Labeling the granulocyte C5a receptor with a unique photoreactive probe.
Human C5a anaphylatoxin is a complement-derived chemotactic factor that binds to specific receptors that are found in the granulocyte plasma membrane. These receptors, or a specific subunit of these receptors, can be covalently labeled with a unique photoreactive analog of human C5a. This photoaffinity probe, p-azidobenzoyl-2-mercapto-N-ethylamide-C5a (ABMEA-SC5a), was synthesized by coupling p-azidobenzoyl-2-mercapto-N-ethylamide-2'-thiopyridine disulfide to human C5a after it had been partially reduced with dithiothreitol. Both direct and competitive binding studies demonstrated that a radioiodinated ABMEA-SC5a derivative retained the capacity to specifically bind to either neutrophil or U937 cell C5a receptors. Half-maximal binding of the photoreactive analog was observed at a concentration of 1 to 2 nM, a value that is comparable to that observed when 125I-C5a is employed as the ligand. The covalent adducts that were formed after irradiation of 125I-ABMEA-SC5a that had been prebound to either neutrophil or U937 cell plasma membranes were found to have an apparent molecular mass of 52,000 daltons when analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis techniques. These findings demonstrate that the C5a receptors found on human neutrophils and other granulocytes are not only functionally similar, but biochemically similar as well.
D000345:Affinity Labels / Q000378:metabolism*; D001386:Azides / Q000138:chemical synthesis* / Q000494:pharmacology; D002460:Cell Line; D002462:Cell Membrane / Q000378:metabolism; D003182:Complement C5 / Q000378:metabolism*; D015936:Complement C5a; D006801:Humans; D007202:Indicators and Reagents; D007457:Iodine Radioisotopes; D007700:Kinetics; D008970:Molecular Weight; D009504:Neutrophils / Q000378:metabolism*; D010782:Photolysis; D044087:Receptor, Anaphylatoxin C5a; D011951:Receptors, Complement / Q000378:metabolism*
D000345:Affinity Labels; D001386:Azides; D003182:Complement C5; D007202:Indicators and Reagents; D007457:Iodine Radioisotopes; D044087:Receptor, Anaphylatoxin C5a; D011951:Receptors, Complement; D015936:Complement C5a; C045671:4-azidobenzoyl-2-mercapto-N-ethylamide-2'-thiopyridine disulfide