Purification of the active C5a receptor from human polymorphonuclear leukocytes as a receptor - G sub i complex

Journal Article · Fri Feb 01 00:00:00 EST 1991 · Proceedings of the National Academy of Sciences of the United States of America; (United States)

DOI:https://doi.org/10.1073/pnas.88.3.971· OSTI ID:5043285

Rollins, T E; Siciliano, S; Kobayashi, S; Cianciarulo, D N; Bonilla-Argudo, V; Collier, K; Springer, M S ^[1]

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1. Merck Sharp and Dohme Research Lab., Rahway, NJ (United States)

The authors have isolated, in an active state, the C5a receptor from human polymorphonuclear leukocytes. The purification was achieved in a single step using a C5a affinity column in which the C5a molecule was coupled to the resin through its N terminus. The purified receptor, like the crude solubilized molecule, exhibited a single class of high-affinity binding sites with a K{sub d} of 30 pM. Further, the binding of C5a retained its sensitivity to guanine nucleotides, implying that the purified receptor contained a guanine nucleotide-binding protein (G protein). SDS/PAGE revealed the presence of three polypeptides with molecular masses of 42, 40, and 36 kDa, which were determined to be the C5a-binding subunit and the {alpha} and {beta} subunits of G{sub i}, respectively. The 36- and 40-kDa polypeptides were identified by immunoblotting and by the ability of pertussis toxin to ADP-ribosylate the 40-kDa molecule. These results confirm their earlier hypothesis that the receptor exists as a complex with a G protein in the presence or absence of C5a. The tight coupling between the receptor and G protein should make possible the identification of the G protein(s) involved in the transduction pathways used by C5a to produce its many biological effects.

• OSTI ID: 5043285
• Journal Information: Proceedings of the National Academy of Sciences of the United States of America; (United States), Vol. 88:3; ISSN 0027-8424
• Country of Publication: United States
• Language: English

Related Subjects

59 BASIC BIOLOGICAL SCIENCES
COMPLEMENT
RADIORECEPTOR ASSAY
MEMBRANE PROTEINS
ELECTROPHORESIS
RECEPTORS
PURIFICATION
BIOCHEMICAL REACTION KINETICS
IODINE 125
LEUKOCYTES
MAN
PHOSPHORUS 32
ANIMALS
BETA DECAY RADIOISOTOPES
BETA-MINUS DECAY RADIOISOTOPES
BIOLOGICAL MATERIALS
BLOOD
BLOOD CELLS
BODY FLUIDS
DAYS LIVING RADIOISOTOPES
ELECTRON CAPTURE RADIOISOTOPES
INTERMEDIATE MASS NUCLEI
INTERNAL CONVERSION RADIOISOTOPES
IODINE ISOTOPES
ISOTOPE APPLICATIONS
ISOTOPES
KINETICS
LIGHT NUCLEI
MAMMALS
MATERIALS
NUCLEI
ODD-EVEN NUCLEI
ODD-ODD NUCLEI
ORGANIC COMPOUNDS
PHOSPHORUS ISOTOPES
PRIMATES
PROTEINS
RADIOISOTOPES
REACTION KINETICS
TRACER TECHNIQUES
VERTEBRATES
550201* - Biochemistry- Tracer Techniques