Purification and characterization of two collagenase inhibitors from mouse sarcoma 180 conditioned medium.
We have previously shown that mouse sarcoma 180 cells produce vascular endothelial growth factor [VEGF; Rosenthal et al., 1990, Growth Factors, 4: 53-59], an endothelial mitogen that stimulates angiogenesis. Recent reports have implicated metalloproteinases and their inhibitors in the regulation of vascular morphogenesis, tumor invasion, and metastasis. We report here that mouse sarcoma 180 cells produce two collagenase inhibitors. These inhibitors were purified by heparin-Sepharose affinity chromatography, gel filtration, and C4 reverse phase h.p.l.c. Analytical gel electrophoresis of the purified inhibitors (MS-22 and MS-31) revealed molecular masses of 22,000 and 31,000 Da under reducing conditions, and 20,000 and 30,000 Da under nonreducing conditions, respectively. The NH2-terminal amino acid sequence of MS-22 was identical to that of tissue inhibitor of metalloproteinases type 2 (TIMP-2) produced by human melanoma cells [Stetler-Stevenson et al., 1989, J. Biol. Chem. 264: 17374-17378) over the first 30 amino acids. The NH2-terminal amino acid sequence of MS-31 was identical to that of murine TIMP-1 [Gewert et al., 1989, EMBO J 6:651-657]. Statistical analysis of the amino acid composition data of these two mouse sarcoma 180-derived collagenase inhibitors confirms the identification of MS-22 as TIMP-2 and MS-31 as TIMP-1.
Rosenthal
R A
RA
Department of Surgery, Children's Hospital, Boston, Massachusetts 02115.
Moses
M A
MA
Shintani
Y
Y
Megyesi
J F
JF
Langer
R
R
Folkman
J
J
D000595:Amino Acid Sequence; D000818:Animals; D002846:Chromatography, Affinity; D002850:Chromatography, Gel; D002851:Chromatography, High Pressure Liquid; D017077:Culture Media, Conditioned; D004591:Electrophoresis, Polyacrylamide Gel; D006023:Glycoproteins / Q000737:chemistry* / Q000302:isolation & purification* / Q000494:pharmacology; D061965:Matrix Metalloproteinase Inhibitors; D051379:Mice; D008969:Molecular Sequence Data; D012510:Sarcoma 180 / Q000378:metabolism*; D017386:Sequence Homology, Amino Acid; D019714:Tissue Inhibitor of Metalloproteinases; D014407:Tumor Cells, Cultured
D017077:Culture Media, Conditioned; D006023:Glycoproteins; D061965:Matrix Metalloproteinase Inhibitors; D019714:Tissue Inhibitor of Metalloproteinases