[32] Use of cross-linking reagents for detection of subunit interactions of membrane-bound Na+,K+-ATPase

1988 ◽  
pp. 345-350 ◽  
Author(s):  
Wu-Hsiung Huang ◽  
Sham S. Kakar ◽  
Sankaridrug M. Periyasamy ◽  
Amir Askari
Keyword(s):  
Cross Linking ◽  
Subunit Interactions ◽  
Membrane Bound

scholarly journals Characterization of the stilbenedisulphonate binding site on band 3 Memphis variant II (Pro-854→Leu)

1996 ◽  
Vol 317 (2) ◽  
pp. 509-514 ◽  
Author(s):  
James M. SALHANY ◽  
Renee L. SLOAN ◽  
Lawrence M. SCHOPFER
Keyword(s):  
Binding Site ◽  
Conformational Changes ◽  
Covalent Binding ◽  
Band 3 ◽  
Blood Group Antigen ◽  
Cross Linking ◽  
Anion Exchange Protein ◽  
Membrane Bound ◽  
And Control

Band 3 Memphis variant II is a mutant anion-exchange protein associated with the Diego a+ blood group antigen. There are two mutations in this transporter: Lys-56 → Glu within the cytoplasmic domain, and Pro-854 → Leu within the membrane-bound domain. The Pro-854 mutation, which is thought to give rise to the antigenicity, is located within the C-terminal subdomain of the membrane-bound domain. Yet, there is an apparent enhancement in the rate of covalent binding of H2DIDS (4,4´-di-isothiocyanatodihydro-2,2´-stilbenedisulphonate) to ‘lysine A’ (Lys-539) in the N-terminal subdomain, suggesting widespread conformational changes. In this report, we have used various kinetic assays which differentiate between conformational changes in the two subdomains, to characterize the stilbenedisulphonate site on band 3 Memphis variant II. We have found a significantly higher H2DIDS (a C-terminal-sensitive inhibitor) affinity for band 3 Memphis variant II, due to a lower H2DIDS ‘off’ rate constant, but no difference was found between mutant and control when DBDS (4,4´-dibenzamido-2,2´-stilbenedisulphonate) (a C-terminal-insensitive inhibitor) ‘off’ rates were measured. Furthermore, there were no differences in the rates of covalent binding to lysine A, for either DIDS (4,4´-di-isothiocyanato-2,2´-stilbenedisulphonate) or H2DIDS. However, the rate of covalent intrasubunit cross-linking of Lys-539 and Lys-851 by H2DIDS was abnormally low for band 3 Memphis variant II. These results suggest that the Pro-854 → Leu mutation causes a localized conformational change in the C-terminal subdomain of band 3.

scholarly journals VirB8 homolog TraE from plasmid pKM101 forms a hexameric ring structure and interacts with the VirB6 homolog TraD

2018 ◽  
Vol 115 (23) ◽  
pp. 5950-5955 ◽  
Author(s):  
Bastien Casu ◽  
Charline Mary ◽  
Aleksandr Sverzhinsky ◽  
Aurélien Fouillen ◽  
Antonio Nanci ◽  
...  
Keyword(s):  
Molecular Mass ◽  
Secretion System ◽  
High Molecular Mass ◽  
Full Length ◽  
Size Exclusion ◽  
Cross Linking ◽  
X Ray ◽  
Exclusion Chromatography ◽  
Plasmid Pkm101 ◽  
Membrane Bound

Type IV secretion systems (T4SSs) are multiprotein assemblies that translocate macromolecules across the cell envelope of bacteria. X-ray crystallographic and electron microscopy (EM) analyses have increasingly provided structural information on individual T4SS components and on the entire complex. As of now, relatively little information has been available on the exact localization of the inner membrane-bound T4SS components, notably the mostly periplasmic VirB8 protein and the very hydrophobic VirB6 protein. We show here that the membrane-bound, full-length version of the VirB8 homolog TraE from the plasmid pKM101 secretion system forms a high-molecular-mass complex that is distinct from the previously characterized periplasmic portion of the protein that forms dimers. Full-length TraE was extracted from the membranes with detergents, and analysis by size-exclusion chromatography, cross-linking, and size exclusion chromatography (SEC) multiangle light scattering (MALS) shows that it forms a high-molecular-mass complex. EM and small-angle X-ray scattering (SAXS) analysis demonstrate that full-length TraE forms a hexameric complex with a central pore. We also overproduced and purified the VirB6 homolog TraD and show by cross-linking, SEC, and EM that it binds to TraE. Our results suggest that TraE and TraD interact at the substrate translocation pore of the secretion system.

scholarly journals Lentil seed aquaporins form a hetero-oligomer which is phosphorylated by a Mg2+-dependent and Ca2+-regulated kinase

2000 ◽  
Vol 352 (1) ◽  
pp. 183-190 ◽  
Author(s):  
Pol HARVENGT ◽  
Ariane VLERICK ◽  
Bruno FUKS ◽  
Ruddy WATTIEZ ◽  
Jean-Marie RUYSSCHAERT ◽  
...  
Keyword(s):  
Stress Responses ◽  
Lens Culinaris ◽  
Kinase Activity ◽  
Cross Linking ◽  
Protein Storage Vacuoles ◽  
Membrane Bound ◽  
Flow Through ◽  
Growth Development ◽  
Chemical Cross Linking ◽  
Aquaporin Family

In plants, aquaporins regulate the water flow through membranes during growth, development and stress responses. We have isolated two isoforms of the aquaporin family from the protein-storage vacuoles of lentil (Lens culinaris Med.) seeds. Chemical cross-linking experiments showed that both isoforms belong to the same oligomer in the membrane and are phosphorylated by a membrane-bound protein kinase. We assigned the kinase activity to a 52kDa protein that is magnesium-dependent and calcium-regulated.

scholarly journals Rat and human neutrophil N-formyl-peptide chemotactic receptors. Species difference in the glycosylation of similar 35-38 kDa polypeptide cores

1991 ◽  
Vol 277 (1) ◽  
pp. 67-72 ◽  
Author(s):  
J J Remes ◽  
U E Petäjä-Repo ◽  
H J Rajaniemi
Keyword(s):  
Ligand Binding ◽  
Species Difference ◽  
Cross Linking ◽  
Complex Type ◽  
High Affinity ◽  
Affinity Ligand ◽  
Pngase F ◽  
Formyl Peptide ◽  
Membrane Bound ◽  
High Affinity Ligand

Rat and human neutrophil N-formyl-peptide chemotactic receptors were subjected to glycosidase and proteinase treatments to determine the extent and species differences of glycosylation and the carbohydrate requirement in the high-affinity ligand binding. N-Formyl-Nle-Leu-Phe-Nle-125I-Tyr-Lys was attached to rat and human neutrophils either before or after glycosidase and proteinase treatments, and the labelled receptors were solubilized after glutaraldehyde cross-linking and analysed by SDS/PAGE and autoradiography. Both the rat and human N-formyl-peptide chemotactic receptors contain only N-linked oligosaccharides, as demonstrated by their sensitivity to peptide N-glycosidase F (PNGase F) and resistance to O-glycanase treatment. The N-linked oligosaccharides seem to be of the complex type rather than the high-mannose or hybrid type and lack terminal sialic acid, as demonstrated by their resistance to endoglycosidases D and H and neuraminidase treatments. This sensitivity pattern was similar in both species, and the shift in the molecular size of the receptors to 35-38 kDa after PNGase F treatment occurred through one intermediate product, suggesting that both receptors contain a similar 35-38 kDa polypeptide core with two N-linked complex-type oligosaccharides, the heterogeneity of which is responsible for the species difference in receptor size. Papain treatment alone or followed by PNGase F produced in both species a 33-36 kDa membrane-bound fragment that was still able to bind the ligand, suggesting that the oligosaccharides are located on the approx. 2 kDa papain-cleavable polypeptide fragment of the receptors. The cleavage sites for both papain and PNGase F were hidden in occupied receptors, suggesting a conformational or topographical change in these upon ligand binding. Scatchard analyses and cross-linking experiments demonstrated that carbohydrates are not required for high-affinity ligand binding and that the 33-36 kDa membrane-bound papain fragment of both receptors contains the ligand-binding site.

The effects of ergosterol and alcohols on germ-tube formation and chitin synthase in Candida albicans

1982 ◽  
Vol 60 (1) ◽  
pp. 15-20 ◽  
Author(s):  
Yoke Yin Chiew ◽  
Patrick A. Sullivan ◽  
Maxwell G. Shepherd
Keyword(s):  
Candida Albicans ◽  
Germ Tube ◽  
Tube Formation ◽  
Chitin Synthase ◽  
Uridine Diphosphate ◽  
Subunit Interactions ◽  
Protease Activation ◽  
Membrane Bound ◽  
Inhibitory Components ◽  
Protease Action

Ergosterol inhibited germ-tube formation and the membrane-bound enzyme chitin synthase in Candida albicans. The sterol solvents methanol and ethanol stimulated chitin synthase activity but inhibited germination. Methanol increased both the rate of protease activation of the chitin synthase proenzyme and the Vmax of the enzyme. The activation was not due to a change in the protease action on the proenzyme. The methanol activation was reversible and therefore the extraction of inhibitory components from the membrane was not causing the activation. Methanol had no effect on the N-acetylglucosamine activation sites. Analysis of the N-acetylglucosamine activation by an iteration programme gave Ka values of 0.3 mM for the high affinity sites and 20 mM for the low affinity sites. The degree of cooperativity with respect to the substrate uridine diphosphate N-acetylglucosamine was not changed by the presence of methanol and therefore the solvent did not affect the subunit–subunit interactions of the enzyme. Arrhenius plots of chitin synthase have discontinuities and methanol did not affect the transition temperature indicating that the structure of the membrane in the immediate vicinity of the enzyme was unchanged.

scholarly journals Transglutaminase-mediated Intramolecular Cross-linking of Membrane-bound α-Synuclein Promotes Amyloid Formation in Lewy Bodies

2009 ◽  
Vol 284 (40) ◽  
pp. 27252-27264 ◽  
Author(s):  
Zoltán Nemes ◽  
Goran Petrovski ◽  
Maarten Aerts ◽  
Kjell Sergeant ◽  
Bart Devreese ◽  
...  
Keyword(s):  
Lewy Bodies ◽  
Cross Linking ◽  
Amyloid Formation ◽  
Membrane Bound

scholarly journals Interaction Interface of Mason-Pfizer Monkey Virus Matrix and Envelope Proteins

2020 ◽  
Vol 94 (20) ◽  
Author(s):  
Jan Prchal ◽  
Jakub Sýs ◽  
Petra Junková ◽  
Jan Lipov ◽  
Tomáš Ruml
Keyword(s):  
Nuclear Magnetic Resonance ◽  
Human Immunodeficiency Virus ◽  
Magnetic Resonance ◽  
Cytoplasmic Tail ◽  
Cross Linking ◽  
Terminal Part ◽  
Immunodeficiency Virus ◽  
The Matrix ◽  
Membrane Bound

ABSTRACT Retroviral envelope glycoprotein (Env) is essential for the specific recognition of the host cell and the initial phase of infection. As reported for human immunodeficiency virus (HIV), the recruitment of Env into a retroviral membrane envelope is mediated through its interaction with a Gag polyprotein precursor of structural proteins. This interaction, occurring between the matrix domain (MA) of Gag and the cytoplasmic tail (CT) of the transmembrane domain of Env, takes place at the host cell plasma membrane. To determine whether the MA of Mason-Pfizer monkey virus (M-PMV) also interacts directly with the CT of Env, we mimicked the in vivo conditions in an in vitro experiment by using a CT in its physiological trimeric conformation mediated by the trimerization motif of the GCN4 yeast transcription factor. The MA protein was used at the concentration shifting the equilibrium to its trimeric form. The direct interaction between MA and CT was confirmed by a pulldown assay. Through the combination of nuclear magnetic resonance (NMR) spectroscopy and protein cross-linking followed by mass spectrometry analysis, the residues involved in mutual interactions were determined. NMR has shown that the C terminus of the CT is bound to the C-terminal part of MA. In addition, protein cross-linking confirmed the close proximity of the N-terminal part of CT and the N terminus of MA, which is enabled in vivo by their location at the membrane. These results are in agreement with the previously determined orientation of MA on the membrane and support the already observed mechanisms of M-PMV virus-like particle transport and budding. IMPORTANCE By a combination of nuclear magnetic resonance (NMR) and mass spectroscopy of cross-linked peptides, we show that in contrast to human immunodeficiency virus type 1 (HIV-1), the C-terminal residues of the unstructured cytoplasmic tail of Mason-Pfizer monkey virus (M-PMV) Env interact with the matrix domain (MA). Based on biochemical data and molecular modeling, we propose that individual cytoplasmic tail (CT) monomers of a trimeric complex bind MA molecules belonging to different neighboring trimers, which may stabilize the MA orientation at the membrane by the formation of a membrane-bound net of interlinked Gag and CT trimers. This also corresponds with the concept that the membrane-bound MA of Gag recruits Env through interaction with the full-length CT, while CT truncation during maturation attenuates the interaction to facilitate uncoating. We propose a model suggesting different arrangements of MA-CT complexes between a D-type and C-type retroviruses with short and long CTs, respectively.

scholarly journals Cloning and characterization of Fc alpha Rb, a novel Fc alpha receptor (CD89) isoform expressed in eosinophils and neutrophils

Blood ◽  
1996 ◽  
Vol 88 (11) ◽  
pp. 4229-4238 ◽  
Author(s):  
TB van Dijk ◽  
M Bracke ◽  
E Caldenhoven ◽  
JA Raaijmakers ◽  
JW Lammers ◽  
...  
Keyword(s):  
Amino Acids ◽  
Phosphatidyl Inositol ◽  
Surface Expression ◽  
Cross Linking ◽  
Wild Type ◽  
Human Eosinophil ◽  
Transmembrane Glycoprotein ◽  
Membrane Bound ◽  
Intracellular Region

Abstract The Fc receptor for IgA (Fc alpha R, CD89) is a transmembrane glycoprotein found on monocytes, macrophages, neutrophils, and eosinophils. Here we describe the characterization of a novel isoform of the Fc alpha R cloned from a human eosinophil cDNA library. This clone, Fc alpha Rb, lacks the exon encoding the transmembrane/intracellular region of wild type Fc alpha R, which is replaced by 23 new amino acids. Expression of Fc alpha Rb mRNA could be detected in eosinophils and neutrophils. IIA1.6 murine pro-B cells transfected with Fc alpha Rb cDNA secrete high levels of the protein, but also a substantial amount of Fc alpha Rb is expressed at the cell membrane. Membrane-bound Fc alpha Rb binds IgA-coated beads equally well as wild type Fc alpha R. Surface expression is not affected by phosphatidyl inositol phospholipase C, indicating that glycosyl phosphatidyl inositol-linkage of Fc alpha Rb is not likely. In IIA1.6 cells expressing Fc alpha Rb and FcR gamma, which is necessary for signal transduction by wild type Fc alpha R, no tyrosine phosphorylation or Ca(2+)-mobilization could be observed after receptor cross-linking. These results indicate that Fc alpha Rb is likely to have a different function than wild-type Fc alpha R receptor.

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