Matrix Metalloproteinase Protocols (Methods in Molecular by Ed. I. Clark

By Ed. I. Clark

Best specialists aspect confirmed laboratory suggestions for the research of MMPs. The tools comprise these for the expression and purification of MMPs and TIMPs, for the detection of MMPs and TIMPs at either the protein and mRNA degrees, and for the assay of MMP and TIMP actions in a large choice of situations. each one process comprises step by step directions and notes version purposes and pitfalls to prevent. A selective assessment of the MMP area spells out the place the sphere has been, the place it really is, and the place it's going. finished and hugely useful, Matrix Metalloproteinase Protocols brings jointly the lengthy and richly deserved adventure of grasp experimentalists that might permit not just beginners to wake up to hurry quick, but in addition upload to the repertoire of winning ideas in professional laboratories.

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And Chambon, P. (1990) A novel metalloproteinase gene specifically expressed in stromal cells of breast carcinomas. Nature 348, 699–704. 83. , Segain, J. , and Basset, P. (1993) The 28-kDa N-terminal domain of mouse stromelysin-3 has the general properties of a weak metalloproteinase. J. Biol. Chem. 268, 15,435–15,441. 84. , and Weiss, S. J. (1994) Hydrolytic inactivation of a breast carcinoma cell-derived serpin by human stromelysin-3. J. Biol. Chem. 269, 25,849–25,855. 85. Pei, D. and Weiss, S.

And Uitto, J. (1983) Proteinases in human polymorphonuclear leukocytes. Purification and characterization of an enzyme which cleaves denatured collagen and a synthetic peptide with a Gly-Ile sequence. Eur. J. Biochem. 134, 129–137. 75. Wilhelm, S. , Collier, I. , Marmer, B. , Eisen, A. , Grant, G. , and Goldberg, G. I. (1989) SV40-transformed human lung fibroblasts secrete a 92-kDa type IV collagenase which is identical to that secreted by normal human macrophages. J. Biol. Chem. 264, 17,213–17,221.

To clone collagenase-3, we utilized degenerate oligonucleotide primers spanning two highly conserved amino acid sequences in known MMPs and reverse-transcribed RNA from human breast carcinomas. The conserved structural motifs corresponded to the activation locus (Pro-Arg-Cys-Gly-Val-Pro-Asp) containing the Cys residue essential for the maintenance of the latency of these enzymes, and the Zn-binding site (ValAla-Ala-His-Glu-Phe-Gly-His) present in the catalytic domain of all MMPs. After synthesizing two degenerate oligonucleotides encoding these conserved motifs and performing RT of total RNA from a mammary carcinoma, a band of the expected size (about 400 bp) was obtained and cloned.

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