Purification and photoaffinity labeling of the I-Ak histocompatibility molecule.

Details

Serval ID
serval:BIB_92FB738650C5
Type
Article: article from journal or magazin.
Collection
Publications
Title
Purification and photoaffinity labeling of the I-Ak histocompatibility molecule.
Journal
Journal of Immunological Methods
Author(s)
Luescher I.F., Unanue E.R.
ISSN
0022-1759 (Print)
ISSN-L
0022-1759
Publication state
Published
Issued date
1990
Peer-reviewed
Oui
Volume
135
Number
1-2
Pages
233-245
Language
english
Notes
Publication types: Journal Article ; Research Support, Non-U.S. Gov't ; Research Support, U.S. Gov't, P.H.S.
Publication Status: ppublish
Abstract
Photoaffinity labeling was used to evaluate optimal conditions for purification of I-A k histocompatibility molecules in functionally active form. We assessed the biological activity of I-A k primarily by its binding of the hen egg-white lysozyme (HEL) peptide from residues 46-61. [125I]iodo,4-azidosalicyloly(HEL)46-61 (IASA-46-61)-labeled I-A k on B cell hybridoma membranes and their detergent solubilisates, at the alpha chain. Following extensive detergent dialysis, the intensity of this labeling remained unchanged in the case of MEGA 8 and MEGA 9 detergents, but decreased in the case of deoxycholate and n-octylglucoside. Conditions for affinity purifications were assessed on one hand by determining the dissociation conditions of I-A k from various monoclonal antibodies and by determining the denaturation of I-A k under these conditions. Effective dissociation in the absence of detectable denaturation was observed for 10.3.6.2 and 40.LH monoclonal antibody at pH 3.5 and to a lesser extent at low concentrations of ammonium thiocyanate and guanidine thiocyanate at neutral pH. I-A k purified from cell membranes using MEGA 8 and MEGA 9 detergent mixtures and acid elution from 10.3.6.2 Sepharose was efficiently labeled by IASA-46-61. Thus I-A k was active in antigen presentation to a T cell hybridoma when reconstituted in planar membranes. In contrast to I-A k on cell membranes, purified I-A k in detergent showed extensive labeling of the beta chain. The overall labeling intensity and the extent of beta chain labeling substantially changed upon addition of certain lysophosphatides.
Keywords
Affinity Labels, Antibodies, Monoclonal/immunology, Azides, Cell Membrane/chemistry, Chromatography, Affinity/methods, Deoxycholic Acid/pharmacology, Detergents, Electrophoresis, Polyacrylamide Gel, Fatty Acids, Glucosamine/analogs & derivatives, Glucosides/pharmacology, Histocompatibility Antigens Class II/isolation & purification, Hydrogen-Ion Concentration, Muramidase, Peptide Fragments, Sorbitol/analogs & derivatives, Tumor Cells, Cultured
Pubmed
Web of science
Create date
28/01/2008 11:19
Last modification date
20/08/2019 14:55
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