No abstract

Human rheumatoid synovial cells in culture secrete both 72-kDa progelatinase and a complex consisting of 72-kDa progelatinase and a 24-kDa inhibitor of metalloproteinases, TIMP-2. In addition, the culture medium contains TIMP-1, the classical inhibitor of metalloproteinases, with a molecular mass of 30 kDa. TIMP-1 does not form a complex with free 72-kDa progelatinase.Free progelatinase and progelatinase complexed with TIMP-2 can be activated with the organomercury compound p-aminophenylmercury acetate. The activated complex shows less than 10% the enzyme activity of activated free gelatinase.The progelatinase -TIMP-2 complex could be shown to be an inhibitor for other metalloproteinases, such as gelatinase and collagenase secreted by human rheumatoid synovia fibroblasts, as well as for the corresponding enzymes from human neutrophils.The tissue inhibitor of metalloproteinases (TIMP-1) is a glycoprotein with a molecular mass of about 30 kDa, purified initially from culture medium conditioned by normal skin fibroblasts, and later from various human sources, including plasma, amniotic fluid and synovial fluid [l -71.The TIMP-1 gene has been localized to the x chromosome [8]. The secreted protein consists of 184 amino acids and possesses six disulfide bonds and two glycosylation sites containing N-linked oligosaccharides [5, 91.TIMP-1 specifically inhibits the extracellular-matrix-degrading metalloproteinases such as collagenase, gelatinase and stromelysin. It has no activity on mammalian membrane metalloproteinase, procollagen peptidase, or on bacterial metalloproteinases such as thermolysin [lo].Originally it was thought that TIMP-1 only binds to active enzymes and not to latent ones. However, it was reported recently, that the 72-kDa progelatinase secreted by several cell types, such as Simian-virus-40-transformed human lung fibroblasts, Harvey murine sarcoma virus-transformed human bronchial epithelial cells, human A2058 melanoma cells and normal skin fibroblasts, exists in a stable, but noncovalent 1 : 1 stoichiometric complex with a 24-kDa inhibitor of metalloproteinases called TIMP-2 [ l l , 121. In addition, a 92-kDa progelatinase -TIMP-1 complex is secreted by human lung fibroblasts [13]. This proteinase is normally produced by macrophages and polymorphonuclear leukocytes (PMNL). PMNL do not produce a tissue inhibitor of metalloproteinases, which may account for its 10-fold-higher specific activity 1131.We found human rheumatoid synovial cells to secrete not only the 72-kDa-progelatinase -TIMP-2 complex and TIMP-1, but in addition free 72-kDa progelatinase. In this communication, we show that TIMP-2, although complexed with the progelatinase, still remains an inhibitor of metalloproteinases. MATERIAL AND METHODS Cell cultureHuman rheumatoid synovium obtained by surgery was washed in Hank's solution and freed from fat and cartilage, cut into pieces (approximately 1 mm3 in size), washed with Hank's solution again, and distributed into 75-cmZ culture flasks. 8 ml Dulbecco's modified Eagle's medium containing ...

Reliable observation, detection and characterisation of polluted soil are of major concern in regions with military activities in order to prepare efficient decontamination. Flexible on-site analysis may be facilitated by biosensor devices. With use of fibre-optic evanescent field techniques, it has been shown that immunoaffinity reactions can be used to determine explosives sensitively. Besides antibodies as molecular recognition elements, high-affinity nucleic acids (aptamers) can be employed. Aptamers are synthetically generated and highly efficient binding molecules that can be derived for any ligand, including small organic molecules like drugs, explosives or derivatives thereof. In this paper we describe the development of specific aptamers detecting the explosives molecule TNT. The aptamers are used as a sensitive capture molecule in a fibre-optic biosensor. In addition, through the biosensor measurements the aptamers could be characterised. The advantages of the aptamer biosensor include its robustness, its ability to discriminate between different explosives molecules while being insensitive to other chemical entities in natural soil and its potential to be incorporated into a portable device. Results can be obtained within minutes. The measurement is equally useful for soil that has been contaminated for a long time and for urgent hazardous spills.

C-terminal truncated membrane-type 2 matrix metalloproteinase (MT2-MMP1-269), comprising prodomain and catalytic domain, was expressed as a soluble protein in Escherichia coli. Unlike the corresponding form of MT1-MMP, which can be isolated as a 31 kDa protein, MT2-MMP1-269 proved to be comparatively instable, and already the freshly isolated preparation displayed several proteins in SDS-PAGE representing MT2-MMP1-269 (33 kDa) and four N-truncated forms with N-termini methionine32 (30 kDa), isoleucine37 (30 kDa), leucine84 (24 kDa), and leucine93 (22 kDa), the catalytic domain. After thawing of frozen preparations the 33 and the 30 kDa proforms were no longer detectable in SDS-PAGE, and only the 24 and 22 kDa forms remained. The catalytic domain of MT2-MMP activated progelatinase A as well as the progelatinase A/TMP-2 complex by cleaving the 72 kDa progelatinase A to yield 67 kDa gelatinase A, which is then transformed into 62 kDa gelatinase A. The 62 kDa form is about twice as active as the 67 kDa form towards the synthetic substrate N-(2,4)-dinitrophenyl-Pro-Gln-Gly-Ile-Ala-Gly-Gln-D-Arg. No significant difference in activity was found between free and complexed gelatinase A forms. the activation of the progelatinase A/TIMP-2 complex proceeds in two steps: At first MT2-MMP is inhibited by the progelatinase A/TIMP-2/MT2-MMP, complex, whereby a ternary complex, progelatinase A/TIMP-2/ MT-2MMP is generated. This ternary complex is then activated by excess MT2-MMP. Our results suggest a mechanism for spatially regulated extracellular gelatinase A activity mediated by activation with membrane-type MMPs; Free gelatinase A is released into the extracellular space, while gelatinase A/TIMP-2 bound to MT-MMP remains anchored on the cell surface.

Incubation of progelatinase B, isolated from human polymorphonuclear leukocytes, with TIMP-1 leads to the formation of the progelatinase B/TIMP-1 complex. This complex behaves like a Janus in a similar manner as we previously described for the progelatinase A/TIMP-2 complex. It shows the properties of TIMP-1 and is a better inhibitor for gelatinase A than for gelatinase B. Treatment with trypsin leads to activation of the binary complex. The activity, however, amounts only to slightly more than 10% of the activity of free gelatinase B, not complexed with TIMP-1. When the progelatinase B/TIMP-1 complex inhibits an active matrix metalloproteinase, a ternary complex is generated that after activation displays a distinct higher proteolytic activity than the active binary complex. The active binary complex cannot be transformed into the active ternary complex.