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A COOH-terminally truncated recombinant form of prostromelysin-1 (MMP-3; EC 3.4.27.17) was activated by incubation at elevated temperature or by the addition of aminophenylmercuric acetate (APMA). By using an inhibitor of mature stromelysin to trap intermediates, it was found that the two methods of activation occurred by different mechanisms. Heat activation was achieved by a single-step bimolecular cleavage which was dependent on the presence of a small amount of mature enzyme. In contrast, APMA activation occurred by a complex multistep mechanism which consisted of intramolecular cleavages within the NH2-terminal pro portion of the molecule followed by a bimolecular cleavage at the NH2-terminus of the mature stromelysin. In spite of the different mechanisms of activation, both methods generate indistinguishable active enzymes.

Objective: To determine protein and activity levels of matrix metalloproteinases 1 and 3 (MMP-1 and MMP-3) in synovial fluid of patients with knee joint injury, primary osteoarthritis, and acute pyrophosphate arthritis (pseudogout). Methods: Measurements were done on knee synovial fluid obtained in a cross sectional study of cases of injury (n=283), osteoarthritis (n=105), and pseudogout (n=65), and in healthy controls (n=35). Activity of MMP-1 and MMP-3 in α2 macroglobulin complexes was measured using specific low molecular weight fluorogenic substrates. ProMMP-1, proMMP-3, and TIMP-1 (tissue inhibitor of metalloproteinase 1) were quantified by immunoassay. Results: Mean levels of proMMP-1, proMMP-3, and TIMP-1 were increased in injury, osteoarthritis, and pseudogout compared with controls. MMP-1 activity was increased in pseudogout and injury groups over control levels, whereas MMP-3 activity was increased only in the pseudogout group. The increase in MMP-1 activity coincided with a decrease in TIMP-1 levels in the injury group. Conclusions: Patients with joint injury have a persistent increase in proMMP-1 and proMMP-3 in synovial fluid and an increase in activated MMPs, which are not inhibited by TIMP. The differences in activation and inhibition patterns between the study groups are consistent with disease specific patterns of MMP activation and/or inhibition in joint pathology. Chemicals / CAS: alpha 2 macroglobulin, 95568-41-5; interstitial collagenase, 9001-12-1; stromelysin, 79955-99-0; tissue inhibitor of metalloproteinase 3, 145809-21-8, 164781-40-2; alpha-Macroglobulins; Matrix Metalloproteinases, EC 3.4.24.-; Tissue Inhibitor of Metalloproteinase-1

Objective: To determine protein and activity levels of matrix metalloproteinases 1 and 3 (MMP-1 and MMP-3) in synovial fluid of patients with knee joint injury, primary osteoarthritis, and acute pyrophosphate arthritis (pseudogout). Methods: Measurements were done on knee synovial fluid obtained in a cross sectional study of cases of injury (n=283), osteoarthritis (n=105), and pseudogout (n=65), and in healthy controls (n=35). Activity of MMP-1 and MMP-3 in α2 macroglobulin complexes was measured using specific low molecular weight fluorogenic substrates. ProMMP-1, proMMP-3, and TIMP-1 (tissue inhibitor of metalloproteinase 1) were quantified by immunoassay. Results: Mean levels of proMMP-1, proMMP-3, and TIMP-1 were increased in injury, osteoarthritis, and pseudogout compared with controls. MMP-1 activity was increased in pseudogout and injury groups over control levels, whereas MMP-3 activity was increased only in the pseudogout group. The increase in MMP-1 activity coincided with a decrease in TIMP-1 levels in the injury group. Conclusions: Patients with joint injury have a persistent increase in proMMP-1 and proMMP-3 in synovial fluid and an increase in activated MMPs, which are not inhibited by TIMP. The differences in activation and inhibition patterns between the study groups are consistent with disease specific patterns of MMP activation and/or inhibition in joint pathology. Chemicals / CAS: alpha 2 macroglobulin, 95568-41-5; interstitial collagenase, 9001-12-1; stromelysin, 79955-99-0; tissue inhibitor of metalloproteinase 3, 145809-21-8, 164781-40-2; alpha-Macroglobulins; Matrix Metalloproteinases, EC 3.4.24.-; Tissue Inhibitor of Metalloproteinase-1

The mechanisms by which epidermal growth factor (EGF) enhances the production of pro-matrix metalloproteinase 3 (proMMP-3/prostromelysin 1) and tissue inhibitor of metalloproteinases (TIMP)-1 were investigated using human uterine cervical fibroblasts. The treatment of the cells with EGF for 24 h resulted in about 5–6-fold increase in the production of proMMP-3 and TIMP-1 compared with the untreated control cells. This increase was accompanied by an increase of proMMP-3 and TIMP-1 mRNAs. However, an about 3- and 2-fold increase in the production of proMMP-3 and TIMP-1, respectively, was observed as early as 1 h after the treatment of the cells with EGF, and it was not accompanied by any apparent increase in proMMP-3 and TIMP-1 mRNAs. This early effect of EGF on the enhanced production of proMMP-3 and TIMP-1 was not inhibited by actinomycin D, even though actinomycin D inhibited the synthesis of the total RNA in both the EGF-treated and untreated cells. These results indicate that EGF enhances the apparent production of proMMP-3 and TIMP-1 by two mechanisms: one by the accelerated translation of their mRNAs; and the other by the enhanced transcription of their genes. The former event takes place much earlier than the latter.