Analysis of thyrotropin receptors by photoaffinity labelling. Orientation of receptor subunits in the cell membrane

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.

Download Full-text

Interaction of autoantibodies to thyrotropin receptor with a hydrophilic subunit of the thyrotropin receptor

Biochemical Journal ◽

10.1042/bj2280111 ◽

1985 ◽

Vol 228 (1) ◽

pp. 111-117 ◽

Cited By ~ 10

Author(s):

E Davies Jones ◽

F A Hashim ◽

Y Kajita ◽

F M Creagh ◽

P R Buckland ◽

...

Keyword(s):

Gradient Centrifugation ◽

Polyacrylamide Gel Electrophoresis ◽

Sodium Dodecyl ◽

Water Soluble ◽

Tsh Receptor ◽

Human Thyroid ◽

Thyrotropin Receptor ◽

Freezing And Thawing ◽

Sucrose Density Gradient Centrifugation ◽

A Subunit

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.

Download Full-text

The covalent nature of the human antithrombin III–thrombin bond

Biochemical Journal ◽

10.1042/bj1890481 ◽

1980 ◽

Vol 189 (3) ◽

pp. 481-489 ◽

Cited By ~ 55

Author(s):

M O Longas ◽

T H Finlay

Keyword(s):

Gel Electrophoresis ◽

Antithrombin Iii ◽

Polyacrylamide Gel Electrophoresis ◽

Sodium Dodecyl ◽

Native Protein ◽

Disulphide Bridge ◽

Terminal Amino Acid ◽

Carboxypeptidase B ◽

Terminal Amino ◽

Covalent Nature

1. Cleavage of the human antithrombin III–thrombin complex with [14C]methoxyamine hydrochloride results in inactive thrombin and 14C-labelled antithrombin III. 2. Discontinuous polyacrylamide-gel electrophoresis of the reduced dissociation fragments of the complex in the presence of sodium dodecyl sulphate reveals two antithrombin III bands that do not resolve during electrophoresis without reduction. The heavy band has the electrophoretic mobility of the native protein. The light band has an apparent mol.wt. that is approx. 4000 less than the molecular weight of native antithrombin III. 3. Treatment of the cleavage products of the complex with carboxypeptidase B yields 1 mumol of arginine, a new C-terminal amino acid, per mumol of thrombin dissociated. The results indicate that during formation of the antithrombin III–thrombin complex, the inhibitor is cleaved at an arginine–X bond; this arginine residue forms a carboxylic ester with the enzyme, while the excised polypeptide remains bound through a disulphide bridge(s).

Download Full-text

The metabolism of benzene by bacteria. Purification and some properties of the enzyme cis-1,2-dihydroxycyclohexa-3,5-diene (nicotinamide–adenine dinucleotide) oxidoreductase (cis-benzene glycol dehydrogenase)

Biochemical Journal ◽

10.1042/bj1360927 ◽

1973 ◽

Vol 136 (4) ◽

pp. 927-934 ◽

Cited By ~ 39

Author(s):

B. C. Axcell ◽

P. J. Geary

Keyword(s):

Molecular Weight ◽

Polyacrylamide Gel Electrophoresis ◽

Sodium Dodecyl ◽

Homogeneous State ◽

Hydrogen Acceptor ◽

Diffusion Analysis ◽

A Subunit ◽

Partial Inactivation ◽

And Diffusion ◽

Cis Isomer

1. cis-Benzene glycol dehydrogenase was purified to a homogeneous state from a species of Pseudomonas grown with benzene as the major carbon source. 2. The enzyme was specific for the cis-isomer of its substrate and required NAD+as hydrogen acceptor. 3. Partial inactivation of the enzyme, which was observed during purification, could be reversed by the addition of Fe2+and GSH. 4. A molecular weight of 440000 was calculated from data obtained by sedimentation-velocity and diffusion analysis in the ultracentrifuge. Sodium dodecyl sulphate polyacrylamide-gel electrophoresis indicated a subunit of molecular weight 110000. 5. p-Chloromercuribenzoic acid and 1,10-phenanthroline were shown to inhibit the enzyme.

Download Full-text

A bifunctional enzyme complex in coenzyme A biosynthesis: purification of pantetheine phosphate adenylyltransferase and dephospho-CoA kinase

Biochemical Journal ◽

10.1042/bj2150153 ◽

1983 ◽

Vol 215 (1) ◽

pp. 153-157 ◽

Cited By ~ 27

Author(s):

D M Worrall ◽

P K Tubbs

Keyword(s):

Gel Electrophoresis ◽

Polyacrylamide Gel Electrophoresis ◽

Sodium Dodecyl ◽

Gel Filtration ◽

Bifunctional Enzyme ◽

Enzyme Complex ◽

Major Band ◽

A Subunit ◽

Dimeric Protein ◽

Coenzyme A Biosynthesis

Pantetheine phosphate adenylyltransferase (EC 2.7.7.3) and dephospho-CoA kinase (EC 2.7.1.24) were purified to near homogeneity from pig liver. The purification steps included the use of Sepharose-linked triazine dyes and affinity elution by CoA. Both activities co-purified at every stage of the 18 000-fold purification. An Mr of 115 000 was obtained by gel filtration on Sephadex G-150, and the final preparation yielded one major band on sodium dodecyl sulphate/polyacrylamide-gel electrophoresis, with a subunit Mr of 57 000. It is concluded that pantetheine phosphate adenylyltransferase and dephospho-CoA kinase exist as a bifunctional dimeric protein, which could be designated CoA synthetase.

Download Full-text

Pathogenesis of Shigella diarrhea. IX. Simplified high yield purification of Shigella toxin and characterization of subunit composition and function by the use of subunit-specific monoclonal and polyclonal antibodies.

Journal of Experimental Medicine ◽

10.1084/jem.160.6.1767 ◽

1984 ◽

Vol 160 (6) ◽

pp. 1767-1781 ◽

Cited By ~ 85

Author(s):

A Donohue-Rolfe ◽

G T Keusch ◽

C Edson ◽

D Thorley-Lawson ◽

M Jacewicz

Keyword(s):

Cell Surface ◽

Hela Cell ◽

Specific Activity ◽

Polyclonal Antibodies ◽

Sodium Dodecyl ◽

Fold Increase ◽

High Yield ◽

B Subunit ◽

A Subunit ◽

High Yields

A simple purification scheme for shigella cytotoxin was devised, resulting in high yields (approximately 50%) and a 1,300-fold increase in specific activity compared with the initial crude bacterial cell lysate. The purified toxin was enterotoxic in ligated rabbit ileal loops and neurotoxic when injected into the peritoneal cavity of mice. Measurement of specific activity of cytotoxin and enterotoxin demonstrated that these two toxicities copurify during the fractionation procedure. On sodium dodecyl sulfate gel electrophoresis, the toxin migrated as two polypeptide subunits, an A subunit of 32,000 mol wt and a B subunit of 6,500 mol wt. Chemical cross-linking experiments demonstrate that the toxin is a complex consisting of one A and five B subunits with a molecular weight of 64,000. Polyclonal rabbit anti-toxin and anti-subunit B antisera were produced as well as subunit-specific mouse monoclonal antibodies. All antibodies preincubated with toxin neutralized cytotoxic effects in HeLa cell monolayers. In contrast, only A subunit-specific antibodies were able to neutralize toxin prebound to the HeLa cell surface. Antibody to the B subunit also inhibited binding of 125I-labeled toxin to these cells by 94% or more. These data demonstrate that the B subunit is involved in shigella toxin binding to the cell surface.

Download Full-text

Inactivated Pseudomonas PE(ΔIII) exotoxin fused to neutralizing epitopes of PEDV S proteins produces a specific immune response in mice

Animal Diseases ◽

10.1186/s44149-021-00021-9 ◽

2021 ◽

Vol 1 (1) ◽

Author(s):

Leqiang Sun ◽

Yajie Tang ◽

Keji Yan ◽

Huanchun Chen ◽

Huawei Zhang

Keyword(s):

Immune Responses ◽

Neutralizing Antibodies ◽

Polyacrylamide Gel Electrophoresis ◽

Sodium Dodecyl ◽

Mucosal Vaccine ◽

Economic Losses ◽

Swine Industry ◽

Diarrhea Virus ◽

B Subunit ◽

Porcine Epidemic Diarrhea

AbstractPorcine epidemic diarrhea (PED) caused by the porcine epidemic diarrhea virus (PEDV), is a severe infectious and devastating swine disease that leads to serious economic losses in the swine industry worldwide. An increased number of PED cases caused by variant PEDV have been reported in many countries since 2010. S protein is the main immunogenic protein containing some B-cell epitopes that can induce neutralizing antibodies of PEDV. In this study, the construction, expression and purification of Pseudomonas aeruginosa exotoxin A (PE) without domain III (PEΔIII) as a vector was performed for the delivery of PEDV S-A or S-B. PE(ΔIII) PEDV S-A and PE(ΔIII) PEDV S-B recombinant proteins were confirmed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and Western blot analysis. The immunogenicity of PEDV S-A and PEDV S-B subunit vaccines were evaluated in mice. The results showed that PEDV-S-B vaccine could not only induce specific humoral and Th1 type-dominant cellular immune responses, but also stimulate PEDV-specific mucosal immune responses in mice. PEDV-S-B subunit vaccine is a novel candidate mucosal vaccine against PEDV infection.

Download Full-text

Degradation of Chloroaromatics: Purification and Characterization of a Novel Type of Chlorocatechol 2,3-Dioxygenase of Pseudomonas putida GJ31

Journal of Bacteriology ◽

10.1128/jb.180.2.296-302.1998 ◽

1998 ◽

Vol 180 (2) ◽

pp. 296-302 ◽

Cited By ~ 78

Author(s):

Stefan R. Kaschabek ◽

Thomas Kasberg ◽

Dagmar Müller ◽

Astrid E. Mars ◽

Dick B. Janssen ◽

...

Keyword(s):

Pseudomonas Putida ◽

Molecular Mass ◽

Polyacrylamide Gel Electrophoresis ◽

Sodium Dodecyl ◽

Gel Filtration ◽

Great Sensitivity ◽

Kinetic Properties ◽

Extradiol Dioxygenase ◽

A Subunit ◽

Extradiol Dioxygenases

ABSTRACT A purification procedure for a new kind of extradiol dioxygenase, termed chlorocatechol 2,3-dioxygenase, that converts 3-chlorocatechol productively was developed. Structural and kinetic properties of the enzyme, which is part of the degradative pathway used for growth ofPseudomonas putida GJ31 with chlorobenzene, were investigated. The enzyme has a subunit molecular mass of 33.4 kDa by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Estimation of the native M r value under nondenaturating conditions by gel filtration gave a molecular mass of 135 ± 10 kDa, indicating a homotetrameric enzyme structure (4 × 33.4 kDa). The pI of the enzyme was estimated to be 7.1 ± 0.1. The N-terminal amino acid sequence (43 residues) of the enzyme was determined and exhibits 70 to 42% identity with other extradiol dioxygenases. Fe(II) seems to be a cofactor of the enzyme, as it is for other catechol 2,3-dioxygenases. In contrast to other extradiol dioxygenases, the enzyme exhibited great sensitivity to temperatures above 40°C. The reactivity of this enzyme toward various substituted catechols, especially 3-chlorocatechol, was different from that observed for other catechol 2,3-dioxygenases. Stoichiometric displacement of chloride occurred from 3-chlorocatechol, leading to the production of 2-hydroxymuconate.

Download Full-text

Characterization of three kinetically distinct forms of glutamate decarboxylase from pig brain

Biochemical Journal ◽

10.1042/bj2310695 ◽

1985 ◽

Vol 231 (3) ◽

pp. 695-703 ◽

Cited By ~ 47

Author(s):

D C Spink ◽

T G Porter ◽

S J Wu ◽

D L Martin

Keyword(s):

Sodium Dodecyl Sulphate ◽

Glutamate Decarboxylase ◽

Polyacrylamide Gel Electrophoresis ◽

Sodium Dodecyl ◽

Kinetic Properties ◽

First Order ◽

Pig Brain ◽

A Subunit ◽

Activation Cycle

Pig brain contains three forms of glutamate decarboxylase with pI values of 5.3, 5.5 and 5.8, referred to as the α-, β- and γ-forms respectively. These forms were purified and kinetically characterized. The major synaptic form of glutamate decarboxylase (the β-form) migrated as a single band on electrophoresis in sodium dodecyl sulphate/polyacrylamide gels with an apparent Mr of 60 000. Sodium dodecyl sulphate/polyacrylamide gel electrophoresis followed by immunoblotting with an affinity-purified antibody to the enzyme indicated a subunit Mr of 60 000 for the α- and γ-forms as well. An extensive kinetic analysis, aided by an integrated equation that describes the inactivation and re-activation cycle of the enzyme, revealed that the three forms of the enzyme differ markedly in kinetic properties. The Km values for L-glutamate were 0.17, 0.45 and 1.24 mM respectively for the α-, β- and γ-forms. The Ki for 4-aminobutyrate, the first-order rate constants for inactivation by L-glutamate and 4-aminobutyrate, the rate constant for re-activation of the apoenzyme by pyridoxal 5′-phosphate and the dissociation constant for pyridoxal 5′-phosphate also differed in a similar way among the three forms; the values were in the order α-form less than β-form less than γ-form.

Download Full-text

Properties of thyrotropin receptor on cloned hybrid human thyroid cells

Acta Endocrinologica ◽

10.1530/acta.0.114s186 ◽

1987 ◽

Vol 116 (1_Suppl) ◽

pp. S186-S192

Author(s):

Jean-Jacques Rémy ◽

Jean Salamero ◽

Jeannine Charreire

Keyword(s):

Binding Sites ◽

Polyacrylamide Gel Electrophoresis ◽

Sodium Dodecyl ◽

Human Thyroid ◽

Relative Molecular Mass ◽

Thyrotropin Receptor ◽

Single Chain ◽

Thyroid Cells ◽

Disulphide Bonds ◽

Dimethyl Suberimidate

Abstract. Purification of the thyrotropin (TSH) binding sites from cloned human thyroid cells (GEJ) was performed after biosynthetic labelling of the cells, affinity chromatography on a human TSH-sepharose column and polyacrylamide gel electrophoresis in sodium dodecyl sulphate (PAGE-SDS). The relative molecular mass (Mr) of the GEJ cell TSH receptor (TSH-R) was approximately 48 000. This was confirmed by crosslinking [125I]TSH to GEJ binding sites with two homo-bifunctional agents: dimethyl suberimidate and disuccinimidyl suberate. Moreover, the absence of a dithiothreitol effect demonstrated that the TSH binding site on GEJ cells is formed by a single chain lacking disulphide bonds.

Download Full-text

Abnormal ratio of membrane immunoglobulin classes in mice with an X-linked B-lymphocyte defect.

Journal of Experimental Medicine ◽

10.1084/jem.142.5.1316 ◽

1975 ◽

Vol 142 (5) ◽

pp. 1316-1321 ◽

Cited By ~ 45

Author(s):

F D Finkelman ◽

A H Smith ◽

I Scher ◽

W E Paul

Keyword(s):

Plasma Membrane ◽

Cell Membrane ◽

B Lymphocyte ◽

Polyacrylamide Gel Electrophoresis ◽

Genetic Defect ◽

Sodium Dodecyl ◽

Cell Types ◽

Antibody Responses ◽

Lymphocyte Function ◽

Membrane Immunoglobulin

CBA/N mice have an X-linked genetic defect in B-lymphocyte function manifested by inability to make antibody responses to T-independent antigens. Plasma membrane immunoglobulin (Ig) on spleen, lymph node, and Peyer's patch cells was analyzed by lactoperoxidase-catalyzed iodination, NP-40 extraction, specific immunoprecipitation, and sodium dodecyl sulfate-polyacrylamide gel electrophoresis. These studies indicated that the X-linked immune defect was associated, in all three cell types, with a decrease in the ratio of cell membrane IgD analog to cell membrane IgM. This suggests either that IgD analog may be important in initiation of T-independent antibody responses or that CBA/N mice lack a subpopulation of B cells specialized to respond to T-independent antigens, and that these cells are relatively rich in plasma membrane IgD analog.

Download Full-text