rotary shadowing technique--are compared. Two types of collagen were used for the comparison: type I collagen, isolated by acid extraction from calf skin, and type IX, isolated from chicken cartilage. An average length of 303 nm was obtained for type I collagen (by measurement of SLS aggregates 247 nm). Type IX collagen forms three different types of SLS crystallites corresponding to molecular lengths of 61, 95 and 206 nm; with the rotary shadowing technique, lengths of 84, 116 and 197 nm were measured. 相似文献
15.
Measurement of the synthesis rates of collagens and total protein in rabbit muscle. 总被引:2,自引:2,他引:0
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1. Collagen- and total-protein-synthesis rates were determined in rabbit muscle by continuous infusion of radioactive proline. 2. The precursor pool of free proline used for collagen synthesis was defined by measuring the specific radioactivity of hydroxy-proline in isolated type I procollagen. The specific radioactivities of type I procollagen were about 40% of those for free proline in the homogenate. 3. The mean ratio (+/- S.E.M.) between the fractional synthesis rates of muscle collagen and total protein was 0.99 +/- 0.10, where the total protein values were based on specific radioactivities of the homogenate free proline pools. 4. Types I, III and V collagen were solubilized by pepsin and isolated by fractional precipitation with NaCl. The fractional synthesis rates of types I and III collagens were very similar. Type V collagen samples had higher specific radioactivities than the other collagens, but this was not necessarily indicative of a higher rate of synthesis because of uncertainty about the cellular origin of this collagen and, hence, the specific radioactivity of its precursor proline pool. 相似文献
16.
Phosphorylation of pig brain microtubule proteins. General properties and partial characterization of endogenous substrate and cyclic AMP-dependent protein kinase. 总被引:3,自引:0,他引:3
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P Sheterline 《The Biochemical journal》1977,168(3):533-539
Collagen synthesis and the activities of prolyl hydroxylase, lysyl hydroxylase, collagen galactosyltransferase and collagen glucosyltransferase were studied in isolated chick-embryo tendon cells after the administration of cortisol acetate to the chick embryos. When the steroid was injected 1 day before isolation of the tendon cells, collagen synthesis was decreased, even though the enzyme activities were not changed. When cortisol acetate was given as repeated injections over a period of 4 days, both collagen synthesis and the enzyme activities decreased. The hydroxylase activities decreased even more than the two collagen glycosyltransferase activities, both in isolated cells and in whole chick embryos. The amount of prolyl hydroxylase protein diminished to the same extent as the enzyme activity, indicating that cortisol acetate inhibits enzyme synthesis. The inhibitory effect of cortisol acetate on collagen synthesis and on the enzyme activities was partially reversible in 3 days. Total protein synthesis was completely restored within this time. Only massive doses of cortisol acetate inhibited collagen synthesis in vitro. Additional experiments indicated that cortisol acetate did not decrease the rate of the enzyme reactions when added directly to the enzyme incubation mixtures. The results suggest that cortisol acetate decreases collagen synthesis both by its direct effect on collagen polypeptide-chain synthesis and by decreasing the activities of enzymes involved in post-translational modifications. 相似文献
17.
Analysis of a crosslinked peptide from calf bone collagen: evidence that hydroxylysyl glycoside participates in the crosslink 总被引:10,自引:0,他引:10
A crosslinked, double-chained peptide has been isolated from calf bone collagen after digestion with crude bacterial collagenase. Initially, the 3H-labelled peptide was isolated from collagen that had been treated with [3H]-NaBH4, but an almost identical peptide was also isolated from collagen without prior reduction. After periodate oxidation of the reduced peptide the two component chains were resolved by further chromatography. Amino acid compositions showed that the peptide probably derived from an intermolecular crosslink between a carboxyterminal sequence of the collagen molecule and a sequence near the aminoterminus that previously has been shown to be the site of a glycosylated hydroxylysine residue. The crosslinking compound in the reduced peptide, hydroxylysinohydroxynorleucine, appeared to have derived mainly by reduction with borohydride of hydroxylysinooxonorleucine, the keto-amine rearranged form of the dehydro crosslink. The remaining hydroxyl group of the crosslink, the one not derived by reduction of the keto group, appeared to be glycosylated. 相似文献
18.
1. The contents of the fibrous proteins collagen and elastin in the pleural and parenchymal regions of bovine lungs were determined. The collagen content was approx. 70% (g/100g of salt-extracted defatted powder) in each tissue, and the elastin content was 28% in pleura and 13.5% in parenchyma. 2. Purification of the insoluble collagen from the pleura and parenchyma of bovine lungs by various methods was attempted. The collagen fractions isolated after incubation of the pulmonary tissues with the proteolytic enzymes collagenase ("collagenase-soluble" fraction) or pancreatic elastase ("elastase-insoluble" fraction) each contained approx. 87% of the total collagen initially present. 3. Both collagen fractions were chemically analysed for their amino acid and carbohydrate contents and were found to be similar to those of the intact interstitial collagens isolated from skin, bone and tendon. 4. The contents of the two aldimine cross-linking compounds, dehydrohydroxylysinonorleucine and dehydrodihydroxylysinonorleucine, were determined in the bovine pulmonary collagen fractions, and were found to decrease with increasing age of the animals, and were similar to the values found in intact collagens from bone and tendon. 相似文献
19.
In vitro studies on adult cardiac myocytes: attachment and biosynthesis of collagen type IV and laminin 总被引:5,自引:0,他引:5
E Lundgren D Gullberg K Rubin T K Borg M J Terracio L Terracio 《Journal of cellular physiology》1988,136(1):43-53
The interactions between adult rat cardiac myocytes and the basement membrane components collagen type IV and laminin were investigated in attachment experiments and biosynthesis studies and by immunofluorescence staining. Adult myocytes attached equally well to native collagen type IV and laminin but did not attach to collagen type IV solubilized with pepsin (P-CIV) or to collagen type I. However, when laminin was used to coat P-CIV, attachment was enhanced. Affinity-purified antibodies against laminin inhibited the attachment of myocytes to dishes coated with native collagen type IV, indicating that cell surface-bound laminin mediated attachment of the cells to this substrate. Immunofluorescence staining of freshly isolated myocytes, using antibodies against laminin or collagen type IV, revealed the presence of laminin but not of collagen type IV on the surface of freshly isolated cells, indicating that during the isolation procedure collagen IV was removed from the cell surface. Metabolic labeling followed by immunoprecipitation demonstrated synthesis of both laminin and collagen type IV in cardiac myocytes as they progressed into culture over a 14-day period. This synthesis was accompanied by the deposition of the collagen type IV and laminin into distinctly different patterns as revealed by immunofluorescence staining. As the cells progressed into culture, newly synthesized laminin formed a network radiating from the center of the reorganizing cell into the pseudopods. The laminin was redistributed and remodeled with time in culture to form a dense layer beneath the cell. Collagen type IV was also synthesized with time in culture, but the pattern was a much finer network as opposed to the denser pattern of laminin staining. These studies demonstrate that adult cardiac myocytes synthesize and remodel the basement membrane as they adapt to the culture environment. 相似文献
20.