Identification of a family of insulin-like growth factor II secreted by cultured rat epithelial-like cell line 18,54-SF: application of a monoclonal antibody.
Somatomedin/insulin-like growth factor (IGF)-like polypeptides (designated SMP) were purified from the serum-free conditioned medium of cultured rat epithelial-like cells, 18,54-SF. A monoclonal antibody (MAb) was produced against partially purified SMP. The antibody was immunoglobulin G1 relatively specific for multiplication-stimulating activity III-2 (rat IGF-II), with a Kd value of 5.6 X 10(-9) M. The antibody showed 100% cross-reactivity with human IGF-II and 10% cross-reactivity with human IGF-I, but did not cross-react with insulin. For purification of SMP, therefore, immunoaffinity chromatography on Sepharose coupled with the MAb was used besides a procedure including ion exchange chromatography, gel filtration, and reverse phase HPLC. The purified SMP (at least five polypeptides) each gave a single peak on reverse phase HPLC and appeared as a single band with an apparent mol wt of 5000-8000 on sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The major components of SMP (designated HP1-SMP and HP3-SMP), which were purified about 100-fold from conditioned medium, stimulated DNA synthesis in human fibroblasts in culture and sulfation in chick embryonic cartilage in culture. These polypeptides showed almost the same cross-reactivity as multiplication-stimulating activity III-2 on RIA with the MAb. The partial amino acid sequences of HP1- and HP3-SMP were determined, and these polypeptides were identified with rat IGF-II.
Tanaka
H
H
Department of Biochemistry, Kanazawa Medical University, Ishikawa, Japan.
Asami
O
O
Hayano
T
T
Sasaki
I
I
Yoshitake
Y
Y
Nishikawa
K
K
D000818:Animals; D000911:Antibodies, Monoclonal / Q000302:isolation & purification; D002460:Cell Line; D002846:Chromatography, Affinity; D002850:Chromatography, Gel; D002852:Chromatography, Ion Exchange; D004848:Epithelium; D000939:Epitopes / Q000032:analysis*; D007335:Insulin-Like Growth Factor II / Q000276:immunology / Q000302:isolation & purification / Q000378:metabolism*; D011863:Radioimmunoassay; D051381:Rats; D013002:Somatomedins / Q000378:metabolism*
D000911:Antibodies, Monoclonal; D000939:Epitopes; D013002:Somatomedins; D007335:Insulin-Like Growth Factor II