Identification, purification, and partial sequence analysis of autotaxin, a novel motility-stimulating protein.
Autotaxin (ATX) is a potent human motility-stimulating protein that has been identified in the conditioned medium from A2058 melanoma cells. This protein has been purified to homogeneity utilizing a strategy involving five column steps. Homogeneity of ATX was verified by two-dimensional gel electrophoresis. The molecular size of ATX is 125 kDa, and it has an isoelectric point of 7.7 +/- 0.2. Purified ATX was digested with cyanogen bromide and trypsin, and the resulting ATX peptides were purified by reverse-phase high performance liquid chromatography. Eleven peptides were subjected to amino acid sequence analysis, and 114 residues were identified. The partial amino acid sequences and the amino acid composition obtained for ATX show that it does not exhibit any significant homology to known growth factors or previously described motility factors. At picomolar concentrations, ATX stimulates both random and directed migration of human A2058 melanoma cells. Pretreatment of the melanoma cells with pertussis toxin abolishes the response to purified ATX, indicating that ATX stimulates motility through a receptor acting via a pertussis toxin-sensitive G protein.
Stracke
M L
ML
Laboratory of Pathology, National Cancer Institute, National Institutes of Health, Bethesda, Maryland 20892.
Krutzsch
H C
HC
Unsworth
E J
EJ
Arestad
A
A
Cioce
V
V
Schiffmann
E
E
Liotta
L A
LA
D000595:Amino Acid Sequence; D002465:Cell Movement; D002851:Chromatography, High Pressure Liquid; D003488:Cyanogen Bromide / Q000737:chemistry; D004591:Electrophoresis, Polyacrylamide Gel; D006801:Humans; D007526:Isoelectric Point; D008545:Melanoma / Q000378:metabolism; D008969:Molecular Sequence Data; D009363:Neoplasm Proteins / Q000235:genetics / Q000302:isolation & purification* / Q000494:pharmacology; D010449:Peptide Mapping; D014357:Trypsin / Q000737:chemistry; D014407:Tumor Cells, Cultured
D009363:Neoplasm Proteins; D014357:Trypsin; D003488:Cyanogen Bromide