Solubilization of the functional C5a receptor from human polymorphonuclear leukocytes.
The C5a receptor has been extracted in an active state from the membranes of human polymorphonuclear leukocytes with the detergents digitonin and beta-dodecyl maltoside. The solubilized receptor exhibits a single class of high affinity binding sites with a Kd = 90 pM, a value similar to that found with intact membranes. Physical studies with the soluble receptor demonstrate that it exists in two forms which differ in molecular mass. Gel filtration experiments with receptor to which C5a has been bound give an apparent molecular mass for the complex of 150-200 kDa. When the experiments were repeated with nonliganded receptor, most of the C5a binding activity eluted with an apparent mass of 150-200 kDa. However, the peak had a pronounced trailing shoulder indicating that, in the nonliganded state, a portion of the receptor population exists in a smaller form, which may be converted to the larger form on binding C5a. The molecular mass of the smaller form, estimated to be 30-70 kDa, is consistent with that of the binding subunit of the receptor. These data imply that the larger form, and therefore the bulk of the solubilized receptor, is oligomeric, a conclusion which is supported by cross-linking studies. When C5a was cross-linked to the soluble receptor two specific complexes with molecular masses of 52 and 95 kDa were formed. The former is the covalent adduct of C5a and the binding subunit of the receptor and the latter appears to be a complex between the 52-kDa species and an additional polypeptide.
Rollins
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Department of Immunology Research, Merck Sharp and Dohme Research Laboratories, Rahway, New Jersey 07065.
Siciliano
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Springer
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MS
D002462:Cell Membrane / Q000378:metabolism; D002850:Chromatography, Gel; D003182:Complement C5 / Q000378:metabolism*; D003902:Detergents; D006801:Humans; D007700:Kinetics; D008970:Molecular Weight; D009504:Neutrophils / Q000378:metabolism*; D044087:Receptor, Anaphylatoxin C5a; D011951:Receptors, Complement / Q000302:isolation & purification / Q000378:metabolism*
D003182:Complement C5; D003902:Detergents; D044087:Receptor, Anaphylatoxin C5a; D011951:Receptors, Complement