Structure of the Mr 140,000 growth hormone-dependent insulin-like growth factor binding protein complex: determination by reconstitution and affinity-labeling.
To determine the structure of the high molecular weight, growth hormone-dependent complex between the insulin-like growth factors (IGF-I and IGF-II) and their binding proteins in human serum, we have reconstituted the complex from its purified component proteins and analyzed it by gel electrophoresis and autoradiography after covalent cross-linking. The proteins tested in reconstitution mixtures were an acid-labile Mr 84,000-86,000 glycoprotein doublet (alpha subunit), an acid-stable Mr 47,000-53,000 glycoprotein doublet with IGF-binding activity (BP-53 or beta subunit), and IGF-I or IGF-II (gamma subunit). In incubations containing any one of the three subunits 125I-labeled and the other two unlabeled, identical 125I-labeled alpha-beta-gamma complexes of Mr 140,000 were formed. Minor bands of Mr 120,000 and 90,000 were also seen, thought to represent a partially deglycosylated form of the alpha-beta-gamma complex, and an alpha-gamma complex arising as a cross-linking artifact. When serum samples from subjects of various growth hormone status were affinity-labeled with IGF-II tracer, a growth hormone-dependent Mr 140,000 band was seen, corresponding to the reconstituted alpha-beta-gamma complex. Other growth hormone-dependent labeled bands, of Mr 90,000 (corresponding to alpha-gamma), Mr 55,000-60,000 (corresponding to labeled beta-subunit doublet), and smaller bands of Mr 38,000, 28,000, and 23,000-25,000 (corresponding to labeled beta-subunit degradation products), were also seen in the affinity-labeled serum samples and in the complex reconstituted from pure proteins. All were immunoprecipitable with an anti-BP-53 antiserum. We conclude that the growth hormone-dependent Mr 140,000 IGF-binding protein complex in human serum has three components: the alpha (acid-labile) subunit, the beta (binding) subunit, and the gamma (growth factor) subunit.
Baxter
R C
RC
Department of Endocrinology, Royal Prince Alfred Hospital, Sydney, NSW, Australia.
Martin
J L
JL
D000172:Acromegaly / Q000097:blood; D000328:Adult; D000345:Affinity Labels / Q000378:metabolism; D002352:Carrier Proteins / Q000187:drug effects / Q000302:isolation & purification* / Q000378:metabolism; D004591:Electrophoresis, Polyacrylamide Gel; D013006:Growth Hormone / Q000494:pharmacology*; D006801:Humans; D018969:Insulin-Like Growth Factor Binding Proteins; D007334:Insulin-Like Growth Factor I / Q000378:metabolism*; D007335:Insulin-Like Growth Factor II / Q000378:metabolism*; D007457:Iodine Radioisotopes; D046911:Macromolecular Substances; D008961:Models, Structural; D008970:Molecular Weight; D013002:Somatomedins / Q000378:metabolism*
D000345:Affinity Labels; D002352:Carrier Proteins; D018969:Insulin-Like Growth Factor Binding Proteins; D007457:Iodine Radioisotopes; D046911:Macromolecular Substances; D013002:Somatomedins; D007334:Insulin-Like Growth Factor I; D007335:Insulin-Like Growth Factor II; D013006:Growth Hormone