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Bold indicates homologous substitutions. The sequence alignment of astacin and BMP Source of Materials Polymerase chain reaction products were purified with a Qiaquick kit Qiagen. We subsequently used site-directed mutagenesis and assay of recombinant enzyme to identify specific lysyl and cysteine residues in the active site of BMP-1 that are important for PCP activity.
These results indicated that Lys 87 and Lys act together to stabilize enzyme-substrate interactions involving the primed side of the substrate. However, the fact that the C65A mutant was well secreted raised the possibility that another cysteine residue could substitute for Cys 65 in bonding to Cys Medium harvested from COS-7 cells transfected with the empty vector contained no immunoreactive proteins.
Green indicates identical residues. This raised the possibility that Cys 85 could form a disulfide bond with a cysteine other than Cys Although the metalloproteinase domains 262237 astacin and BMP-1 are homologous and presumably have a similar tertiary structure, it is not obvious why BMP-1 cleaves scissile bonds between a small side chained residue and an aspartic acid.
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These cysteine residues straddle Cys 65which is conserved across the astacin family. This was in agreement 262377 the suggestion that Cys 85 bonds with Cys These cysteine residues are 262237 64 and Cys 84respectively, and are invariant in all astacin family members In preliminary studies we performed a multiple sequence alignment of 31 members of the astacin family of metalloproteinases using MultAlin 25 data not shown.
Red indicates residues that are different between BMP-1 and astacin and that also occur at functionally significant sites in the proteins. The most conspicuous difference between BMP-1 and other non-PCP astacins was dw occurrence of two unique cysteine residues at the active site of the enzyme.
The study also showed that the introduction of the c-myc tag at the C terminus of BMP-1 did not prohibit subsequent assays of PCP activity.
Assay of Procollagen C-proteinase Recombinant BMP-1 was assayed dz procollagen C-proteinase activity using human 14 C-labeled type I procollagen substrate and analysis of the cleavage products on SDS gels as described 3.
Latent denotes the latent form of BMP Prev Next Table of Contents. The results are summarized in Fig. The concentration of BMP-1 molecules was normalized. Undigested procollagen P ; containing disulfide-bonded chain migrates near the top of the SDS-gel.
Western blot analysis showed that the antibody recognized the C-propeptides after cleavage of procollagen with recombinant BMP-1 and BMP-1myc data not shown.
The pcDNA3 vector containing the cDNA for BMP-1myc was transfected into COS-7 cells, and the conditioned culture medium and the cell lysate were analyzed by Western blotting using the 9E10 antibody which detects the c-myc tag at the C terminus of the molecule and the neoepitope antibody which was raised to a peptide corresponding to the 10 residues at the N terminus of mature BMP In all the known fs of BMP-1 the scissile bond resides between a small side-chained residue and an sd acid.
The preparations were then examined in assays of procollagen C-proteinase.
Previous studies had shown that the addition of the FLAG peptide 266237 the C terminus of BMP-1 has negligible effect on the ability of the molecule to cleave procollagen Pwo DNA polymerase was used to minimize base misincorporation during the polymerase chain reactions.
Both fragments were gel-purified Macherey-Nagelmixed, and reamplified with the Pwo enzyme with the Xcm Forward and Blp Reverse primers.
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Section solely to indicate this fact. Responses Submit a Letter to the Editor. The cleaved C-propeptides CP migrate as a single disulfide-bonded trimer. The zinc-bound water is thought to be polarized for nucleophilic attack of the scissile bond by the glutamic acid residue in the consensus sequence HE XX H.
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This loop contains Lys This included astacin, BMP-1, mammalian tolloid, mammalian tolloid-like-1, mammalian tolloid-like-2, and meprins. Furthermore, because collagen is expressed in soft tissues such as liver and lung during progressive fibro-proliferative diseases e.
All the mutants exhibited reduced PCP activity.