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Human platelet-derived mitogens. I. Identification of insulinlike growth
factors I and II by purification and N alpha amino acid sequence analysis
KP Karey, H Marquardt and DA Sirbasku
Department of Biochemistry and Molecular Biology, University of Texas
Medical School, Houston 77225.
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 Acetic acid extracts of outdated human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.
Volume 74,
Issue 3,
pp. 1084-1092,
08/15/1989
Copyright © 1989 by The American Society of Hematology
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