Synthesis of a new photoreactive C5a analog that permits identification of the ligand binding component of the granulocyte C5a receptor.
Human C5a is a complement derived inflammatory mediator that binds to specific receptors that are found in the granulocyte plasma membrane. A new photoreactive C5a analog, that can be radiolabeled in the aryl azide moiety, has been synthesized by coupling p-azidosalicyl-2-mercapto-N-ethylamide-2'-thiopyridine disulfide to a partially reduced form of C5a. This unique analog (ASAMEA-SC5a) specifically bound to the granulocyte C5a receptor with an apparent Kd of 1.8 nM, a value that is comparable to that of the native ligand. Photoactivation resulted in crosslinking of this probe to the granulocyte C5a receptor. Subsequent reductions of the complex produced a radiolabeled C5a receptor, or a subunit of this receptor that had an apparent molecular weight of 47,000 Daltons on SDS-PAGE.
Johnson
R J
RJ
Department of Pathology, VA Medical Center, San Diego, CA.
Chenoweth
D E
DE
D000345:Affinity Labels / Q000138:chemical synthesis; D001665:Binding Sites; D002460:Cell Line; D003182:Complement C5; D015936:Complement C5a; D006098:Granulocytes / Q000378:metabolism*; D006801:Humans; D008024:Ligands; D008970:Molecular Weight; D009504:Neutrophils / Q000378:metabolism; D010777:Photochemistry; D044087:Receptor, Anaphylatoxin C5a; D011951:Receptors, Complement / Q000378:metabolism*
D000345:Affinity Labels; D003182:Complement C5; D008024:Ligands; D044087:Receptor, Anaphylatoxin C5a; D011951:Receptors, Complement; D015936:Complement C5a