Purification and characterization of two collagenase inhibitors from mouse sarcoma 180 conditioned medium

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Abstract
We have previously shown that mouse sarcoma 180 cells produce vascular endothelial growth factor [VGEF; Rosenthal et al., 1990, Growth Factors, 4: 53–59], endothelial mitogen that stimulates angiogeneis. Recent reports have implicated metallaproteinases and their inhibitors in the regulation of vascular morphogeneis, 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 32,000 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 metalloproiteinases 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 identificationof MS‐22 as TIMP‐2 and MS‐31 as TIMP‐1.