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1.
Analysis of thyrotropin receptors by photoaffinity labelling. Orientation of receptor subunits in the cell membrane. 总被引:1,自引:0,他引:1
Y Kajita C R Rickards P R Buckland R D Howells B Rees Smith 《The Biochemical journal》1985,227(2):413-420
Porcine thyrotropin (TSH) receptors have been purified by Sepharose-TSH affinity chromatography and crosslinked to a 125I-labelled photoactive derivative (N-hydroxysuccinimidyl 4-azidobenzoate; HSAB) of TSH (125I-HSAB-TSH). Purification of the crosslinked complexes on Sephacryl S-300 followed by polyacrylamide-gel electrophoresis in sodium dodecyl sulphate showed that the receptor contained two subunits. One subunit (A) with Mr 45 000 was crosslinked to TSH and the other (B) subunit, Mr 25 000, was linked to the A subunit by a disulphide bridge(s). Other, as yet unidentified, subunits may have been non-covalently associated with the A and B subunits. Analysis of reduced and non-reduced crosslinked TSH receptor-125I-HSAB-TSH on Sephacryl S-300 in the presence and absence of detergent indicated that the A subunit was a hydrophilic peptide. This was confirmed in studies of the release into aqueous solution by reducing agent treatment of 125I-HSAB-TSH crosslinked to the TSH receptor A subunit in thyroid membranes. Similar results were obtained with TSH receptors in human thyroid and guinea pig fat cell membranes. These studies suggest that the hydrophilic A subunit of the receptor forms a binding site for TSH on the outside surface of the cell membrane and that the A subunit is linked to the cell membrane by way of a disulphide bridge to the receptor B subunit. 相似文献
2.
J Furmaniak F A Hashim P R Buckland V B Petersen K Beever R D Howells B R Smith 《FEBS letters》1987,215(2):316-322
An investigation of the properties of TSH receptors on FRTL5 cells using affinity labelling with a 125I-labelled photoactive derivative of TSH is described. Our studies suggest that FRTL5 cells contain 2 principal types of cell surface TSH receptors. One form, probably a precursor, consists of a single polypeptide chain (Mr 120,000) with an intrachain loop of amino acids formed by a disulphide bridge. The other type of receptor consists of a water-soluble A chain (Mr 55,000) linked to an amphiphilic B chain (Mr 35,000) by a disulphide bridge. The 2 chain structure is probably derived from the single chain 120,000 protein by enzymatic cleavage of peptide sequences within the loop of amino acids formed by the intrachain disulphide bridge. 相似文献
3.
The properties of proteoglycan prepared from human articular cartilage by using associative caesium chloride gradients of high and low starting densities. 总被引:3,自引:1,他引:2
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Reduction of human thyroid membranes with dithiothreitol caused the release of a water-soluble glycoprotein which neutralized the thyrotropin (TSH) receptor-binding and thyroid-stimulating activities of Graves' serum. Analysis of the protein by gel filtration and sucrose density gradient centrifugation allowed estimates of 3.45 nm for the Stokes' radius, 3.6 S for the s20,w and 47 000 +/- 5000 (mean +/- S.D.; n = 4) for the Mr. The material released by dithiothreitol treatment could be crosslinked to 125I-labelled TSH coupled to N-hydroxysuccinimidyl 4-azidobenzoate (125I-HSAB-TSH), suggesting that it contained a component of the TSH receptor. Furthermore, analysis of the crosslinked material by sodium dodecyl sulphate/polyacrylamide-gel electrophoresis indicated that it contained the TSH receptor A subunit (Mr 50 000). Several factors suggested therefore that the glycoprotein released by dithiothreitol treatment of human thyroid membranes was the TSH receptor A subunit. In particular, (a) both preparations were hydrophilic and were released from membranes by reduction, (b) they had similar Mr values and (c) both preparations crosslinked to 125I-HSAB-TSH. Material similar to the TSH receptor A subunit was released from thyroid membranes by treatment with papain, probably as a result of cleavage of the receptor A subunit at a site close to the interchain disulphide bridge. A similar mechanism, involving thyroid proteinases, was probably involved in release of material with similar properties to the TSH receptor A subunit during freezing and thawing of human thyroid homogenates. 相似文献
4.
Elimination of the disulphide bridge in fragment B of diphtheria toxin: effect on membrane insertion, channel formation, and ATP binding 总被引:1,自引:0,他引:1
Active diphtheria toxin consists of two disulphide-linked fragments, termed A and B. Fragment B, which contains an internal disulphide bridge, facilitates translocation of the enzymatically active fragment A to the cytosol of eukaryotic cells. In this process cation-selective channels are formed. An in vitro translated full-length mutant lacking the internal disulphide bridge (A-58**) was functionally indistinguishable from its disulphide-containing counterpart (A-58) with respect to trypsin sensitivity, receptor binding, A-fragment translocation, and channel formation. In contrast, the B fragment of A-58** (B-36**) was slightly less trypsin resistant than the S-S-containing B fragment, B-36, and was approximately 300-fold less efficient than B-36 in permeabilizing cells. When first dialysed and then reconstituted with A fragment, B fragment without disulphide bridge yielded a less-active toxin than did wild-type B fragment. We conclude that the disulphide bridge in fragment B is not necessary for toxicity, as earlier believed, and that channel formation may play a role in membrane translocation. 相似文献
5.
Domain structure of proteoheparan sulphate from confluent cultures of human embryonic skin fibroblasts. 总被引:1,自引:1,他引:0
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Radiolabelled proteoheparan sulphates were isolated from confluent monolayers of fibroblasts and from their spent media. The cell-surface-associated proteoglycan (Mr 350 000) has a core protein of Mr 180 000 that is cleaved by reduction of disulphide bonds into polypeptides of Mr 90 000, both of which can bind transferrin [Fransson, Carlstedt, Cöster & Malmström (1984) Proc. Natl. Acad. Sci. U.S.A. 81, 5657-5661]. Thrombin digestion of the proteoglycan yielded two major fragments. The larger one contained the heparan sulphate chains and glycoprotein-type oligosaccharides, whereas the smaller one contained interchain disulphide bond(s) and had affinity for transferrin as well as for octyl-Sepharose. The larger thrombic fragment was cleaved by trypsin into fragments containing the heparan sulphate chains and the oligosaccharides respectively. The smaller proteoheparan sulphate derived from the culture medium (Mr 150 000) had a core protein of Mr 30 000, which contained heparan sulphate-attachment and oligosaccharide-attachment regions, but no domains for binding of transferrin or for hydrophobic interactions. 相似文献
6.
《FEBS letters》1987,220(1):116-120
The lectin from Datura stramonium seeds was separated into three individual isolectins by hydrophobic-interaction chromatography on phenyl-Sepharose. Two of these isolectins are homodimers made up of two A- or two B-subunits, whereas the third is a heterodimer composed of one A- and one B-subunit. Analysis of the homodimeric AA- and BB-isolectins revealed that the A- and B-subunits have similar but not identical Mr values (32 000 and 28 000, respectively), amino acid and carbohydrate compositions. The A-subunit has a higher affinity for N-acetyl-D-glucosamine oligomers than the B-subunit, whereas the latter is more specific for the carbohydrate determinants of some animal glycoproteins such as fetuin, asialofetuin and ovomucoid. 相似文献
7.
A Wilimowska-Pelc 《Acta biochimica Polonica》1985,32(4):351-361
A trypsin inhibitor was extracted from the kale seeds with 0.01 M-HCl, precipitated with ammonium sulphate, and purified by affinity chromatography on immobilized trypsin and ion-exchange chromatography on QAE-Sephadex A-25. The inhibitor, of Mr 8 000, is composed of 64 amino acid residues and contains neither threonine nor methionine. Its isoelectric point is 8.9. In addition to trypsin, the inhibitor acts on subtilopeptidase A and shows a very weak antichymotrypsin activity. The factors modifying the arginine residues inactivate the inhibitor. A modified form of the inhibitor (with a broken reactive site peptide bond) has been isolated in pure form, and its properties were compared with those of the virgin form. 相似文献
8.
B R Westr?m 《Hoppe-Seyler's Zeitschrift für physiologische Chemie》1979,360(12):1869-1878
Characterization of the trypsin-, chymotrypsin- and elastase-inhibiting properties of porcine serum was carried out by gel filtration on Ultrogel, AcA 44, and agarose gel electrophoresis with subsequent processing for protease-inhibiting activity. Moreover, by allowing the fractions obtained from gel filtration to react with antibodies to porcine serum protease inhibitors, the specific inhibiting properties of these inhibitor molecules were identified. At least six protease inhibitors were identified and partially characterized in porcine serum. Two alpha 2 -macroglobulins (alpha 2 Mf and alpha 2 Ms), homologues to human alpha 2 -macroglobulin, with slightly different electrophoretic mobilities, were both found to exhibit trypsin, chymotrypsin and elastase inhibiting activity. Alpha 1 -Protease inhibitor (Mr 51 000), a homologue to human alpha 1 -protease inhibitor (alpha 1 -antitrypsin), also showed trypsin-, chymotrypsin- and elastase-inhibiting properties. Inter-alpha-trypsin inhibitor (Mr 162 000 and 129000), a porcine serum counterpart to human inter-alpha-trypsin inhibitor, showed trypsin- and chymo-trypsin-inhibiting properties. In addition, a specific trypsin inhibitor, alpha 2 -antigrypsin (Mr 58 000), and a specific elastase inhibitor, beta-elastase inhibitor, were characterized in porcine serum, and these seem to have no counterparts in human serum. 相似文献
9.
S E Gennick C G Thomas S N Nayfeh 《Biochemical and biophysical research communications》1986,135(1):208-214
125I-TSH binding to porcine thyroid and guinea pig fat resulted in curvilinear Scatchard plots with similar dissociation constants for the high and low affinity binding components. Antibodies from the sera of patients with Graves' disease inhibited binding to the high and low affinity binding components of both tissues. Covalent cross-linking of 125I-TSH to membranes from each tissue resulted in the specific labeling of two protein bands. The guinea pig fat receptor subunits have Mr values of 52,000 and 38,000, whereas the porcine thyroid receptor subunits have values of 46,000 & 35,000. The labeling of the receptor subunits was inhibited by preincubation with Graves' autoantibodies. Despite possessing a different subunit composition, the receptors from these tissues exhibit similar affinity for TSH and share similar antigenic determinants for Graves' autoantibodies. 相似文献
10.
S Y Ying 《Proceedings of the Society for Experimental Biology and Medicine. Society for Experimental Biology and Medicine (New York, N.Y.)》1987,186(3):253-264
The long-sought, nonsteroidal, gonadal inhibitor of the secretion of FSH has been isolated, characterized, and the primary structure in several species (human, porcine, bovine, murine) has been deduced. Inhibins are proteins consisting of two subunits (18-kDa alpha- and 14-kDa beta-subunits) linked by disulfide bridges and two forms of inhibins were observed in human, porcine, and murine, but only one in bovine. Each form of inhibin (A and B) has a common alpha-subunit, but a highly homologous, distinct beta-subunit (beta A and beta B). The beta-subunits and the alpha-subunit are linked to form inhibins A and B which exert an inhibitory effect on basal FSH secretion, but the dimer formed by either two beta A-subunits or two distinct beta A- and two beta B-subunits (homoactivin-A and activin, respectively) possess FSH-stimulating activity. Inhibin secreted in response to FSH from the pituitary originates primarily from the granulosa cells of the ovary and the Sertoli cells of the testes, thus demonstrating a reciprocal feedback relationship. 相似文献
11.
The large and the small subunits (Mr 50 000 and 10 500 respectively) of human eosinophil peroxidase were isolated by gel filtration under reducing conditions. The subunits were very strongly associated but not apparently cross-linked by disulphide bridges. During storage, the large subunit tended to form aggregates, which required reduction to dissociate them. Amino acid analysis of the performic acid-treated large subunit showed the presence of 19 cysteic acid residues. The small subunit of eosinophil peroxidase had the same Mr value as the small subunit of myeloperoxidase. However, although these subunits have very similar amino acid compositions, they showed different patterns of peptide fragmentation after CNBr treatment. The carbohydrate of eosinophil peroxidase seemed associated exclusively with the large subunit and comprised mannose (4.5%, w/w) and N-acetylglucosamine (0.8%, w/w). The far-u.v.c.d. spectrum of the enzyme indicated the presence of relatively little ordered secondary structure. 相似文献
12.
The purification to homogeneity of a potent growth factor from porcine platelets is described. This cationic mitogen is named porcine platelet-derived growth factor (PDGF) on the basis of close structural, functional and immunological similarities to human PDGF. Porcine PDGF, like its human homologue, is a hydrophobic, disulphide cross-linked protein, which is stable to heat, acid, sodium dodecyl sulphate (SDS), and guanidine. The purified protein has an apparent mol. wt. on SDS-polyacrylamide gels of 38 000, similar to those reported for human PDGF (27 500-35 000). Amino terminal sequence analysis of native porcine PDGF gave a single 15 amino acid residue sequence, of which 11 residues were identical to the amino terminal sequence of the B chain of human PDGF. Gel permeation h.p.l.c. in guanidine solutions of the reduced protein revealed a single species of mol. wt. 17 000 suggesting that native porcine PDGF may be a homodimer of a 17 000 mol. wt. chain. Since porcine PDGF can be purified at low cost from large quantities of fresh platelets, it provides an alternative source of PDGF for structural and functional studies, and could be of use in preparing defined media for cell culture. 相似文献
13.
The complete amino acid sequence of the major Kunitz trypsin inhibitor from the seeds of Prosopsis juliflora. 总被引:1,自引:0,他引:1
A N Negreiros M M Carvalho J Xavier Filho A Blanco-Labra P R Shewry M Richardson 《Phytochemistry》1991,30(9):2829-2833
The major inhibitor of trypsin in seeds of Prosopsis juliflora was purified by precipitation with ammonium sulphate, ion-exchange column chromatography on DEAE- and CM-Sepharose and preparative reverse phase HPLC on a Vydac C-18 column. The protein inhibited trypsin in the stoichiometric ratio of 1:1, but had only weak activity against chymotrypsin and did not inhibit human salivary or porcine pancreatic alpha-amylases. SDS-PAGE indicated that the inhibitor has a Mr of ca 20,000, and IEF-PAGE showed that the pI is 8.8. The complete amino acid sequence was determined by automatic degradation, and by DABITC/PITC microsequence analysis of peptides obtained from enzyme digestions of the reduced and S-carboxymethylated protein with trypsin, chymotrypsin, elastase, the Glu-specific protease from S. aureus and the Lys-specific protease from Lysobacter enzymogenes. The inhibitor consisted of two polypeptide chains, of 137 residues (alpha chain) and 38 residues (beta chain) linked together by a single disulphide bond. The amino acid sequence of the protein exhibited homology with a number of Kunitz proteinase inhibitors from other legume seeds, the bifunctional subtilisin/alpha-amylase inhibitors from cereals and the taste-modifying protein miraculin. 相似文献
14.
It was found that fragments E (Mr = 45 000), DH (Mr = 95 000) and DL (Mr = 82 000) decrease the rate of plasmin hydrolysis of fibrin that is not cross-linked with factor XIII; the most effective inhibitor is fragment DL. The Kd values for the interactions of fragments E, DH and DL with plasmin are equal to 0.15, 0.4 and 0.04 microM, respectively. 相似文献
15.
C J van den Bergh A C Bekkers P De Geus H M Verheij G H de Haas 《European journal of biochemistry》1987,170(1-2):241-246
The cDNA coding for porcine pancreatic prophospholipase A2 (proPLA) has been cloned and expressed in Saccharomyces cerevisiae. Expression and secretion of proPLA could only be obtained after fusing the proPLA to the prepro sequence of the yeast alpha-mating factor. Upon secretion, the fusion protein was cleaved by the KEX2 protease yielding a 140-amino-acid zymogen-like form of the phospholipase A2. This protein was purified in high yield by ion-exchange chromatography. Limited proteolysis with trypsin cleaved the 'zymogen' to yield active phospholipase A2, which was indistinguishable from the authentic porcine pancreatic enzyme. These results show that a protein with a disulphide bridge content as high as 7 per 124 amino acid residues can be correctly processed by the yeast secretory apparatus. 相似文献
16.
Proteolytic cleavage of [3H]nitrobenzylthioinosine-labelled nucleoside transporter in human erythrocytes.
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The transmembrane topology of the nucleoside transporter of human erythrocytes, which had been covalently photolabelled with [3H]nitrobenzylthioinosine, was investigated by monitoring the effect of proteinases applied to intact erythrocytes and unsealed membrane preparations. Treatment of unsealed membranes with low concentrations of trypsin and chymotrypsin at 1 degree C cleaved the nucleoside transporter, a band 4.5 polypeptide, apparent Mr 66 000-45 000, to yield two radioactive fragments with apparent Mr 38 000 and 23 000. The fragment of Mr 38 000, in contrast to the Mr 23 000 fragment, migrated as a broad peak (apparent Mr 45 000-31 000) suggesting that carbohydrate was probably attached to this fragment. Similar treatment of intact cells under iso-osmotic saline conditions at 1 degree C had no effect on the apparent Mr of the [3H]nitrobenzylthioinosine-labelled band 4.5, suggesting that at least one of the trypsin cleavage sites resulting in the apparent Mr fragments of 38 000 and 23 000 is located at the cytoplasmic surface. However, at low ionic strengths the extracellular region of the nucleoside transporter is susceptible to trypsin proteolysis, indicating that the transporter is a transmembrane protein. In contrast, the extracellular region of the [3H]cytochalasin B-labelled glucose carrier, another band 4.5 polypeptide, was resistant to trypsin digestion. Proteolysis of the glucose transporter at the cytoplasmic surface generated a radiolabelled fragment of Mr 19 000 which was distinct from the Mr 23 000 fragment radiolabelled with [3H]nitrobenzylthioinosine. The affinity for the reversible binding of [3H]cytochalasin B and [3H]nitrobenzylthioinosine to the glucose and nucleoside transporters, respectively, was lowered 2-3-fold following trypsin treatment of unsealed membranes, but the maximum number of inhibitor binding sites was unaffected despite the cleavage of band 4.5 to lower-Mr fragments. 相似文献
17.
Two trypsin inhibitors (TI-1, TI-2) were isolated from guinea pig plasma and purified to homogeneity. In amino-acid composition as well as molecular masses, TI-1 (Mr 58,000) and TI-2 (Mr 57,000) are similar to each other and to human and mouse alpha 1-proteinase inhibitors, and mouse con-trapsin. The two inhibitors form equimolar complexes with proteinases. The effectiveness of the inhibitors was characterized by association rate constants under second-order rate conditions. The inhibitory action of TI-1 was rapid for bovine trypsin, porcine pancreatic elastase and guinea pig plasma kallikrein, but slow for bovine thrombin and guinea pig plasmin and not detectable for bovine chymotrypsin and porcine pancreatic kallikrein. The inhibitory action of TI-2 was rapid for trypsin and chymotrypsin, but slow for guinea pig plasma kallikrein and not detectable for other proteinases. These results show that TI-1 and TI-2 are physicochemically similar but functionally distinct from each other and from human alpha 1-proteinase inhibitor that inhibits trypsin, chymotrypsin and elastase. 相似文献
18.
Structural diversity and domain composition of a unique collagenous fragment (intima collagen) obtained from human placenta. 总被引:23,自引:3,他引:20
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Intima collagen was obtained from pepsin digests of human placenta in two forms, which differ to some extent in the size of their constituent polypeptide chains (Mr 50 000-70 000). These chains are connected by disulphide bonds to large aggregates. The aggregates are arranged in a triple-helical conformation with a remarkably high thermal stability (Tm 41-62 degrees C) and are resistant to further proteolytic digestion. Reduction of as little as 5% of the disulphide bonds produces mainly monomeric triple helices (Mr about 160 000) with Tm 32 degrees C. Partially reduced material can be separated into triple-helical and non-collagenous domains by proteolysis. Pepsin releases a collagenous component with chains of Mr 38 000. Bacterial collagenase liberates two non-collagenous segments (Mr 15 000-30 000) rich in cystine. Treatment with collagenase before reduction separates intima collagen into a large fragment composed of collagenous (Tm 41 degrees C) and non-collagenous structures and a single non-collagenous segment. The data support the electron-microscopical model of intima collagen [Furthmayr, Wiedemann, Timpl, Odermatt & Engel (1983) Biochem. J. 211, 303-311], indicating that the basic unit of the fragment consists of a continuous triple helix joining two globular domains. 相似文献
19.
Species specificity of human and murine tumor necrosis factor. A comparative study of tumor necrosis factor receptors 总被引:10,自引:0,他引:10
R A Smith M Kirstein W Fiers C Baglioni 《The Journal of biological chemistry》1986,261(32):14871-14874
Recombinant murine and human tumor necrosis factor (mTNF and hTNF, respectively) were radioiodinated to high specific activity using a solid-phase lactoperoxidase method. A single class of high affinity receptors for 125I-TNF was identified on TNF-sensitive murine L cells and human HeLa S2 cells. Competitive radioligand binding assays were used to study the species specificity of TNF preparations. Unlabeled hTNF competed 30-fold less effectively than mTNF for binding to L cell receptors, whereas mTNF competed to approximately the same extent as hTNF for binding to HeLa cell receptors. A similar species specificity was observed in cytotoxicity assays; hTNF was more cytotoxic for HeLa cells than mTNF. Conversely, mTNF was more growth inhibitory and cytotoxic for L cells than hTNF. mTNF. and hTNF.receptor complexes were compared by gel filtration chromatography and polyacrylamide gel electrophoresis before and after cross-linking with bis[2-(succinimidooxycarbonyloxy)ethyl]sulfone (BSOCOES). These complexes eluted in gel filtration at a position corresponding to a globular protein of 350,000 Mr. Gel autoradiographs of the fractions containing cross-linked complexes showed bands of 95,000 and 75,000 Mr as well as small amounts of higher Mr bands. mTNF and hTNF treated with BSOCOES formed cross-linked dimers and trimers. Therefore, we were unable to determine whether the 95,000 and 75,000 Mr bands represented two distinct subunits of receptors or one subunit to which either a dimer or a monomer of TNF was cross-linked. These results demonstrate species specificity in the TNF-receptor interaction. In addition, the affinity labeling studies in two species give an identical pattern for the TNF X receptor complexes, suggesting that the receptors have similar subunit composition. 相似文献
20.
The genes encoding A1-ATPase A- and B-subunits were cloned from Haloarcula japonica strain TR-1. Nucleotide sequencing analysis of the A1-ATPase gene revealed that the A- and B-subunits consisted of 586 and 473 amino acids, respectively. The deduced amino acid sequences of the A- and B-subunits of Ha. japonica showed high identities with those of Halobacterium salinarum and Haloferax volcanii. The consensus ATP-binding motif was found in the A-subunit. 相似文献