cap alpha. -transforming growth factor secreted by untransformed bovine anterior pituitary cells in culture. I. Purification from conditioned medium
A 6-kDa ..cap alpha..-transforming growth factor (TGF) was purified 100,000-fold to homogeneity from the culture fluid conditioned by normal bovine anterior pituitary-derived cells. Initial purification of the acid-soluble TGF from concentrated conditioned medium was achieved by Bio-Gel P-60 gel filtration (apparent molecular mass of 9 kDa). After the Bio-Gel step, three different steps of reverse-phase fast-protein liquid chromatography on the same Pharmacia C/sub 18/ column, using linear acetonitrile gradients, gave complete purification. The ion-pairing agents used in the three consecutive steps were: 0.1% trifluoroacetic acid, 0.13% heptafluorobutyric acid, and again, 0.1% trifluoroacetic acid at a shallower gradient. Homogeneity was confirmed by reverse-phase high performance liquid chromatography, and by polyacrylamide gel electrophoresis, where TGF visualization was facilitated by autoradiography of /sup 125/I-TGF. The /sup 125/I-TGF bound to epidermal growth factor (EGF) receptors and after elution ran identically to the starting material. The molecular mass of TGF is 6 kDa by polyacrylamide gel electrophoresis and 6.6 kDa by amino acid analysis. The amino acid composition of bovine TGF is similar to that of rat or human ..cap alpha..TGF and distinct from epidermal growth factor. Colony-stimulating activity was lost after purification, but the TGF retained its ability to stimulate thymidine uptake by quiescent cells. This mitogenic activity could be blocked completely by anti-EGF-receptor monoclonal antibodies, indicating that the activity was mediated through the EGF-receptor.
- Research Organization:
- Univ. of Toronto, Ontario
- OSTI ID:
- 6845157
- Journal Information:
- J. Biol. Chem.; (United States), Vol. 261:31
- Country of Publication:
- United States
- Language:
- English
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59 BASIC BIOLOGICAL SCIENCES
PITUITARY GLAND
RADIORECEPTOR ASSAY
PROTEINS
PURIFICATION
ANIMAL CELLS
CATTLE
CULTURE MEDIA
ELECTROPHORESIS
GROWTH
IODINE 125
LIQUID COLUMN CHROMATOGRAPHY
MONOCLONAL ANTIBODIES
RECEPTORS
ANIMALS
ANTIBODIES
BETA DECAY RADIOISOTOPES
BODY
CHROMATOGRAPHY
DAYS LIVING RADIOISOTOPES
DOMESTIC ANIMALS
ELECTRON CAPTURE RADIOISOTOPES
ENDOCRINE GLANDS
GLANDS
INTERMEDIATE MASS NUCLEI
IODINE ISOTOPES
ISOTOPE APPLICATIONS
ISOTOPES
MAMMALS
MEMBRANE PROTEINS
NUCLEI
ODD-EVEN NUCLEI
ORGANIC COMPOUNDS
ORGANS
RADIOISOTOPES
RUMINANTS
SEPARATION PROCESSES
TRACER TECHNIQUES
VERTEBRATES
550601* - Medicine- Unsealed Radionuclides in Diagnostics
550201 - Biochemistry- Tracer Techniques