Purification and characterization of a low molecular weight transforming growth factor from the urine of melanoma patients.
A low Mr human transforming growth factor (TGF) present in melanoma patients' urine has been purified approximately 200,000-fold to apparent homogeneity. Initial purification of an acid-soluble fraction of urine was achieved by Bio-Gel P-30 gel filtration chromatography in 1 M acetic acid. TGF activities were demonstrated in the Mr ranges of 30,000 and 6,000-10,000. These competed with epidermal growth factor (EGF) for binding to A431 membrane receptors and induced anchorage-independent growth of untransformed fibroblasts. The low Mr TGF activity obtained from P-30 chromatography was purified to apparent homogeneity by two sequential reverse-phase high performance liquid chromatography steps with a mu Bondapak C18 column first using a linear gradient of acetonitrile going from 0-60% in 120 min and then by rechromatography of the activity over the same column using a shallower gradient of acetonitrile going from 20-40% in 160 min. The isoelectric point of the melanoma patient-derived urinary TGF was determined to be 6.2, which is distinct from that for human EGF. Amino acid composition analysis of the purified urinary TGF (uTGF) revealed that it is composed of at least 42 amino acid residues with a minimum estimated Mr of 4,545. Compositional analysis further revealed distinct similarities and differences between the uTGF, human EGF and TGFs secreted by various transformed human and rodent cell lines.
Kim
M K
MK
Warren
T C
TC
Kimball
E S
ES
D000596:Amino Acids / Q000032:analysis; D000818:Animals; D001667:Binding, Competitive; D002294:Carcinoma, Squamous Cell; D002460:Cell Line; D002462:Cell Membrane / Q000378:metabolism; D002471:Cell Transformation, Neoplastic; D004815:Epidermal Growth Factor / Q000378:metabolism; D066246:ErbB Receptors; D006801:Humans; D007668:Kidney / Q000166:cytology / Q000187:drug effects; D007700:Kinetics; D008545:Melanoma / Q000652:urine*; D008970:Molecular Weight; D009363:Neoplasm Proteins / Q000652:urine*; D010455:Peptides / Q000302:isolation & purification / Q000494:pharmacology / Q000652:urine*; D051381:Rats; D011956:Receptors, Cell Surface / Q000378:metabolism; D015291:Transforming Growth Factors
D000596:Amino Acids; D009363:Neoplasm Proteins; D010455:Peptides; D011956:Receptors, Cell Surface; D004815:Epidermal Growth Factor; D015291:Transforming Growth Factors; D066246:ErbB Receptors