Bovine spleen, a convenient source for purifying a type I interferon receptor.
Bovine spleen was investigated for the presence of receptors for radioiodinated human interferon-alpha 2 (125I-labeled HuIFN-alpha 2). Membranes were prepared by homogenization and differential centrifugation and analyzed for labeled IFN binding with recently developed tissue membrane assays. The characteristics of IFN binding included an affinity constant (Ka) of 3.1 +/- 0.99 X 10(10) M-1 and a receptor content of 8.4 +/- 0.74 fmoles/mg (wet weight) of bovine spleen membranes. The labeled IFN-receptor complex on these membranes was chemically cross-linked with 1.0 mM ethylene glycol bis(succinimidyl succinate (EGS), and subjected to SDS-PAGE and autoradiography. The formation of a 137-kD complex observed on autoradiographs was inhibited in a dose-dependent manner by unlabeled HuIFN-alpha 2 at concentrations that inhibited the binding of labeled IFN to the membranes. The products of the cross-linking reaction were purified by gel filtration on a column of Ultragel AcA34 in the presence of SDS and examined by SDS-PAGE and autoradiography. In addition to the 137-kD complex, several low-molecular-weight species were observed in the column profile of radioactivity which migrated to the bottom of 10% polyacrylamide gels. The fractions containing the 137-kD complex were pooled, concentrated, and utilized as a substrate for endoglycosidase digestion assays. Endoglycosidase H (EndoH) had no affect on the migration of the 137-kD complex while peptide:N-glycosidase F (PNGase F) increased the migration of the 137-kD band to a position with an Mr of 105 kD.(ABSTRACT TRUNCATED AT 250 WORDS)
D000818:Animals; D002417:Cattle; D006026:Glycoside Hydrolases; D007370:Interferon Type I / Q000378:metabolism; D008970:Molecular Weight; D011971:Receptors, Immunologic / Q000302:isolation & purification* / Q000378:metabolism; D017471:Receptors, Interferon; D013154:Spleen / Q000276:immunology* / Q000378:metabolism
D007370:Interferon Type I; D011971:Receptors, Immunologic; D017471:Receptors, Interferon; D006026:Glycoside Hydrolases