Identification and purification of truncated insulin-like growth factor I from porcine uterus. Evidence for high biological potency.
We report the completion of the purification of uterine-derived growth factors (UDGF) described previously by this laboratory [Ikeda, T., & Sirbasku, D.A. (1984) J. Biol. Chem. 259, 4049-4064]. During isolation, the mitogenic activity was monitored by using the human MCF-7 breast cancer cells in serum-free Ham's F12 and Dulbecco's modified Eagle's medium (1:1, v/v) containing 15 mM 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (pH 7.2), 200 micrograms/mL bovine serum albumin, and 10 micrograms/mL human transferrin. This medium sustained growth for several days in response to a single addition of growth factor. The isolation of UDGF began with acetic acid extraction followed by sulfopropyl-Sephadex chromatography, Bio-Gel P-10 molecular sieve fractionation, and a series of reverse-phase high-pressure liquid chromatography separations. Purifications [[(1.0-8.5) X 10(6)]-fold] of three mitogens (5-20 ng each) were achieved. The mitogens were shown by protein microsequencing to be DES 1----3 to DES 1----6 forms of insulin-like growth factor I (truncated IGF-I). An Mr estimated by 125I labeling, urea-sodium dodecyl sulfate-polyacrylamide gel electrophoresis, and autoradiography was consistent with a DES 1----3(4) N alpha truncation. Immunoadsorption and radioimmunoassay confirmed immunological properties equivalent to IGF-I. Radioreceptor assays showed truncated IGF-I was functionally equivalent to recombinant IGF-I. The ED50 values of DES 1----3 IGF-I and recombinant IGF-I for MCF-7 cell growth were 0.8-6.0 and 30-150 pg/mL, respectively. With Balb/c 3T3 mouse fibroblasts, the ED50 of DES 1----3 IGF-I was 100 times lower than that of IGF-I. We conclude that the major acid-stable low-Mr mitogenic activities isolated from uterus are very potent forms of truncated IGF-I capable of stimulating growth of epithelial and mesenchymal cells.
Ogasawara
M
M
Department of Biochemistry and Molecular Biology, University of Texas Medical School, Houston 77225.
Karey
K P
KP
Marquardt
H
H
Sirbasku
D A
DA
D000595:Amino Acid Sequence; D000818:Animals; D002455:Cell Division / Q000187:drug effects; D002460:Cell Line; D002478:Cells, Cultured; D002851:Chromatography, High Pressure Liquid; D005260:Female; D006801:Humans; D007334:Insulin-Like Growth Factor I / Q000302:isolation & purification* / Q000494:pharmacology; D007335:Insulin-Like Growth Factor II / Q000494:pharmacology; D051379:Mice; D008807:Mice, Inbred BALB C; D008969:Molecular Sequence Data; D008970:Molecular Weight; D011863:Radioimmunoassay; D011994:Recombinant Proteins / Q000494:pharmacology; D013002:Somatomedins / Q000302:isolation & purification*; D013552:Swine; D014599:Uterus / Q000502:physiology*
D011994:Recombinant Proteins; D013002:Somatomedins; D007334:Insulin-Like Growth Factor I; D007335:Insulin-Like Growth Factor II