Isolation from bovine elastic tissues of collagen type VI and characterization of its form in vivo.

Foetal-bovine nuchal ligament and aorta, together with adult-bovine aorta and pregnant uterus, were extracted under dissociative conditions in the absence and in the presence of a reducing agent. A collagenous glycoprotein of Mr 140000 [designated component 140K(VI)], identified in these extracts as the major periodate/Schiff-positive component, was shown to be related to collagen type VI. Digestion of non-reduced extracts with pepsin yielded periodate/Schiff-positive peptides that, on the basis of their electrophoretic mobilities, amino acid analyses and peptide 'maps', were identical with type VI collagen fragments prepared by standard procedures. It is concluded that collagen type VI occurs in vivo as molecule comprising three chains of Mr 140000 in which the helical domains account for about one-third of each polypeptide. Biosynthetic experiments with nuchal-ligament fibroblasts in culture demonstrated that a bacterial-collagenase-sensitive [3H]fucose-labelled glycoprotein, Mr 140000, was immunoprecipitated from culture medium by a specific antibody to the pepsin-derived form of collagen type VI. This result suggests that the collagenous polypeptides [140K(VI) components] represent the biosynthetic precursors of type VI collagen that do not undergo processing to smaller species before deposition in the extracellular matrix. Analyses of 5M-guanidinium chloride extracts of tissues with markedly different elastin contents and at different stages of development suggested that there was no relationship between collagen type VI and elastic-fibre microfibrils, a conclusion supported by the observation that the immunoprecipitated glycoprotein, Mr 140000, was distinct from the glycoprotein MFPI, Mr 150000, believed to be a constituent of these microfibrils [Sear, Grant & Jackson (1981) Biochem. J. 194, 587-598].     

Isolation and characterization of acidic structural glycoproteins in pulmonary tissues.

1. Extraction of the pleural and parenchymal regions of bovine lungs with salt and organic solvents gave powders that contained glycoproteins in addition to collagen and elastin. The contents of these glycoproteins (g/100 of powder) was 4% in pleura and 15.5% in parenchyma. 2. Attempts were made to purify these glycoproteins fromboth tissues by various methods. 3. Both tissues contained heterogeneous mixtures that were similar solubilities, had similar amino acid and carbohydrate compostions. 4. The compostions of the pulmonary glycoproteins resembled those isolated from other connective tissues by other workers.     

Isolation and partial characterization of bovine gingival AB collagen

Summary Limited proteolysis with pepsin solubilized 25% of the insoluble gingival matrix as mainly soluble collagenous material. Fractional salt precipication at neutral pH resulted in the separation of types III and I at 1.8 and 2.6 M NaCl, respectively. In addition, a collagenous fraction accounting for 2% of the solubilized collagen and precipitating at 4.5 M NaCl was shown to be identical with type V collagen. Isolation and partial characterization of the constituent-α-chains of the 4.5 M PPT by gel filtration, ion exchange and hydroxylapatite chromatography as well as disc electrophoresis showed that gingival type V collagen contains αA and αB chains in a ratio αB/αA of 1.73–1.8. Electron microscopic examination of ATP-precipitates showed that this collagen type gave only one kind of SLS aggregates with asymmetric band pattern characteristically different from that of type I collagen. The data provide evidence that gingival AB collagen is a heteropolymer in which the αA and αB chains are assembled in the same macromolecule in a 1∶2 ratio.     

Isolation and characterization of a fluorescent material in bovine achilles tendon collagen

A fluorescent material in bovine achilles tendon collagen was isolated and characterized by ultraviolet spectroscopy, fluorescence spectroscopy and nmr spectroscopy. The data suggest that the compound is a 3-hydroxypyridinium derivative with three amino acid side chains. The name “pyridinoline” is proposed. Pyridinoline is a novel type crosslink of collagen.     

Isolation and characterization of type III collagen from bovine cardiac muscle

• 1.1. Bovine cardiac muscle collagen was solubilized by limited digestion with pepsin and subsequently fractionated by differential salt precipitation.
• 2.2. Chromatographie studies on CM-cellulose and agarose of pepsin-soluble collagen and its salt-precipitated fractions showed it to be composed of type I collagen and a species with a mol. wt of 285,000 daltons, having an amino acid composition characteristic of type III collagen.
• 3.3. The molecular structure of the 285,000-dalton component was confirmed as [α1(III)]₃ after reduction with dithiothreitol to a chains having the same amino acid composition.
• 4.4. Cyanogen bromide peptides of pepsin-soluble collagen which co-eluted on CM-cellulose and were identified as α1(I)-CB8 and α1(III)-CB8 showed a molar ratio of 2:1 indicating that 25% of the collagen molecules solubilized are [α1(III)]₃.

     

Polyamine synthesis in mammalian tissues. Isolation and characterization of spermidine synthase from bovine brain.

Spermidine synthase (EC 2.5.1.16) was purified to apparent homogeneity (about 11 000-fold) from bovine brain by affinity chromatography, with S-adenosyl-(5')-3-thiopropylamine linked to Sepharose as the adsorbent. The enzyme preparation was free from S-adenosylmethionine decarboxylase (EC 4.1.1.50) and spermine synthase (EC 2.5.1.22) activities. The native enzyme had an apparent Mr of 70 000, was composed of two subunits of equal size, and had an isoelectric point at pH 5.22. The apparent Km values for putrescine and decarboxylated adenosylmethionine [S-adenosyl-(5')-3-methylthiopropylamine] were 40 microM and 0.3 microM respectively. Cadaverine and 1,6-diaminohexane could replace putrescine as the aminopropyl acceptor, although the reaction rates were only 6% and 1% respectively of that obtained with putrescine. Ethyl, propyl and carboxymethyl analogues of decarboxy-S-adenosylmethionine could act as propylamine donors. Both the reaction products, spermidine and 5'-methylthioadenosine, were mixed-type inhibitors of the enzyme. On the basis of initial-velocity and product-inhibition studies, a ping-pong reaction mechanism for the spermidine synthase reaction was ruled out.     

Polyamine synthesis in mammalian tissues. Isolation and characterization of spermine synthase from bovine brain

Spermine synthase, a propylamine transferase, which catalyses the biosynthesis of spermine from S-methyladenosylhomocystemine and spermidine has been purified to an apparent homogeneity (about 6000-fold) from bovine brain using spermine-Sepharose affinity chromatography. The enzyme preparation was free from S-adenosylmethionine decarboxylase and spermidine synthase activities. The molecular Stokes radius of the enzyme was calculated to be 4.16 nm. The enzyme has an apparent molecular weight of approximately 88 000, composing of two subunits of equal size. The enzyme showed a broad pH optimum between 7.0 and 8.0 and an acidic isoelectric point at pH 5.10. The apparent Km values for S-methyladenosylhomocysteamine was 0.6 microM and about 60 microM for spermidine. The enzyme showed strict specificity to spermidine as the propylamine acceptor. Both the reaction products, spermine and 5'-methylthioadenosine inhibited the enzyme activity, methylthioadenosine being a powerful competitive inhibitor with respect to S-methyladenosylhomocysteamine (Ki value of about 0.3 microM). Putrescine also inhibited competitively with respect to spermidine (Ki value of about 1.7 mM). Spermine synthase had no requirements for metal or other cofactors.     

Type IV collagen from chicken muscular tissues. Isolation and characterization of the pepsin-resistant fragments

Type IV collagen has been isolated from adult chicken gizzard after limited pepsin digestion and subsequent differential salt fractionation in acidic and neutral conditions. After denaturation, three fragments (called F1, F2, and F3) were isolated by agarose gel filtration and carboxymethylcellulose chromatography. F1 and F2 possessed apparent molecular weights of 53 000 and 50 000, respectively, and were consistently isolated in a 2:1 proportion. F3 was larger and after reduction of disulfide bonds gave rise to three fragments (called F3A, F3B, and F3C) of apparent molecular weights 68 000, 40 000, and 29 000. No alpha-chain-sized components of Type IV collagen were observed. A native fraction containing F1 and F2, but no F3, was isolated after extraction using less pepsin and an additional salt fractionation in acidic conditions. F1 and F2 in the native form were not separated by carboxymethylcellulose or diethylaminoethylcellulose chromatography performed in nondenaturating conditions or by differential salt precipitation in acidic or neutral conditions; these results suggest that F1 and F2 arise as a single native component of structure (F1)2F2. The fraction containing F1 and F2 also gave rise to a single segment long spacing crystallite pattern and to a circular dichroism spectrum which was typical for a native collagen. F1 and F2 were also isolated from chicken heart, blood vessels, and skeletal muscle, whereas from bovine aorta, using the same isolation procedures, two alpha-chain-sized components were obtained, which appeared to be similar to the two Type IV chains recently described by other groups. The data suggest that (i) pepsin fragmentation of type IV collagen from chicken tissues occurs in a different manner compared to Type IV collagen from mammalian tissues and (ii) for the chicken there must be at least two Type IV chains which are assembled into a single native molecule.     

Isolation and characterization of a collagen fibril-associated dermatan sulphate proteoglycan from bovine lung

Dermatan sulphate proteoglycans have been extracted from bovine lung with 2.0 M CaCl2 and isolated using CsCl density gradient centrifugation, DEAE ion-exchange chromatography, gel chromatography and preparative sodium dodecyl sulphate-polyacrylamide gel electrophoresis. Ultrastructurally these proteoglycans are specifically associated with collagen fibrils. Dermatan sulphate (Mr 15.10(3)-35.10(3), with a strong prevalence for the higher Mr) is link via an O-glycosidic bond to a protein core, which is rich in Asx, Glx and Leu. Of the total uronic acid, 91% is iduronic acid. A part of the glucuronic acid residues is located near the protein core and a large cluster of disaccharides is devoid of glucuronic acid residues. An inhibition enzyme immunoassay has been developed to quantitate the proteoglycan. A model for the interaction between dermatan sulphate proteoglycans and collagen fibrils is proposed.     

Isolation and characterization of bovine stefin B.

A new cysteine proteinase inhibitor (CPI) was isolated from bovine thymus. According to the amino acid sequence it belongs to the stefin family. It appears as a monomer and a dimer with monomer M(r) of 11,178 and pI values 5.6 for the monomer and 5.2 and 5.6 for the dimer. Ki for the interaction with papain was determined to be 0.12 nM. The most interesting feature of bovine stefin B is the replacement of the highly conserved QVVAG region in stefins with the QLVAG sequence without interfering its inhibitory properties.     

The chemical composition of bovine vitreous-humour collagen fibres.

The insoluble protein fraction was prepared from the central and posterior peripheral fraction of bovine vitreous humour. The collagen present in this fraction was solubilized by pepsin and fractionated by gel chromatography. Analysis of the solubilized collagen fractions showed that the alpha-chain component had an amino acid composition and yielded a series of CNBr-cleavage peptides that showed it was very similar to type II collagen obtained from articular cartilage. Bovine vitreous-humour collagen alpha-chains differed, however, from those of cartilage collagen in that they had a lower alanine content and differed in their susceptibility to cleavage by CNBr. Satisfactory cleavage was obtained after two CNBr treatments involving reduction and alkylation. In addition, significant quantities of other peptides constituents were present in the vitreous-humour collagen fractions, and the galactose and glucose content of the alpha-chain fraction was more than double that of the same fraction obtained from articular cartilage. Although the origin of the additional peptide constituents in the vitreous-humour collagen preparations is not known, the results obtained indicate that they are probably not derived from a distinct type of alpha-chain component but may be terminal peptides covalently linked to the alpha 1 type-II helical portions of the collagen. The differences in the chemical composition of the vitreous-humour collagen indicate that vitreous-humour fibres are composed of a special type-II collagen.     

Isolation and characterization of bovine factor VII.

Factor VII (proconvertin) has been purified approximately 5 x 10(5)-fold from bovine plasma with an overall yield of 30%. The isolation procedure involves barium sulfate adsorption and elution, DEAE-Sephadex batchwise adsorption and elution, benzamidine-agarose column chromatography, heparin-agarose column chromatography, and preparative polyacrylamide gel disc electrophoresis. The final product was homogeneous when examined by gel electrophoresis in the presence of sodium dodecyl sulfate. A minimal molecular weight of 45,500 was determined by sedimentation equilibrium. The molecular weight estimated by sodium dodecyl sulfate gel electrophoresis was 54,000. Factor VII is composed of a single polypeptide chain possessing an amino-terminal sequence of Ala-Asn-Gly-Phe-Leu-. The amino acid and carbohydrate compositions of factor VII are also reported.     

Isolation of thymone A from bovine thymus partial chemical and biological characterization

An homogeneous fraction of structural glycoproteins (SGP) purified from the connective matrix of rabbit skin, exerts a strong inhibitory influence on the protein syntheses that occur in cultured fibroblasts both from rabbit and human skin. The effect is dose dependent from 0.7 to 8.4 × 10^?6 M. The inhibition of collagen synthesis parallels that of the bulk of proteins.     

Isolation and characterization of cyanogen bromide peptides from the collagen of bovine articular cartilage

Significant amounts of native collagen can be extracted from bovine articular cartilage after removal of the acid mucopolysaccharides by controlled proteolysis. The fraction thus solubilized upon denaturation gives rise to three identical alpha chains. Cleavage of these chains with CNBr generated nine peptides, all of which contain glycine as one-third of their total amino acid residues. Two of the smaller peptides CB-1 and CB-2 contain partially hydroxylated proline. A similar CNBr digest of intact cartilage also gives a series of peptides identical with those obtained from the soluble cartilage collagen. The absence of cross-linking peptides, the fact that only few beta components are seen in articular cartilage collagen and the similarity in peptide pattern between the two collagen fractions investigated, suggests that this collagen is stabilized by a different cross-linking mechanism, possibly involving an association with the tissue proteoglycans.     

Isolation of bovine type X collagen and immunolocalization in growth-plate cartilage.

The accessibility of the cysteine residues of actin from rabbit muscles to the thiol-targeted reagent 7-dimethylamino-4-methyl-(N-maleimidyl)coumarin (DACM) was investigated. Under conditions where the actin is in the unpolymerized form (G-actin), the most reactive thiol group was Cys-257, suggesting that it was located on the surface of the actin molecule. The selective modification of Cys-374 for this reagent as reported by Sutoh [(1982) Biochemistry 21, 3654-3661] was not observed. Cys-10, Cys-217 and Cys-374 were much less reactive and only gradually became extensively modified when the concentration of DACM approached 5 molar equivalents of actin. Presumably these thiol groups were located further inward away from the surface or situated in a different environment that rendered them less reactive. On the other hand, Cys-285 was completely inaccessible and presumably was buried. The lack of preferential labelling of Cys-374 by DACM is incompatible with the finding with iodoacetic acid as the reagent as reported by Elzinga & Collins [(1975) J. Biol. Chem. 250, 5897-5905]. This discrepancy, however, might well be due to the different reagents employed. The DACM-G-actin largely retained its competence for polymerization. Upon polymerization of G-actin, practically all the thiol groups became inaccessible to DACM, suggesting that a drastic change occurred in the conformation of actin units in the transition of monomers to filamentous actin.