Abstract
The putative matrix metalloproteinase mouse stromelysin-3 was expressed from Escherichia coli and from a mouse myeloma cell line. In the former case a single major protein of 58-kDa was detectable by immunoblotting, but no proteolytic activity could be elicited by zymography or trypsin or organomercurial treatment as would be expected for a typical matrix metalloproteinase. In the latter case immunodetectable proteins of 55-58 and 27-28-kDa were produced. The effect of trypsin or organomercurial treatment of the 55-58-kDa forms was to generate a 51-kDa form and lower molecular mass fragments. Upon zymographic analysis only the 27-28-kDa forms showed caseinolytic activity. N-terminal sequencing and immunoblotting analysis with antibodies specific to distinct domains of stromelysin-3 indicated that the 27-28-Da stromelysin-3 forms had lost the predicted propeptide and the majority of the C-terminal domain. The purified 28-kDa form of stromelysin-3 could weakly degrade a number of extracellular matrix proteins and was inhibited by TIMP. However, the evidence that mature full-length stromelysin-3 is a metalloproteinase could not be substantiated and the precise role of this protein in vivo remains to be elucidated. By partial analogy with interstitial collagenase, one hypothesis is that stromelysin-3 with an intact C-terminal domain has specific properties for an as yet undefined substrate.
Highlights
(Wolf et al, 1992; Muller et al, 1993)
From the percentage Analysis of the culture medium of myeloma cellstransfected with stromelysin-3 cDNA in the correct orientation by immunoblotting using mAb 4C10, known to react with the Nterminal portionf stromelysin-3, showed that a number of closely grouped bands of about 58 kDa anda band of about 28kDa were present (Fig. lA, lane 3 )
This is in comparisontotherecombinantform of bothhumanand mouse stromelysin-3 expressed by E. coli, which could be detected as a single major band of 58 kDa (Fig. lA, lanes 1 and 2)
Summary
Column was washed with 25 mM sodium cacodylate, pH 6.0, 0.5 M NaC1, 10 mM CaC12,0.02% azide to background Azmbefore elution of stromelysin-3 with 25 mM Tris-HC1, pH 7.5, 10 mM imidazole, 0.5 M NaC1, 10 mM CaClZ,0.02% azide. From the percentage Analysis of the culture medium of myeloma cellstransfected with stromelysin-3 cDNA in the correct orientation by immunoblotting using mAb 4C10, known to react with the Nterminal portion (catalytic domaino)f stromelysin-3, showed that a number of closely grouped bands of about 58 kDa (mobility distorted by the presenceof albumin) anda band of about 28kDa were present (Fig. lA, lane 3 ). This is in comparisontotherecombinantform of bothhumanand mouse stromelysin-3 expressed by E. coli, which could be detected as a single major band of 58 kDa (Fig. lA, lanes 1 and 2). In the caseof the starting materialit was not possible to obtain an accurate value for caseinase activity due to the presence of large amounts of albumin from the culture medium, which interferes with the assay
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