Isolation and subunit composition of tuftsin receptor.
Tuftsin (Thr-Lys-Pro-Arg) receptor was purified to apparent homogeneity by affinity chromatography, using a pentapeptide analog (Thr-Lys-Pro-Pro-Arg) that binds the receptor more than 4 times as avidly as tuftsin. The analog was covalently linked to a solid support (Affi-Gel 10). Rabbit peritoneal granulocyte membrane solubilized with 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonate was applied to the affinity column, the column was washed with 0.1 M ammonium carbonate (pH 7.9) and 0.1 M ammonium acetate (pH 5), and bound material was eluted with 20 nM tuftsin or pentapeptide. The eluate was concentrated and subjected to gel filtration; this yielded one major peak of [3H]tuftsin binding activity corresponding to approximately 500 kDa and a minor peak at approximately 250 kDa. Rechromatography of either peak resulted in the appearance of the same major and minor peaks. NaDodSO4/PAGE of the affinity-purified material under nonreducing conditions showed only two silver-staining bands. Electroblotting followed by [3H]tuftsin overlay and fluorography showed two adjacent radioactive bands corresponding in mobility to the silver-stained bands. Under reducing conditions, NaDodSO4/PAGE yielded molecular mass values 62 kDa and 52 kDa for the two tuftsin receptor subunits. Electron microscopy revealed a homogeneous population of spherical molecules with diameters of 104 A.
Bump
N J
NJ
Lee
J
J
Wleklik
M
M
Reichler
J
J
Najjar
V A
VA
D000818:Animals; D002846:Chromatography, Affinity; D002850:Chromatography, Gel; D004591:Electrophoresis, Polyacrylamide Gel; D046911:Macromolecular Substances; D008854:Microscopy, Electron; D008970:Molecular Weight; D009504:Neutrophils / Q000032:analysis; D011817:Rabbits; D011971:Receptors, Immunologic / Q000302:isolation & purification*; D014405:Tuftsin / Q000378:metabolism*
D046911:Macromolecular Substances; D011971:Receptors, Immunologic; C029695:tuftsin receptor; D014405:Tuftsin