Recombinant porcine collagenase: purification and autolysis.

Collagenase is a member of the matrix metalloproteinase family whose members are all capable of degrading extracellular matrix components. The mature form of porcine collagenase has been expressed in Escherichia coli using the pAX5 expression vector. The fusion protein consists of beta-galactosidase at the N-terminus joined to a collagen hinge region and a blood-coagulation factor Xa cleavage site linked to an active form of collagenase. Recombinant collagenase was biologically active in the form of a fusion protein; this was cleaved with factor Xa to yield collagenase with the authentic N terminus (phenylalanine) found in vivo and purified in a single step on a peptide hydroxamic acid affinity column. On purification the recombinant porcine collagenase undergoes autolysis at a number of different bonds in the region connecting the active site domain with the C-terminal hemopexin-like domain. This may represent a loop region of poor secondary structure, making it susceptible to relatively nonspecific cleavage. The N-terminal fragment retains a reduced level of collagenolytic activity, along with that against casein and gelatin.

Tissue inhibitor of metalloproteinases (TIMP-1) stimulates the secretion of collagenase from human skin fibroblasts.

This study aimed to investigate possible feedback mechanisms in the control of collagenase (matrix metalloproteinase-1) and tissue inhibitor of metalloproteinases (TIMP-1). Procollagenase, active collagenase, TIMP-1 and collagenase-TIMP complex were applied to human skin and synovial fibroblasts for 48 hours, the cells were washed and the resulting collagenase and TIMP-1 secretion was measured over the following 72 hours using ELISAs. Of the additions, only TIMP-1 showed any measurable effect, stimulating collagenase secretion over a range of 1-8 micrograms/ml. Endotoxin contamination was ruled out as an explanation and the active conformation of the TIMP-1 molecule was shown to be necessary.

The measurement of collagenase, tissue inhibitor of metalloproteinases (TIMP), and collagenase-TIMP complex in synovial fluids from patients with osteoarthritis and rheumatoid arthritis.

OBJECTIVE: To compare the levels of collagenase, tissue inhibitor of metalloproteinases (TIMP), and collagenase-TIMP complex in synovial fluid (SF) from patients with rheumatoid arthritis (RA) and patients with osteoarthritis (OA). This study aims to clarify existing data from previously used enzyme or inhibitor activity assays performed following separation by gel filtration, by using a 1-step enzyme-linked immunosorbent assay (ELISA) for each component. METHODS: Total collagenase, free TIMP, and collagenase-TIMP complex were measured using a newly developed, specific double-antibody sandwich ELISA: RESULTS: Levels of both collagenase and TIMP were significantly higher in RA patients (collagenase 1,560 +/- 150 ng/ml [mean +/- SEM], TIMP 1,610 +/- 130 ng/ml; n = 80) than OA patients (collagenase 420 +/- 90 ng/ml, TIMP 1,050 +/- 60 ng/ml; n = 80), with the difference being especially striking for collagenase. Sixteen RA fluids had detectable levels of collagenase-TIMP complex, compared with only 3 OA fluids. CONCLUSION: The level of total collagenase in SF is greater in RA than OA, while levels of free TIMP show more overlap between the 2 diseases; this may simply reflect the increased inflammation seen in RA, or it may reflect a different disease mechanism.

Monoclonal antibodies against human fibroblast collagenase and the design of an enzyme-linked immunosorbent assay to measure total collagenase.

Monoclonal antibodies have been raised against purified human fibroblast collagenase and characterised. One of these antibodies has been used in combination with a polyclonal anticollagenase antibody in a double antibody sandwich ELISA to measure collagenase. The assay range was 2-50 ng/ml collagenase. The assay measures total collagenase, i.e. pro- and active enzyme as well as collagenase in complex with TIMP. The level of collagenase has been measured in sera samples from patients with rheumatoid arthritis and compared with age- and sex-matched controls. The levels measured were: rheumatoid arthritis, 69 +/- 29 ng/ml; normal, 49 +/- 14 ng/ml.

Polyclonal and monoclonal antibodies against human tissue inhibitor of metalloproteinases (TIMP) and the design of an enzyme-linked immunosorbent assay to measure TIMP.

Both polyclonal and monoclonal antibodies have been raised against purified human fibroblast tissue inhibitor of metalloproteinases (TIMP) and characterised. Combinations of antibodies were tested for their suitability in a double antibody sandwich ELISA to measure TIMP. Two combinations were applicable to the immunoassay: (i) a monoclonal capture antibody with a polyclonal detecting antibody; (ii) two monoclonal antibodies. The assay range was (i) 2-50 ng/ml and (ii) 5-50 ng/ml of human TIMP. The levels of TIMP in several human body fluids were measured using assay (ii), and values in ng/ml with standard deviations (n-1) obtained as follows: serum, 299 +/- 62; plasma, 109 +/- 35; amniotic fluid, 724 +/- 62; cerebrospinal fluid, 144 +/- 156; saliva, 209 +/- 103.