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 /sup 125/I-C5a is employed as the ligand. The covalent adducts that were formed after irradiation of /sup 125/I-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.
- OSTI ID:
- 6380953
- Journal Information:
- J. Biol. Chem.; (United States), Journal Name: J. Biol. Chem.; (United States) Vol. 12; ISSN JBCHA
- Country of Publication:
- United States
- Language:
- English
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59 BASIC BIOLOGICAL SCIENCES
AFFINITY
AZIDES
BETA DECAY RADIOISOTOPES
BIOCHEMICAL REACTION KINETICS
BIOLOGICAL MATERIALS
BLOOD
BLOOD CELLS
BODY FLUIDS
CELL CONSTITUENTS
CELL MEMBRANES
CHEMICAL REACTIONS
COMPLEMENT
COVALENCE
DAYS LIVING RADIOISOTOPES
DECOMPOSITION
ELECTRON CAPTURE RADIOISOTOPES
ELECTROPHORESIS
INTERMEDIATE MASS NUCLEI
IODINE 125
IODINE ISOTOPES
ISOTOPE APPLICATIONS
ISOTOPES
KINETICS
LABELLING
LEUKOCYTES
LIGANDS
MATERIALS
MEMBRANES
MOLECULAR WEIGHT
NEUTROPHILS
NITROGEN COMPOUNDS
NUCLEI
ODD-EVEN NUCLEI
PHOTOCHEMICAL REACTIONS
PHOTOLYSIS
RADIOISOTOPES
REACTION KINETICS
RECEPTORS
TRACER TECHNIQUES