Use of the heterobifunctional cross-linker m-maleimidobenzoyl N-hydroxysuccinimide ester to affinity label cholecystokinin binding proteins on rat pancreatic plasma membranes.
The binding of 125I-cholecystokinin-33 (125I-CCK-33) to its receptors on rat pancreatic membranes was decreased by modification of membrane protein sulfhydryl groups. Sulfhydryl modifying reagents also caused an accelerated release of bound 125I-CCK-33 from its receptor. Because of the presence of an essential sulfhydryl group(s) in CCK receptor binding we studied the application of the heterobifunctional (SH,NH2) cross-linker, m-maleimidobenzoyl N-hydroxysuccinimide ester (MBS), to affinity label 125I-CCK-33 binding proteins on rat pancreatic plasma membranes. Analysis of the cross-linked products by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and autoradiography revealed that this heterobifunctional cross-linker affinity labeled a major Mr = 80,000-95,000 protein previously identified as part of the CCK receptor on the basis of affinity labeling using homobifunctional and heterobifunctional photoreactive cross-linkers. Additional proteins of Mr greater than 200,000, and Mr = 130,000-140,000 were affinity labeled using MBS. The efficiency of the cross-linking reaction between 125I-CCK-33 and its membrane binding proteins with MBS was significantly greater than that obtained with NH2-directed homobifunctional reagents such as disuccinimidyl suberate. The efficiency of cross-linking could be dramatically improved by reduction of membrane proteins with low-molecular weight thiols prior to binding and cross-linking. The differential labeling patterns of the CCK binding proteins obtained with chemical cross-linkers of similar length but different chemical reactivity underscores the need for caution in predicting native receptor structure from affinity labeling data alone. Using the same pancreatic plasma membrane preparation and 125I-insulin, the Mr = 125,000 alpha-subunit of the insulin receptor was affinity labeled using MBS as cross-linker, demonstrating its utility in identifying other peptide hormone receptors.
D000345:Affinity Labels / Q000494:pharmacology*; D000818:Animals; D002462:Cell Membrane / Q000378:metabolism; D002766:Cholecystokinin / Q000378:metabolism*; D007328:Insulin / Q000031:analogs & derivatives / Q000378:metabolism; D007700:Kinetics; D008297:Male; D008565:Membrane Proteins / Q000302:isolation & purification; D008970:Molecular Weight; D010179:Pancreas / Q000378:metabolism*; D051381:Rats; D011972:Receptor, Insulin / Q000187:drug effects / Q000378:metabolism; D011956:Receptors, Cell Surface / Q000187:drug effects / Q000378:metabolism*; D011949:Receptors, Cholecystokinin; D013388:Succinimides / Q000494:pharmacology*; D013439:Sulfhydryl Reagents / Q000494:pharmacology
D000345:Affinity Labels; D007328:Insulin; D008565:Membrane Proteins; D011956:Receptors, Cell Surface; D011949:Receptors, Cholecystokinin; D013388:Succinimides; D013439:Sulfhydryl Reagents; C008147:insulin, iodo-; C013097:3-maleimidobenzoyl N-hydroxysuccinimide; D002766:Cholecystokinin; D011972:Receptor, Insulin