Demonstration of activation-specific interactions among B lymphocyte membrane proteins by a photoreactive cross-linking agent.
The formation of plasma membrane protein complexes during B cell activation was examined by covalently stabilizing these complexes with a photolabile, cleavable, cross-linking reagent, dithiobisphenylazide (DTPA). Analysis by SDS polyacrylamide gel electrophoresis indicates that LPS induces a time-dependent rearrangement in membrane proteins of splenic B cells. These rearrangements were localized to the plasma membrane by examination of surface-labeled detergent lysates and purified plasma membrane preparations. DTPA-stabilized rearrangements could be detected as early as 15 min and were stable between 3 and 24 hr after mitogen addition. These changes are consistent with the formation of different functional protein complexes during B cell activation. LPS does not induce membrane protein rearrangement in thymocytes, nor does Con A do so in B cells. Therefore, the changes appear to be correlated with the transmission of a relevant activation signal. Activation of B cells with 8- bromoguanosine ( 8BrGuo ), an intracellular mitogen, did not result in protein rearrangement until 12 hr after mitogen addition, suggesting that initial stages of signal transduction by 8BrGuo and LPS follow separate pathways. A common "activated state" of the membrane is established by 12 hr. Inhibition of de novo protein synthesis by cycloheximide did not interfere with formation of new protein complexes during early stages of activation (15 min and 3 hr). These data indicate that covalent cross-linking with DTPA can be used to demonstrate activation-specific interactions among proteins on the surface membrane of B cells.
D000818:Animals; D001386:Azides / Q000494:pharmacology*; D001402:B-Lymphocytes / Q000032:analysis / Q000276:immunology* / Q000378:metabolism; D003432:Cross-Linking Reagents; D003513:Cycloheximide / Q000494:pharmacology; D004591:Electrophoresis, Polyacrylamide Gel; D006151:Guanosine / Q000031:analogs & derivatives / Q000494:pharmacology; D008070:Lipopolysaccharides / Q000494:pharmacology; D008213:Lymphocyte Activation / Q000187:drug effects; D008297:Male; D008565:Membrane Proteins / Q000032:analysis / Q000276:immunology / Q000378:metabolism*; D051379:Mice; D008809:Mice, Inbred C3H; D008970:Molecular Weight; D014176:Protein Biosynthesis; D013601:T-Lymphocytes / Q000276:immunology / Q000378:metabolism
D001386:Azides; D003432:Cross-Linking Reagents; D008070:Lipopolysaccharides; D008565:Membrane Proteins; D006151:Guanosine; C025369:4,4'-dithiobis(phenylazide); C035169:8-bromoguanosine; D003513:Cycloheximide