Partial purification of an IGF-II receptor/binding protein from the erythroleukemia cell line K562.
We previously reported that insulin-like growth factor II (IGF-II) stimulated clonal growth of an erythroleukemia cell line, K562, in semi-solid agar, an effect not mimicked by insulin-like growth factor I (IGF-I), as IGF-I receptors are generally not expressed in this cell line. Affinity crosslinking of intact K562 cells with 125I-IGF-II revealed that the labeled hormone predominantly bound to a protein with a molecular weight of approximately 75 K. We report here the partial purification of the 75 K IGF-II binding protein from K562 cells. Triton X-100-solubilized K562 cells were subjected to Sephacryl-400, followed by Sephacryl-200 chromatography. Fractions of interest were collected and applied to a Sepharose-IGF-II column or an immuno-affinity column. The immuno-affinity column was prepared using an antiserum against placental membrane-derived material eluted from the Sephacryl-400 column in the elution volume, corresponding to the IGF-II binding protein from K562 cells. An affi-gel 10 affinity column, prepared with a protein A purified IgG fraction of this antiserum (antibody-29), retarded proteins showing binding specificity for IGF-II, with apparent molecular weights of 76 K, 87 K, and 70 K under reducing conditions. These protein bands were similar to the proteins retarded in the IGF-II affinity column, when evaluated by affinity crosslinking and SDS-PAGE. Fractionation of the purified material from the antibody-29 affinity column on Superose 12 revealed 6 protein peaks. Affinity crosslinking of the peak fractions from FPLC resulted in single bands with a molecular weight of 75 K under reducing conditions with variable specificity for IGF-II.
Zhang
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Q
Department of Molecular Medicine, Karolinska Institute, Stockholm, Sweden.
Hall
K
K
Tally
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M
D000818:Animals; D000906:Antibodies; D015153:Blotting, Western; D002460:Cell Line; D002846:Chromatography, Affinity; D002850:Chromatography, Gel; D003432:Cross-Linking Reagents; D004591:Electrophoresis, Polyacrylamide Gel; D006801:Humans; D018969:Insulin-Like Growth Factor Binding Proteins / Q000302:isolation & purification* / Q000378:metabolism; D007335:Insulin-Like Growth Factor II / Q000302:isolation & purification / Q000378:metabolism*; D007700:Kinetics; D004915:Leukemia, Erythroblastic, Acute; D008223:Lymphoma; D051381:Rats; D017527:Receptor, IGF Type 2 / Q000302:isolation & purification* / Q000378:metabolism; D011994:Recombinant Proteins / Q000302:isolation & purification / Q000378:metabolism; D013388:Succinimides / Q000494:pharmacology; D014407:Tumor Cells, Cultured
D000906:Antibodies; D003432:Cross-Linking Reagents; D018969:Insulin-Like Growth Factor Binding Proteins; D017527:Receptor, IGF Type 2; D011994:Recombinant Proteins; D013388:Succinimides; D007335:Insulin-Like Growth Factor II; C019358:disuccinimidyl suberate