Murine interleukin 5 receptor isolated by immunoaffinity chromatography: comparison of determined N-terminal sequence and deduced primary sequence from cDNA and implication of a role of the intracytoplasmic domain.
The murine interleukin 5 receptor (IL-5R) was identified by utilizing an immobilized IL-5 and an immobilized monoclonal antibody against the murein IL-5R (designated H7 mAb). The H7 mAb immunoaffinity-purified materials from the extract of cell-surface radioiodinated T88-M cells (an IL-5-dependent early B cell line) using 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonate (CHAPS) were reacted with an immobilized IL-5 matrix. SDS-PAGE of the adsorbed fraction revealed a single band at approximately 60 kDa. The binding of the 60 kDa protein to the immobilized IL-5 matrix was inhibited by the excess IL-5. The CHAPS-extract depleted of the 60 kDa protein by the absorption with H7 mAb did not contain any IL-5 binding proteins. Immunoaffinity procedure provided a final 7400-fold purification, based on an estimation of the content of the 60 kDa protein (approximate purity: 20%) from the silver-stained pattern of SDS-PAGE. Actin was copurified with the 60 kDa protein at an approximate ratio of 1:1, suggesting that the intracytoplasmic domain of the IL-5R may interact with actin. Furthermore, soluble IL-5R (molecular mass: 50 kDa) was purified by the H7 mAb-immunoaffinity chromatography. The purified soluble IL-5R was capable of inhibiting the binding of IL-5 to T88-M cells. Preparative SDS-PAGE followed by electroblotting onto a membrane permitted the determination of the N-terminal sequence of the IL-5R. The determined N-terminal sequence of the IL-5R and the deduced primary sequence from recently isolated cDNA were compared.
Yamaguchi
N
N
Department of Biology, Kumamoto University Medical School, Japan.
Hitoshi
Y
Y
Takaki
S
S
Murata
Y
Y
Migita
M
M
Kamiya
T
T
Minowada
J
J
Tominaga
A
A
Takatsu
K
K
D000199:Actins / Q000378:metabolism; D000595:Amino Acid Sequence; D000818:Animals; D000911:Antibodies, Monoclonal; D001402:B-Lymphocytes / Q000737:chemistry*; D002460:Cell Line; D002462:Cell Membrane / Q000737:chemistry; D002846:Chromatography, Affinity / Q000379:methods; D003593:Cytoplasm / Q000737:chemistry / Q000378:metabolism; D007163:Immunosorbent Techniques; D008213:Lymphocyte Activation; D051379:Mice / Q000235:genetics; D008969:Molecular Sequence Data; D008970:Molecular Weight; D011971:Receptors, Immunologic / Q000737:chemistry / Q000302:isolation & purification* / Q000378:metabolism; D018123:Receptors, Interleukin; D053649:Receptors, Interleukin-5; D011994:Recombinant Proteins; D016415:Sequence Alignment
D000199:Actins; D000911:Antibodies, Monoclonal; D011971:Receptors, Immunologic; D018123:Receptors, Interleukin; D053649:Receptors, Interleukin-5; D011994:Recombinant Proteins