title: Purification of an equine apotransferrin variant (thyromedin) essential for thyroid hormone dependent growth of GH1 rat pituitary tumor cells in chemically defined culture.
abstract: Pituitary tumor cells require thyroid hormones for growth in vivo [Sorrentino, J. M., Kirkland, W. L., & Sirbasku, D. A. (1976) J. Natl. Cancer Inst. 56, 1155-1158]. In vitro, GH1 rat pituitary tumor cells were studied in a serum-free defined medium (PCM-10) formulated with Ham's F12 and Dulbecco's modified Eagle's media (1:1, v/v) supplemented with 2.2 g/L sodium bicarbonate, 15 mM 4-(2-hydroxy-ethyl)-1-piperazineethanesulfonic acid (pH 7.2), 10 micrograms/mL human transferrin, 50 microM ethanolamine, 10 micrograms/mL insulin, 10 ng/mL selenous acid, 0.1 nM 3,5,3'-triiodothyronine (T3) and 500 micrograms/mL bovine serum albumin and in the same medium without T3 (PCM-0). The cells only grew in PCM-10 when low concentrations of horse serum were added. Attempts to replace the serum factor requirement with known growth factors and adhesion proteins were unsuccessful. The Mr 65,000-72,000 serum factor regulating T3-induced growth (thyromedin) was purified to homogeneity and identified as equine transferrin R and/or D by amino acid sequencing. The ED50 in PCM-10 was 17-40 micrograms/mL (260-620 nM) while in PCM-0 half-maximum growth was not achieved at 200 micrograms/mL. Concentrations of 75 micrograms/mL in PCM-10 caused 80% of serum-stimulated growth rate. Removal of iron from thyromedin, and assay in iron salts reduced PCM-10, increased the specific activity 110-270-fold to ED50 150 ng/mL (2.3 nM); at 1.0 micrograms/mL, growth in PCM-10 was 16-fold greater than in PCM-0. Iron saturation of thyromedin caused total loss of biological activity. We conclude that the horse transferrin variant isolated in this report is active as apotransferrin.
authors.affiliation: Department of Biochemistry and Molecular Biology, University of Texas Medical School, Houston 77225.
authors.forename: D A
authors.initials: DA
authors.lastname: Sirbasku
authors.forename: B H
authors.initials: BH
authors.lastname: Stewart
authors.forename: R
authors.initials: R
authors.lastname: Pakala
authors.forename: J E
authors.initials: JE
authors.lastname: Eby
authors.forename: H
authors.initials: H
authors.lastname: Sato
authors.forename: J M
authors.initials: JM
authors.lastname: Roscoe
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D001059:Apoproteins / Q000302:isolation & purification / Q000494:pharmacology*; D002455:Cell Division / Q000187:drug effects*; D002460:Cell Line; D002846:Chromatography, Affinity; D002852:Chromatography, Ion Exchange; D003470:Culture Media; D004591:Electrophoresis, Polyacrylamide Gel; D014644:Genetic Variation; D006736:Horses; D007700:Kinetics; D008969:Molecular Sequence Data; D008970:Molecular Weight; D010911:Pituitary Neoplasms; D051381:Rats; D014168:Transferrin / Q000302:isolation & purification / Q000494:pharmacology*; D014284:Triiodothyronine / Q000494:pharmacology*
chemical_list: D001059:Apoproteins; D003470:Culture Media; D014168:Transferrin; C028284:apotransferrin; C066910:thyromedin; D014284:Triiodothyronine