Purification and characterization of an insulin-like growth factor II variant from human plasma.
An insulin-like growth factor II variant (IGF-II variant) was purified from Cohn fraction IV1 of human plasma by ion exchange, gel filtration, and reversed-phase high pressure liquid chromatography. The amino-terminal sequence of the first 35 amino acid residues showed a replacement of Ser-29 of IGF-II with the tetrapeptide Arg-Leu-Pro-Gly of IGF-II variant. Peptides isolated and sequenced after digestion with endoproteinase Asp-N and endoproteinase Glu-C disclosed no differences with the sequence predicted from an IGF-II variant cDNA clone isolated by Jansen, M., van Shaik, F. M. A., van Tol, H., Van den Brande, J. L., and Sussenbach, J. S. (1985) FEBS Lett., 179, 243-246. The molecular ion of intact IGF-II variant was 7809.4 mass units, as measured by plasma desorption mass spectrometry. This is in close agreement with the molecular ion of 7812.8 mass units calculated from the determined sequence and indicates the entire amino acid sequence had been accounted for. Binding of IGF-II variant to purified insulin-like growth factor I (IGF-I) receptors demonstrated a 2-3-fold lower affinity for this receptor compared with IGF-I or IGF-II. The dissociation constants for IGF-I, IGF-II, and IGF-II variant are 0.23, 0.38, and 0.80 nM, respectively. In a growth assay, the concentration of IGF-II and IGF-II variant required to stimulate the half-maximal growth of MCF-7 cells was 4 and 13 nM, respectively. Finally, the amount of IGF-II variant that can be purified by this method constitutes approximately 25% of the total IGF-II isolated from Cohn fraction IV1 of human plasma.
Hampton
B
B
Molecular Biology Laboratory, Jerome H. Holland Laboratory, American Red Cross, Rockville, Maryland 20855.
Burgess
W H
WH
Marshak
D R
DR
Cullen
K J
KJ
Perdue
J F
JF
D000595:Amino Acid Sequence; D000818:Animals; D002462:Cell Membrane / Q000378:metabolism; D002850:Chromatography, Gel; D002851:Chromatography, High Pressure Liquid; D003001:Cloning, Molecular; D004247:DNA / Q000235:genetics; D004591:Electrophoresis, Polyacrylamide Gel; D005260:Female; D014644:Genetic Variation; D006801:Humans; D007334:Insulin-Like Growth Factor I / Q000032:analysis / Q000302:isolation & purification; D007335:Insulin-Like Growth Factor II / Q000032:analysis* / Q000235:genetics / Q000302:isolation & purification; D008969:Molecular Sequence Data; D010920:Placenta / Q000378:metabolism; D011247:Pregnancy; D051381:Rats; D011956:Receptors, Cell Surface / Q000378:metabolism; D017451:Receptors, Somatomedin; D013002:Somatomedins
D011956:Receptors, Cell Surface; D017451:Receptors, Somatomedin; D013002:Somatomedins; D007334:Insulin-Like Growth Factor I; D007335:Insulin-Like Growth Factor II; D004247:DNA