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1.
We studied the ultrastructural distribution of laminin, type IV collagen, and the amino terminal pro-peptide of type III collagen (type III pN-collagen) in normal human lymph nodes. After fixation with freshly prepared 4% paraformaldehyde mixed with 0.1% glutaraldehyde, cryoultramicrotomy proved to preserve the antigenicity of these proteins better than embedding in Lowicryl K4M. Sections were treated with rabbit antibodies against the 7S domain of human type IV collagen, the fragment P1 of human laminin, and the amino terminal pro-peptide of human type III pro-collagen, followed by anti-rabbit IgG conjugated to 10-nm colloidal gold. Laminin and type IV collagen were seen in the basement membrane structures of the blood vessels and in the walls of sinuses. The amorphous material between the collagenous fibers in locations corresponding to reticular fibers also contained laminin and type IV collagen. The amino terminal pro-peptide of type III pro-collagen was present in the collagenous fibers in reticular fibers and in the walls of blood vessels and sinuses. Therefore, a significant number of the type III collagen molecules in these fibers must have retained their amino terminal pro-peptide. These results indicate that the basement membrane proteins laminin and type IV collagen are genuine components of reticular fibers, as suggested earlier by immunohistochemical studies at the light microscopic level.  相似文献   

2.
Protein deficiency and tryptophane load against its background lead to the acid-soluble collagen synthesis in the rat skin. The amino acid composition of the collagen differs from the norm. This is accompanied by changes in the free amino acid pool of blood serum and liver, under tryptophane load the free amino acids pool of the liver increasing twice as high. At the same time protein deficiency increases and tryptophane load decreases the level of tRNA amino acylation with tryptophane in the animal liver. Thus, protein deficiency and tryptophane load against its background cause deep changes in the protein biosynthesis.  相似文献   

3.
Collagen fractions content and level of collagen catabolites in body fluids were determined in normal, hypo- and hyperthyroid guinea pigs. An increase of urinary excretion of hydroxyproline and hydroxylysine as well as concentration of these amino acids in blood serum was found in hyperthyroidism, and a decrease was shown in hyperthyroid guinea pigs. Hyperthyroidism stimulated an increase of neutral-salt-soluble and acid-soluble collagen in skin and liver, and a decrease of insoluble collagen in skin as well as increase of all collagen fractions in bone samples. Hypothyroidism induced a decrease of all collagen fractions in skin and liver, and an increase of acid-soluble and insoluble collagen in bone samples.  相似文献   

4.
The feeding of excessive leucine and phenylalanine doses to rats against a background of protein deficiency is shown to cause changes in the liver tRNA aminoacylation as well as in the synthesis of acid-soluble skin collagen differing from the normal level in the amino acid composition, elution profiles and content of disulphide groups.  相似文献   

5.
In normal lung growth, post-pneumonectomy lung growth, and in possibly several lung disorders, there are marked alterations in the density of collagen and changes in the rate of synthesis of collagen relative to the synthesis of other lung proteins. To provide a technology to begin to understand these changes at the molecular level, polysomes were prepared from rabbit lung and translated in a heterologous cell-free system including rabbit reticulocyte 0.5 M KCl ribosomal wash fraction and liver tRNA. Collagen was shown in the cell-free product by collagenase sensitivity, hydroxylation of incorporated proline by peptidyl prolyl hydroxylase, agarose gel chromatography, and sodium dodecyl sulfate acrylamide gel electrophoresis. The cell-free system was optimized with respect to K+, Mg2+, amino acids, and ribosomal wash fraction and used under conditions where total protein synthesis and collagen synthesis are linear with respect to time and amount of polysomes. Under these conditions, collagen synthesis was directed almost entirely by polysomes derived from the endoplasmic reticulum. Polysomes isolated from late fetal lung directed collagen synthesis at twice the rate (per polysome) as those polysomes isolated from adult lung. Similar changes were seen if lung tRNA replaced liver tRNA and if lung ribosomal wash fraction replaced reticulocyte wash fraction. Although these changes in cell-free lung collagen synthesis with tissue explants, further studies will have to be carried out to determine whether, in fact, age-related alterations in control of lung collagen synthesis are truly explained by these findings.  相似文献   

6.
The genetic control of the murine immune response to denatured beef type II collagen was examined in an antigen-presenting cell-dependent T cell proliferation assay. It was found that the response to collagen is under the control of H-2-linked Ir genes, possibly operating at the level of the antigen-presenting macrophage. Utilizing in vitro cross-stimulation with synthetic collagen-like polypeptides, the data suggest that the antigenic determinants stimulating collagen immune T cells are more dependent on primary amino acid sequence and less on conformation. The implications of such findings for Ir gene specificity and other related immunologic functions is discussed.  相似文献   

7.
The determination of the collagen and glycosaminoglycan (GAG) contents of native and particularly bioengineered tissues is of considerable interest because the collagen-to-GAG ratio determines the water content of the tissue, which is crucial regarding its mechanical properties. (1)H NMR spectroscopy subsequent to the hydrolysis of the sample by aqueous 6 M DCl at 353 K is used to determine the GAG and collagen contents simultaneously. Under these strongly acidic conditions the biopolymers of the extracellular matrix, collagen, and GAG are fragmented into their individual monomers, that is, free amino acids from collagen and monosaccharides from the polymer repeat units of GAGs. The amino acid amount can be easily determined in the presence of an internal standard by (1)H NMR spectroscopy because amino acids proved to be stable under acidic conditions. The carbohydrates are subject to charring in the presence of concentrated DCl, but glucosamine and galactosamine were found to be sufficiently stable for quantification under the chosen conditions.  相似文献   

8.
1. The solubilization of collagen and protein-polysaccharides from the developing cartilage of normal and lathyritic chicks was studied by using mild extraction procedures. One-third of the protein-polysaccharides could be solubilized in salt solutions at neutral pH from normal cartilage, whereas 95-100% could be extracted from the cartilage of animals that were severely lathyritic. Likewise, whereas in normal animals the collagen of cartilage was essentially insoluble in salt solutions at neutral pH, in lathyritic animals it was almost completely soluble. 2. The increased solubility of the collagen of cartilage from lathyritic animals enabled sufficient material to be collected so that the pure alpha1 chains of the collagen were isolated by repeated reconstitution, precipitation and CM-cellulose column chromatography. The purified alpha1 component was characterized by its relatively high content of hydroxylysine (14 residues/1000 amino acids). 3. About 37% of the collagen from the cartilage of normal chick embryos could be extracted as the gelatin at pH7.4 in lithium chloride solution. This was accompanied by the extraction of approx. 14% of the protein-polysaccharide content. 4. The protein-polysaccharides and the collagen from normal animals could be extracted from the cartilage relatively independently of one another under mild conditions. These same components obtained from lathyritic animals easily separated from one another after solubilization. This provided evidence that the two components are probably not covalently cross-linked. 5. The collagen of cartilage extracted as a gelatin from normal animals contained a high proportion of alpha chains compared with beta dimers, similar to the lathyritic collagen of cartilage and other tissues, and similar to the gelatin extracted from normal chick bone.  相似文献   

9.
We report a case of mild osteogenesis imperfecta in a 56-year-old male undergoing aortic valve replacement surgery. The primary defect in this patient was the substitution of arginine for glycine 85 in one of the two chains of alpha 1(I) procollagen. The thermal stability of the type I collagen synthesized by the patient's cultured skin fibroblasts was examined by enzymatic digestion. Digestion of the mutant type I collagen with trypsin and chymotrypsin at increasing temperatures sequentially generated three discrete collagenous fragments, approximately 90, 170, and 230 amino acids shorter than normal type I collagen. This incremental thermal denaturation is indicative of cooperative melting blocks within the type I collagen. This is the first demonstration of such cooperative blocks of melting in intact, essentially normal post-translationally modified type I collagen. This direct evidence for cooperative melting domains of uncut type I collagen suggests that discrete blocks of amino acids function as core sites stabilizing the collagen helix. The location of mutations of the alpha chains of type I collagen relative to these discrete blocks of amino acids may influence the severity of the disease phenotype.  相似文献   

10.
A Fertala  A L Sieron  E Adachi  S A Jimenez 《Biochemistry》2001,40(48):14422-14428
Single amino acid substitutions in collagen II cause heterogeneous cartilage disorders including some chondrodysplasias and certain forms of heritable osteoarthritis. In this study, we examined molecular interactions between normal collagen II and collagen IX, and the effect of a Cys substitution for Arg-alpha1-519 in collagen II on these interactions. Binding assays showed that the association equilibrium constant of collagen IX-collagen II interaction is 15 x 10(6) M(-1). Specificity of the interaction was analyzed by the binding of collagen IX to recombinant collagen II variants lacking fragments of 234 amino acids corresponding to particular D-periods. The results indicated that the C-terminal half of collagen II, which includes the D3 and D4 periods, has a high affinity for collagen IX, and that the nontriple helical telopeptides of collagen II are not essential for the specific binding of collagen IX. Computer analysis of the surface of the mutated collagen II and binding assays showed that a Cys substitution for Arg-alpha1-519 changes electrostatic properties around the mutation site, increases the affinity of mutant collagen II for collagen IX, and possibly alters the specificity of the interaction. Thus, the results indicate that interactions between collagen II and collagen IX are site specific and that single amino acid substitutions in collagen II may change the molecular interactions with collagen IX that could destabilize the cartilaginous matrix.  相似文献   

11.
The comparative changes in the amino acid composition of calf skin collagen after gamma-irradiation (doses from 100 to 1,000 Gy) in aqueous solutions under different gas atmospheres (O2, N2O, H2, vacuum) were investigated. The radiochemical yields of collagen amino acid residues destruction were determined. Under O2 (OH X, O2-) most of amino acids are destroyed with higher yields than under N2O. Leucine, valine, isoleucine, phenylalanine, arginine were the exception because of their high reaction rate constants with OH X and hydroxylation reactions. Under H2 (e-aq, H) and in vacuum (e-aq, OH X) the mechanism of collagen radiolysis changed due to its aggregation; the destruction of those amino acids which have high reaction rate constants with water radiolysis products was mainly observed (phenylalanine, tyrosine, histidine).  相似文献   

12.
The extruded tubules of the sea cucumber possess the amino acid composition, intermolecular crosslinking and fibre banding patterns typical of mammalian collagen fibres. Despite this resemblance, particularly the crosslinking normally associated with high tensile strength, the tubule fibres are extremely extensible. We suggest that the molecules are crosslinked through the normal head-to-tail mechanism but that further transverse crosslinking between the irregular fibrils is inhibited by the surrounding sheath of glycosaminoglycan, which also serves the purpose of a lubricant thus allowing slippage of adjacent fibrils under tension.  相似文献   

13.
《The Journal of cell biology》1989,109(4):1849-1856
The levels of type X collagen in mineralizing normal chicken epiphyses and nonmineralizing rachitic chicken tibial epiphyses were measured and compared. Qualitative immunoperoxidase studies with anti-chick type X collagen monoclonal antibodies on sections from normal and rachitic cartilage demonstrated that the type X collagen levels in rachitic growth plates are reduced. Northern hybridization of mRNA and biosynthetic studies have confirmed that type X collagen synthesis in rickets is also decreased. In hypocalcemic rickets, the level of type X collagen mRNA is reduced by 80% whereas the level of type X collagen mRNA is only reduced by 50% in normocalcemic rickets. These observations provide additional evidence that type X collagen is involved in the process of cartilage mineralization and also suggest that the partial recovery of type X collagen synthesis in normocalcemic rickets may be related to the elevated plasma concentration of calcium. Calcium concentration may therefore play an important role in the control of type X collagen synthesis.  相似文献   

14.
The spondyloepiphyseal dysplasias (SEDs) and spondyloepimetaphyseal dysplasias (SEMDs) are a heterogeneous group of skeletal dysplasias (dwarfing disorders) characterized by abnormal epiphyses, with and without varying degrees of metaphyseal irregularities, flattened vertebral bodies, and myopia. To better define the underlying cause of these disorders, we have analyzed the collagens from costal cartilage from several of these patients, using SDS-polyacrylamide gel electrophoresis (SDS-PAGE) and high-performance liquid chromatography (HPLC) of intact chains and cyanogen bromide (CNBr) peptides and amino acid analysis. In almost all of the patients in this study group, the type II collagen exhibited a slower electrophoretic mobility when compared with that in normal controls. The mobility of many, but not all, of the CNBr peptides was also retarded. Peptides near the amino terminus were almost always altered, while the mobility of peptides close to the carboxyl terminus were normal in all but the severely affected cases. Analysis of the CNBr peptides on an HPLC sieving column confirmed that the electrophoretically abnormal peptides were of a higher molecular weight than were control peptides. Amino acid analysis indicated that the abnormal collagens have a higher ratio of hydroxylysine to lysine than does control collagen, suggesting that overmodification may be involved in the altered mobility. Our results are consistent with a defect in the collagen helix that results in overmodification of the molecule from that point toward the amino terminus. We propose that some forms of SED and SEMD are associated with abnormalities in type II collagen that results in delayed helix formation and consequent overmodification of the collagen. Cases of SED fit onto a continuous spectrum of clinical severity that correlates positively with both the extent of alteration and the proximity of the defect to the carboxyl terminus.  相似文献   

15.
In order to characterise the function of the novel fibrillar type XXVII collagen, a series of mice expressing mutant forms of the collagen were investigated. Mice harboring a glycine to cysteine substitution in the collagenous domain were phenotypically normal when heterozygote and displayed a mild disruption of growth plate architecture in the homozygous state. Mice expressing an 87 amino acid deletion in the collagenous domain of collagen XXVII were phenotypically normal as heterozygotes whereas homozygotes exhibited a severe chondrodysplasia and died perinatally from a lung defect. Animals expressing the 87 amino acid deletion targeted specifically to cartilage were viable but severely dwarfed. The pericellular matrix of proliferative chondrocytes was disrupted and the proliferative cells exhibited a decreased tendency to flatten and form vertical columns. Collagen XXVII plays an important structural role in the pericellular extracellular matrix of the growth plate and is required for the organisation of the proliferative zone.  相似文献   

16.
17.
Non-collagenous phosphoproteins, almost all of which can be extracted in EDTA at neutral pH in the presence of proteinase inhibitors, are identified in the matrix of chicken bone, and are therefore not covalently bound to collagen. Similarly, all the peptides containing gamma-carboxyglutamic acid are present in the EDTA extract and none in the insoluble residue, confirming that none is covalently linked to chicken bone collagen. However, organic phosphorus is also found to be present in chicken bone collagen, principally in the alpha2-chains. Of the total protein-bound organic phosphorus present in chicken bone matrix, approx. 80% is associated with the non-collagenous proteins and 20% with collagen. The soluble non-collagenous proteins contain both O-phosphoserine and O-phosphothreonine and these account for essentially of their organic phosphorus content. In contrast, collagen contains neither O-phosphoserine nor O-phosphothreonine. Indeed, no phosphorylated hydroxy amino acid, phosphoamidated amino acid or phosphorylated sugar could be identified in purified components of collagen, which contain approximately four to five atoms of organic phosphorus per molecule of collagen. Peptides containing organic phosphorus were isolated from partial acid hydrolysates and enzymic digests of purified collagen components, which contain an as-yet-unidentified cationic amino acid. These data, the very high concentrations of glutamic acid in the phosphorylated peptides, and the pH-stability of the organic phosphorus moiety in intact collagen chains strongly suggest that at least part of the organic phosphorus in collagen is present as phosphorylated glutamic acid. This would indicate that the two major chemically different protein fractions in chicken bone matrix that contain organic phosphorus may represent two distinct metabolic pools of organic phosphorus under separate biological control.  相似文献   

18.
The Disproportionate micromelia (Dmm) mouse has a three nucleotide deletion in Col2a1 in the region encoding the C-propeptide which results in the substitution of one amino acid, Asn, for two amino acids, Lys-Thr. Western blot and immunohistochemical analyses failed to detect type II collagen in the cartilage matrix of the homozygous mice and showed reduced levels in the matrix of heterozygous mice. Type II collagen chains localized intracellularly within the chondrocytes of homozygote and heterozygote tissues. These findings provide evidence that the expression of type II procollagen chains containing the defective C-propeptide results in an intracellular retention and faulty secretion of type II procollagen molecules. A complete absence of mature type II collagen from the homozygote cartilage and an insufficiency of type II collagen in the heterozygote cartilage explains the Dmm mouse phenotype. The integrity of the C-propeptide is thus crucial for the biosynthesis of normal type II collagen by chondrocytes.  相似文献   

19.
A patient with Ehlers-Danlos syndrome Type VIIB was found to have an interstitial deletion of 18 amino acids in approximately half of the pro-alpha 2(I) chains of Type I procollagen. Analysis of pepsin-solubilized tissue and fibroblast collagen revealed an abnormal additional chain, alpha 2(I)', which migrated in sodium dodecyl sulfate-5% polyacrylamide gel electrophoresis between the normal alpha 1(I) and alpha 2(I) chains. The apparent ratio of normal alpha 1(I):mutant alpha 2(I)':normal alpha 2(I) was 4:1:1. Procollagen studies and enzyme digestion studies of native mutant collagen suggested defective removal of the amino propeptide. Sieve chromatography of CNBr peptides from purified alpha 2(I)' chains revealed the absence of the normal amino telopeptide fragment CB 1 and the appearance of a larger new peptide of approximately 60 residues (CB X). Compositional and sequencing studies of this peptide identified normal amino propeptide sequences. However, the most carboxyl-terminal tryptic peptide of CB X differed substantially in composition and sequence from the expected and was found to have an interstitial deletion of 18 amino acids corresponding to the N-telopeptide of the pro-alpha 2(I) chain. This deletion removes the normal sites of cleavage of the N-proteinase and also removes a critical cross-linking lysine residue. The 18 amino acids deleted correspond exactly to the residues encoded by exon 6 of the pro-alpha 2(I) collagen gene (COL 1 A2), and, therefore, the protein defect may be due to a genomic deletion, or alternatively, an RNA splicing defect.  相似文献   

20.
Collagens present in the connective tissues of the extracellular matrix of fibrosarcoma were isolated and characterized. The fibrosarcoma was induced in rats by the administration of 3-methylcholanthrene. The results obtained were compared with normal muscle. An excess amount of type V collagen was found to be produced by the fibrosarcoma tissue compared to the normal muscle. Type V collagen from fibrosarcoma was characterized on the basis of solubility behavior in sodium chloride solutions, electrophoretic mobility on SDS-polyacrylamide gels, elution pattern of phosphocellulose chromatography and amino acid composition.  相似文献   

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