Purification and characterization of alpha 1-antichymotrypsin-like protease inhibitor that regulates prohormone thiol protease involved in enkephalin precursor processing.

Hook, Vivian; Purviance, R.; Azaryan, Anahit V.; Hubbard, G L; Krieger, Timothy J.

doi:10.1016/s0021-9258(20)80763-7

Cited by 35 publications

(5 citation statements)

References 63 publications

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“…Chymotrypsin possesses two pairs of disulfide bonds that may influence the electrophoretic mobility of the enzyme under nonreducing con -ditions (29). Under reducing conditions, chymotrypsin possesses an apparent molecular weight of 25 kDa (7). These results demonstrate the sensitive detection of both chymotrypsin and trypsin by peptide-fluorescent substrates copolymerized in the in-gel zymogram assay.…”

Section: Resultsmentioning

confidence: 75%

Green Fluorescent Protein as a Quantitative Reporter of Relative Promoter Activity in E. coli

et al. 2000

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Green fluorescent protein (GFP) has become a valuable tool for the detection of gene expression in prokaryotes and eukaryotes. To evaluate its potential for quantitation of relative promoter activity in E. coli, we have compared GFP with the commonly used reporter gene lacZ, encoding beta-galactosidase. We cloned a series of previously characterized synthetic E. coli promoters into GFP and beta-galactosidase reporter vectors. Qualitative and quantitative assessments of these constructs show that (a) both reporters display similar sensitivities in cells grown on solid or liquid media and (b) GFP is especially well suited for quantitation of promoter activity in cells grown on agar. Thus, GFP provides a simple, rapid and sensitive tool for measuring relative promoter activity in intact E. coli cells.

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Section: Resultsmentioning

confidence: 75%

Green Fluorescent Protein as a Quantitative Reporter of Relative Promoter Activity in E. coli

et al. 2000

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“…The second example is the inhibition of a putative cysteine proteinase, bovine prohormone thiol protease (PTP), by a bovine antichymotrypsin-like molecule with an Arg-Thr reactive center (41,42). This protease requires dithiothreitol for activity; it is inhibited by iodoacetate, p-hydroxymercuribenzoate, mercuric chloride, cystatin and chymostatin; and it binds to concanavalin A (43).…”

Section: Discussionmentioning

confidence: 99%

Cross-Class Inhibition of the Cysteine Proteinases Cathepsins K, L, and S by the Serpin Squamous Cell Carcinoma Antigen 1: A Kinetic Analysis

et al. 1998

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The human squamous cell carcinoma antigens (SCCA) 1 and 2 are tandemly arrayed genes that encode two high-molecular-weight serine proteinase inhibitors (serpins). Although these proteins are 92% identical, differences in their reactive site loops suggest that they inhibit different types of proteinases. Our previous studies show that SCCA2 inhibits chymotrypsin-like serine proteinases [Schick et al. (1997) J. Biol. Chem. 272, 1849-1855]. We now show that, unlike SCCA2, SCCA1 lacks inhibitory activity against any of the more common types of serine proteinases but is a potent cross-class inhibitor of the archetypal lysosomal cysteine proteinases cathepsins K, L, and S. Kinetic analysis revealed that SCCA1 interacted with cathepsins K, L, and S at 1:1 stoichiometry and with second-order rate constants >/= 1 x 10(5) M-1 s-1. These rate constants were comparable to those obtained with the prototypical physiological cysteine proteinase inhibitor, cystatin C. Also relative to cystatin C, SCCA1 was a more potent inhibitor of cathepsin K-mediated elastolytic activity by forming longer lived inhibitor-proteinase complexes. The t1/2 of SCCA1-cathepsin S complexes was >1155 min, whereas that of cystatin C-cathepsin complexes was 55 min. Cleavage between the Gly and Ser residues of the reactive site loop and detection of a stable SCCA1-cathepsin S complex by sodium dodecyl sulfate-polyacrylamide gel electrophoresis suggested that the serpin interacted with the cysteine proteinase in a manner similar to that observed for typical serpin-serine proteinase interactions. These data suggest that, contingent upon their reactive site loop sequences, mammalian serpins, in general, utilize their dynamic tertiary structure to trap proteinases from more than one mechanistic class and that SCCA1, in particular, may be involved in a novel inhibitory pathway aimed at regulating a powerful array of lysosomal cysteine proteinases.

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“…There are now a number of well-documented instances of inhibition of cysteine proteinases by serpins. These include inhibition of cathepsins K, L, and S by the serpin squamous cell carcinoma antigen 1, , inhibition of prohormone thiol proteinase by α 1 -antichymotrypsin, and inhibition of members of the caspase family, including caspase 1 (interleukin-1β converting enzyme), caspase 3, and caspase 8 by the viral serpin crmA and caspases 1, 4, and 8 by the human serpin PI9 . Both the cathepsins and the caspases differ structurally from serine proteinases that have either the trypsin or subtilisin folds.…”

Section: 8 Inhibition Of Cysteine Proteinasesmentioning

confidence: 99%

Serpin Structure, Mechanism, and Function

2002

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Purification and characterization of alpha 1-antichymotrypsin-like protease inhibitor that regulates prohormone thiol protease involved in enkephalin precursor processing.

Cited by 35 publications

References 63 publications

Green Fluorescent Protein as a Quantitative Reporter of Relative Promoter Activity in E. coli

Green Fluorescent Protein as a Quantitative Reporter of Relative Promoter Activity in E. coli

Cross-Class Inhibition of the Cysteine Proteinases Cathepsins K, L, and S by the Serpin Squamous Cell Carcinoma Antigen 1: A Kinetic Analysis

Serpin Structure, Mechanism, and Function

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