DS 26237 PDF

DS 26237 PDF

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The levels of BMP-1myc were quantitated by laser densitometry of enhanced chemiluminescence fluorograms exposed to pre-flashed film. The far right lane contains latent BMP-1myc, which migrates at a slower position than the reduced and oxidized form of BMP Secondary antibodies were either horseradish peroxidase conjugated to anti-mouse or anti-rabbit IgG and were detected by the enhanced chemiluminescence method Supersignal West Dura Extended Duration; Pierce.

We propose that the disulfide-bonded side chain of Cys 66 is fundamental to the PCP activity of BMP-1, because it backs onto the P1 residue in procollagen. Media were removed after 48 h and replaced by DMEM without serum and conditioned for another 24 h.

Responses Submit 262377 Letter to the Editor. Undigested procollagen P ; containing disulfide-bonded chain migrates near the top of the SDS-gel. Assay of Procollagen C-proteinase Recombinant BMP-1 was assayed for 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.

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Sequence Analysis In preliminary studies we performed a multiple sequence alignment of 31 members of the astacin family of metalloproteinases using MultAlin 25 data not shown. Furthermore, the culture medium from COS-7 cells transfected with the empty vector contained no immunoreactive proteins, which shows that the endogenous levels of BMP-1 were very low. The product was digested using appropriate restriction enzymes, gel-purified, and introduced in place of the corresponding wild-type fragment in BMP-1myc.

The C63G and C65A mutants migrated exclusively as the slower migrating reduced form.

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Cell lysates contained only the latent form of BMP Although the metalloproteinase domains 2637 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 22637 chained residue and an aspartic acid.

To learn more about how BMP-1 exhibits PCP activity we mapped the primary structure of BMP-1 onto the x-ray crystal structure of astacin and identified residues in the metalloproteinase domain of BMP-1 for subsequent site-directed mutagenesis studies.

Latent denotes the latent form of BMP Previous Section Next Section. This raised the possibility that Cys 85 could form a disulfide bond with a cysteine other than Cys A minor change was that laser densitometry of film exposed to dried gels was replaced by image plate quantitation of the 14 226237 proteins. The concentration of BMP-1 molecules was normalized. Cleavage occurs between a specific alanine or glycine residue depending on the procollagen chain and an invariant aspartic acid residue in each of the three chains of procollagen.

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Residue numbers are labeled with an asteriskin blocks of 10 residues and specific for BMP-1 and astacin. In brief, a peptide corresponding to the 10 N-terminal residues of the mature BMP-1 protein after removal of the prodomain was conjugated to keyhole limpet hemocyanin 22637 subsequently used to immunize two separate rabbits. Both fragments were gel-purified Macherey-Nagelmixed, and reamplified with the Pwo enzyme with the Xcm Forward and Blp Reverse primers.

The neoepitope anti-peptide antibody detected only the mature enzyme, which occurred in the culture medium and not in the cell lysate. In some experiments small levels of latent BMP-1 were detectable in the culture medium, which showed fs cleavage of the prodomain of BMP-1 was not a prerequisite for secretion.

These results indicated that Lys 87 and Lys act together to stabilize enzyme-substrate interactions involving the primed side of the substrate. We showed that substitution of alanine for Glu 94 eliminated the PCP activity of BMP-1 and confirmed that this glutamic acid residue is essential for catalytic activity of this enzyme. All the mutants exhibited reduced PCP activity. Small quantities of latent BMP-1myc were detected in some samples. We wanted to know whether BMP-1myc cleaved procollagen at 262237 physiological site.

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Bold indicates homologous substitutions. The importance of BMP-1 in tissue assembly is exemplified in the BMP-1 knockout mouse, which dies soon after birth from failure of ventral body wall closure rs with abnormal collagen 262337 5.

Human type I procollagen 0. Subsites are shown as arched lines. Nevertheless, these experiments demonstrated that, in COS-7 cells, removal of the prodomain is not a prerequisite for secretion of the protein.

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 This indicated that the Cys 63 and Cys dx mutant molecules have a more open conformation than the wild-type molecule. Articles by Kadler, K.

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The absence of the Cys 63 -Cys 66 disulfide bond in 1 the Cys dd mutant, 2 the Cys 63 mutant because Cys 66 has a free thiol group in this mutantand 3 the Cys 65 mutant because Cys 85 has bonded with Cys 63 thus leaving a free thiol group on Cys 66 alters the chemical structure of the S1 site and decreases PCP activity.

Also, BMP-1 molecules in which the other two cysteine residues on the active site edge strand, i. Furthermore, if these cells synthesized endogenous BMP-1, it was undetectable in assays of procollagen C-proteinase and in Western blotting analyses using a neoepitope antibody that recognizes the N terminus of mature BMP Cleavage of type I procollagen by recombinant BMP-1myc analyzed under reducing conditions.

It has been suggested that the equivalent disulfide bond in BMP-1 is formed between Cys 65 and Cys 85