scholarly journals Location of subunits within the acetylcholine receptor by electron image analysis of tubular crystals from Torpedo marmorata.

1987 ◽  
Vol 105 (1) ◽  
pp. 9-18 ◽  
Author(s):  
E Kubalek ◽  
S Ralston ◽  
J Lindstrom ◽  
N Unwin
Keyword(s):  
Monoclonal Antibody ◽  
Binding Sites ◽  
Alpha Subunit ◽  
Torpedo Marmorata ◽  
Fab Fragment ◽  
Alpha Subunits ◽  
Immunogenic Region ◽  
Electron Image ◽  
Alpha Beta ◽  
Tubular Crystals

The binding sites on the nicotinic acetylcholine receptor of labels specific for the alpha-, beta-, and delta-subunits were determined by electron image analysis, using tubular crystals of receptors grown from the postsynaptic membranes of Torpedo marmorata electric organ. The labels were alpha-bungarotoxin (which attaches to the acetylcholine binding sites on the pair of alpha-subunits), Fab35 (a monoclonal antibody Fab fragment directed against the main immunogenic region of the alpha-subunit), Fab111 (a monoclonal antibody Fab fragment directed against a cytoplasmic site on the beta-subunit), and wheat germ agglutinin (which binds to N-acetylglucosamine residues on the delta-subunit). These labels, bound to receptors in the crystals, were located by comparing labeled with native structures, averaged in each case over more than 5,000 molecules. From the assignments made, we find that the clockwise arrangement of subunits around the receptor, viewed from the synaptic face, is: alpha, beta, alpha, gamma, and delta; that the main immunogenic region is at (or close to) the side of the alpha-subunit; and that the two acetylcholine binding sites are at the synaptic end of the alpha-subunits, 27-28 A from the central axis and approximately 53 A apart. In the crystal lattice, neighboring molecules are paired so that their delta- and alpha-subunits are juxtaposed, an organization that appears to relate closely to the grouping of receptors in vivo.

scholarly journals Monoclonal antibody-defined functional epitopes on the adhesion- promoting glycoprotein complex (CDw18) of human neutrophils

Blood ◽  
1986 ◽  
Vol 67 (4) ◽  
pp. 1007-1013 ◽  
Author(s):  
WJ Wallis ◽  
DD Hickstein ◽  
BR Schwartz ◽  
CH June ◽  
HD Ochs ◽  
...  
Keyword(s):  
Monoclonal Antibody ◽  
Active Site ◽  
Bacterial Infections ◽  
Quaternary Structure ◽  
Alpha Subunit ◽  
Human Neutrophils ◽  
Glycoprotein Complex ◽  
Dose Dependent Fashion ◽  
Alpha Beta ◽  
Dose Dependent

Abstract We have evaluated the functional and immunochemical activities of three monoclonal antibodies (MoAbs) minimally reactive with adherence- defective neutrophils (PMN) from a patient with recurrent bacterial infections. In studies with normal PMN, MoAbs OKM1 and 60.1 both precipitate the same 165kd alpha-subunit (alpha M) within an alpha-beta heterodimer complex (CD11). The CD11 complex is part of a larger complex composed of four glycoproteins (CDw18) precipitated by MoAb 60.3, with properties suggesting that the CDw18 complex is equivalent to the Mac-1, LFA-1, p150, 95 glycoprotein family implicated in adherence-dependent leukocyte functions. PMN adherence to endothelium, spreading on surfaces, aggregation, and phagocytosis of zymosan particles were all inhibited in a dose-dependent fashion by MoAb 60.1 (analogous to previous studies with MoAb 60.3) while MoAb OKM1 had no effect. These findings unify previously disparate observations and suggest that a functionally active site on the adherence promoting glycoprotein complexes CD11 and CDw18 is distant from the alpha M epitope recognized by MoAb OKM1 but closely associated with the alpha M epitope recognized by MoAb 60.1 and the beta-epitope (or epitope created by alpha-beta quaternary structure) recognized by MoAb 60.3.

scholarly journals The crystal structure of the Fab fragment of a rat monoclonal antibody against the main immunogenic region of the human muscle acetylcholine receptor

2000 ◽  
Vol 267 (8) ◽  
pp. 2389-2397 ◽  
Author(s):  
Maria Kontou ◽  
Demetres D Leonidas ◽  
Efstratia H. Vatzaki ◽  
Panayiota Tsantili ◽  
Avgi Mamalaki ◽  
...  
Keyword(s):  
Crystal Structure ◽  
Monoclonal Antibody ◽  
Acetylcholine Receptor ◽  
Human Muscle ◽  
Fab Fragment ◽  
Immunogenic Region

Epitope-selective, monoclonal-antibody-based immunoradiometric assays of predictable specificity for differential measurement of choriogonadotropin and its subunits.

1985 ◽  
Vol 31 (8) ◽  
pp. 1322-1328 ◽  
Author(s):  
S Schwarz ◽  
P Berger ◽  
G Wick
Keyword(s):  
Monoclonal Antibody ◽  
Monoclonal Antibodies ◽  
Conformational Epitope ◽  
Alpha Subunit ◽  
Beta Subunits ◽  
Differential Measurement ◽  
Analytical Strategy ◽  
Alpha Subunits ◽  
Human Choriogonadotropin ◽  
Beta Hcg

Abstract Knowing the epitope specificities of our monoclonal antibodies (MCA) to human choriogonadotropin (hCG), we could design three classes of two-site immunoradiometric assays (IRMA): a combination of two MCA recognizing two separate alpha-epitopes (alpha-MCA) provides a system (i.e., an alpha-IRMA) that measures holo-hCG plus free alpha-subunits plus follitropin, lutropin, and thyrotropin, whereas a beta-IRMA, consisting of two beta-MCA, quantifies holo-hCG plus free beta-subunits. The amount of either of the two subunits can be calculated by subtracting the amount of holo-hCG determined in parallel in a holo-hCG-IRMA. In the latter, one of the alpha- or beta-MCA may be either cross-combined or, preferably, paired with an MCA specific for a conformational epitope. These analytical specificities, predicted from our previously established epitope map of hCG, could be experimentally verified. With these IRMAS we could demonstrate that in certain choriocarcinoma cell lines the earliest and quantitatively predominant tumor marker is the free alpha-subunit. Similar results showing an unbalanced secretion of hCG and its subunits were obtained for patients with related tumors. These findings challenge the present diagnostic practice of relying solely on "beta-hCG" radioimmunoassays and at the same time offer a novel analytical strategy.

scholarly journals Light-dependent GTP-binding proteins in squid photoreceptors

1990 ◽  
Vol 272 (1) ◽  
pp. 79-85 ◽  
Author(s):  
P R Robinson ◽  
S F Wood ◽  
E Z Szuts ◽  
A Fein ◽  
H E Hamm ◽  
...  
Keyword(s):  
Monoclonal Antibody ◽  
G Protein ◽  
G Proteins ◽  
Alpha Subunit ◽  
Protein G ◽  
Adp Ribosylation ◽  
Alpha Subunits ◽  
Gtp Binding ◽  
Molecular Masses ◽  
Lines Of Evidence

Previous biochemical and electrophysiological evidence suggests that in invertebrate photoreceptors, a GTP-binding protein (G-protein) mediates the actions of photoactivated rhodopsin in the initial stages of transduction. We find that squid photoreceptors contain more than one protein (molecular masses 38, 42 and 46 kDa) whose ADP-ribosylation by bacterial exotoxins is light-sensitive. Several lines of evidence suggest that these proteins represent distinct alpha subunits of G-proteins. (1) Pertussis toxin and cholera toxin react with distinct subsets of these polypeptides. (2) Only the 42 kDa protein immunoreacts with the monoclonal antibody 4A, raised against the alpha subunit of the G-protein of vertebrate rods [Hamm & Bownds (1984) J. Gen. Physiol. 84. 265-280]. (3) In terms of ADP-ribosylation, the 42 kDa protein is the least labile to freezing. (4) Of the 38 kDa and 42 kDa proteins, the former is preferentially extracted with hypo-osmotic solutions, as demonstrated by the solubility of its ADP-ribosylated state and by the solubility of the light-dependent binding of guanosine 5′-[gamma-thio]triphosphate. The specific target enzymes for the observed G-proteins have not been established.

scholarly journals Monoclonal antibody-defined functional epitopes on the adhesion- promoting glycoprotein complex (CDw18) of human neutrophils

Blood ◽  
1986 ◽  
Vol 67 (4) ◽  
pp. 1007-1013 ◽  
Author(s):  
WJ Wallis ◽  
DD Hickstein ◽  
BR Schwartz ◽  
CH June ◽  
HD Ochs ◽  
...  
Keyword(s):  
Monoclonal Antibody ◽  
Active Site ◽  
Bacterial Infections ◽  
Quaternary Structure ◽  
Alpha Subunit ◽  
Human Neutrophils ◽  
Glycoprotein Complex ◽  
Dose Dependent Fashion ◽  
Alpha Beta ◽  
Dose Dependent

We have evaluated the functional and immunochemical activities of three monoclonal antibodies (MoAbs) minimally reactive with adherence- defective neutrophils (PMN) from a patient with recurrent bacterial infections. In studies with normal PMN, MoAbs OKM1 and 60.1 both precipitate the same 165kd alpha-subunit (alpha M) within an alpha-beta heterodimer complex (CD11). The CD11 complex is part of a larger complex composed of four glycoproteins (CDw18) precipitated by MoAb 60.3, with properties suggesting that the CDw18 complex is equivalent to the Mac-1, LFA-1, p150, 95 glycoprotein family implicated in adherence-dependent leukocyte functions. PMN adherence to endothelium, spreading on surfaces, aggregation, and phagocytosis of zymosan particles were all inhibited in a dose-dependent fashion by MoAb 60.1 (analogous to previous studies with MoAb 60.3) while MoAb OKM1 had no effect. These findings unify previously disparate observations and suggest that a functionally active site on the adherence promoting glycoprotein complexes CD11 and CDw18 is distant from the alpha M epitope recognized by MoAb OKM1 but closely associated with the alpha M epitope recognized by MoAb 60.1 and the beta-epitope (or epitope created by alpha-beta quaternary structure) recognized by MoAb 60.3.

scholarly journals Expression of wild-type and mutated rabbit osteopontin in Escherichia coli, and their effects on adhesion and migration of P388D1 cells

1995 ◽  
Vol 307 (1) ◽  
pp. 257-265 ◽  
Author(s):  
K Nasu ◽  
T Ishida ◽  
M Setoguchi ◽  
Y Higuchi ◽  
S Akizuki ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Fluid Phase ◽  
Intradermal Injection ◽  
Polymorphonuclear Leucocyte ◽  
Alpha Subunit ◽  
Wild Type ◽  
Alpha Subunits ◽  
Leucocyte Migration ◽  
And Migration ◽  
Chemotactic Effect

Recombinant wild-type rabbit osteopontin (rOP) and the protein with an aspartate-to-glutamate transposition induced by a point mutation in the rabbit OP cDNA within the Gly-Arg-Gly-Asp-Ser (GRGDS) sequence were expressed in Escherichia coli and purified to homogeneity. P388D1 cells bound rOP in a saturable manner. rOP induced adhesion and haptotaxis of P388D1 cells, whereas mutated rabbit OP (rOPmut) did not. Anti-rOP IgG F(ab′)2 and synthetic GRGDS peptide inhibited rOP-mediated adhesion and haptotaxis of P388D1 cells. Fibronectin (FN)-mediated adhesion of P388D1 cells was markedly inhibited in the presence of fluid-phase rOP. Adhesion of P388D1 cells to rOP was significantly inhibited by anti-[alpha-subunits of VLA4 (alpha 4) and VLA5 (alpha 5)] monoclonal antibodies (mAbs), but not by anti-[alpha-subunit of vitronectin (VN) receptor (alpha V) or Mac-1 (alpha M)] mAb. Adhesion of P388D1 cells to FN and VN was significantly inhibited by anti-alpha V mAb but not anti-alpha 4, -alpha 5 or -alpha M mAb. Haptotaxis of P388D1 cells to rOP was significantly inhibited by anti-alpha V mAb, but not by anti-alpha 4, -alpha 5 and alpha M mAbs, whereas that to FN showed no inhibition with all three mAbs. Haptotaxis of P388D1 cells to VN was significantly inhibited by anti-alpha 5 and -alpha V mAbs but not by anti-alpha 4 and -alpha M mAbs. Similar features of inhibition of adhesion and haptotaxis of P388D1 cells to human OP were observed by mAbs. rOP had no chemotactic effect on P388D1 cells. Significant polymorphonuclear leucocyte migration was observed 3-12 h after intradermal injection of rOP into rabbits.

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