Purification and sequence analysis of two rat tissue inhibitors of metalloproteinases.
Two protein inhibitors of metalloproteinases (TIMP) were isolated from medium conditioned by the clonal rat osteosarcoma line UMR 106-01. Initial purification of both a 30-kDa inhibitor and a 20-kDa inhibitor was accomplished using heparin-Sepharose chromatography with dextran sulfate elution followed by DEAE-Sepharose and CM-Sepharose chromatography. Purification of the 20-kDa inhibitor to homogeneity was completed with reverse-phase high-performance liquid chromatography. The 20-kDa inhibitor was identified as rat TIMP-2. The 30-kDa inhibitor, although not purified to homogeneity, was identified as rat TIMP-1. Amino terminal amino acid sequence analysis of the 30-kDa inhibitor demonstrated 86% identity to human TIMP-1 for the first 22 amino acids while the sequence of the 20-kDa inhibitor was identical to that of human TIMP-2 for the first 22 residues. Treatment with peptide:N-glycosidase F indicated that the 30-kDa rat inhibitor is glycosylated while the 20-kDa inhibitor is apparently unglycosylated. Inhibition of both rat and human interstitial collagenase by rat TIMP-2 was stoichiometric, with a 1:1 molar ratio required for complete inhibition. Exposure of UMR 106-01 cells to 10(-7) M parathyroid hormone resulted in approximately a 40% increase in total inhibitor production over basal levels.
Roswit
W T
WT
Department of Medicine, Washington University School of Medicine, St. Louis, Missouri 63130.
McCourt
D W
DW
Partridge
N C
NC
Jeffrey
J J
JJ
D000595:Amino Acid Sequence; D000818:Animals; D002417:Cattle; D002460:Cell Line; D002846:Chromatography, Affinity; D002851:Chromatography, High Pressure Liquid; D002852:Chromatography, Ion Exchange; D004591:Electrophoresis, Polyacrylamide Gel; D006023:Glycoproteins / Q000737:chemistry / Q000302:isolation & purification* / Q000494:pharmacology; D006801:Humans; D007700:Kinetics; D003012:Microbial Collagenase / Q000037:antagonists & inhibitors; D008969:Molecular Sequence Data; D008970:Molecular Weight; D010281:Parathyroid Hormone / Q000494:pharmacology; D051381:Rats; D012689:Sequence Homology, Nucleic Acid; D019714:Tissue Inhibitor of Metalloproteinases
D006023:Glycoproteins; D010281:Parathyroid Hormone; D019714:Tissue Inhibitor of Metalloproteinases; D003012:Microbial Collagenase