Human platelet-derived mitogens. I. Identification of insulinlike growth factors I and II by purification and N alpha amino acid sequence analysis.
Human platelet lysates contained potent mitogenic activities for MCF-7 human breast-cancer cells in serum-free-defined media. Because these activities were not replaced by known platelet mitogens, such as platelet-derived growth factor or transforming growth factor beta, we sought to identify the breast cancer cell mitogens by purification and N alpha amino-acid sequencing. Acetic acid extracts of outdated human platelets were concentrated by ammonium sulfate precipitation and fractionated on Sephadex G-50 and Bio-Gel P-10 columns in 0.5 mol/L acetic acid. Two major activities were resolved by molecular sieve methods and fractionated further by reverse-phase high-performance liquid chromatography (HPLC). Purifications (70,000 to 870,000-fold) were accomplished yielding mol wt 7,400 products that were homogeneous as determined by iodination, sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), and autoradiography. The factors were identified as insulinlike growth factor I (IGF-I) and II (IGF-II) and truncated IGF-I by N alpha amino acid microsequencing. In dose-response experiments, platelet-derived IGF-I and IGF-II promoted multiple divisions of the MCF-7 cells with ED50 values of 12 and 100 pg/mL, respectively. The specific activities and other bioassay characteristics of platelet-derived IGF-I and IGF-II were similar to those of recombinant-produced human growth factors. This is the first report of the purification of insulinlike growth factors from human platelet lysates.
Karey
K P
KP
Department of Biochemistry and Molecular Biology, University of Texas Medical School, Houston 77225.
Marquardt
H
H
Sirbasku
D A
DA
D000595:Amino Acid Sequence; D001943:Breast Neoplasms / Q000378:metabolism / Q000473:pathology; D002452:Cell Count; D002850:Chromatography, Gel; D002851:Chromatography, High Pressure Liquid; D006801:Humans; D007334:Insulin-Like Growth Factor I / Q000302:isolation & purification*; D007335:Insulin-Like Growth Factor II / Q000302:isolation & purification*; D008969:Molecular Sequence Data; D008970:Molecular Weight; D010982:Platelet-Derived Growth Factor / Q000302:isolation & purification*; D013002:Somatomedins / Q000302:isolation & purification*; D013010:Sonication; D013347:Subcellular Fractions / Q000032:analysis; D014407:Tumor Cells, Cultured / Q000378:metabolism / Q000473:pathology
D010982:Platelet-Derived Growth Factor; D013002:Somatomedins; D007334:Insulin-Like Growth Factor I; D007335:Insulin-Like Growth Factor II