The kinetics of hydrolysis of some synthetic substrates containing neutral hydrophilic groups by pig pepsin and chicken liver cathepsin D.

Irvine, G. Brent; Blumsom, Nigel L.; Elmore, D. T.

doi:10.1042/bj2110237

Cited by 6 publications

(4 citation statements)

References 21 publications

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“…2 and 3 show that no cleavage occurred in 1 hr at pH 5.5 or 6.0 but that 4 hr at pH 6.0 gave appreciable conversion. This is similar to the observation by Irvine et al (28). The lag phase was most pronounced at low concentrations of substrate.…”

supporting

confidence: 81%

Cathepsin D-mediated processing of procollagen: lysosomal enzyme involvement in secretory processing of procollagen.

Helseth¹,

Veis²

1984

Proc. Natl. Acad. Sci. U.S.A.

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The proteolytic removal of the extension COOH-terminal propeptide from procollagen has been examined in vitro. A crude enzyme activity was identified in a whole-chicken-embryo extract that acted at acid pH and appeared to be similar to one identified previously [Davidson, J. M., McEneany, L. S. G. & Bornstein, P. (1979) Eur. J. Biochem. 100,[551][552][553][554][555][556][557][558]. This activity was inhibitable by pepstatin but not by leupeptin, suggesting that it might be cathepsin D. Cathepsin D was purified 907-fold from chicken livers by affinity chromatography on pepstatin-aminohexyl-Sepharose 4B and was found to remove the COOH propeptides from procollagen. At pH 6.0, the site of cleavage appeared to shift from the COOH telopeptide to the COOH telopeptide/propeptide junction, based upon the difference in electrophoretic migration of the cleavage products, although determining the actual cleavage site will require end-group analysis. A model for the involvement of cathepsin D in the in vivo processing of procollagen is presented.The enzymes responsible for the sequential removal of the extension propeptides from procollagen, procollagen NH2-terminal proteinase and procollagen COOH-terminal proteinase, have been actively studied, but only the former has been well characterized (1). In vivo it appears that the COOH-terminal propeptide (COOH propeptide) is removed first, with the NH2-terminal propeptide (NH2 propeptide) removed later, possibly after the intermediate has assembled into extracellular fibrils (2-4). The site of COOH propeptide removal has not been well defined. In cell culture experiments, the apparent in vivo pathway is reversed. The COOH propeptide is not cleaved from the product that accumulates in cell culture media, yet conversion is complete in organ culture (5), suggesting that an intact extracellular matrix is required for conversion.In searching for a neutral metalloproteinase that is a COOH-terminal proteinase (6), we observed a cathepsin Dlike activity, which removed the COOH propeptide from procollagen at acid pH. We report here that the cleavage of procollagen with purified cathepsin D is specific for the COOH propeptide and that cleavage occurs at or near the authentic cleavage site when digestion is carried out near pH 6.0. We postulate a role for cathepsin D in the in vivo conversion of procollagen to collagen. MATERIALS AND METHODSProcollagen was isolated from 17-day chicken-embryo tendon fibroblasts (7,8). After tendon dissection and dissociation with collagenase and trypsin, fibroblasts were incubated (2 x 107 cells per ml) for 6 hr in modified Krebs medium II in the presence of 1 ,uCi (1 Ci = 37 GBq) of a mixture of 15 14C-labeled amino acids (New England Nuclear) per ml. Procollagen and partially processed collagen from which the The publication costs of this article were defrayed in part by page charge payment. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. §1734 solely to indicate this fact. 3302NH2 propeptide was removed (...

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supporting

confidence: 81%

Cathepsin D-mediated processing of procollagen: lysosomal enzyme involvement in secretory processing of procollagen.

Helseth¹,

Veis²

1984

Proc. Natl. Acad. Sci. U.S.A.

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“…Rather than using a single time point when a protein band was no longer visible on an SDS–PAGE gel or Western blot (time to disappearance based on the human eye), protein bands on SDS–PAGE gels were quantified by densitometry ( Bindslev-Jensen et al 2003 ; Brussock and Currier 1990 ; Cantu and Nelson 1994 ; Syrovy and Hodny 1991 ) over a digestion time course, and the pattern of protein degradation was modeled using a negative exponential equation (pseudo-first-order decline). Studies on the pepsinolysis of proteins and peptides have often supported a pseudo-first-order pattern of digestion (e.g., Baderschneider et al 2002 ; Belorgey et al 1996 ; Garrett et al 2004 ; Irvine et al 1983 ; Matthyssens et al 1972 ; Sachdev and Fruton 1975 ; Terada at al. 1974 ).…”

Section: Kinetic Data Analysismentioning

confidence: 99%

Digestion Assays in Allergenicity Assessment of Transgenic Proteins

Herman¹,

Storer²,

Gao³

2006

Environ Health Perspect

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The food-allergy risk assessment for transgenic proteins expressed in crops is currently based on a weight-of-evidence approach that holistically considers multiple lines of evidence. This approach recognizes that no single test or property is known to distinguish allergens from nonallergens. The stability of a protein to digestion, as predicted by an in vitro simulated gastric fluid assay, currently is used as one element in the risk assessment process. A review of the literature on the use of the simulated gastric fluid assay to predict the allergenic status of proteins suggests that more extensive kinetic studies with well-characterized reference proteins are required before the predictive value of this assay can be adequately judged.

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“…Table I shows the residue combinations which constitute the most probable primary cleavage sites in proteins for cathepsins B, L, and D as well as those residue combinations that are non-substrates for the enzymes. The data were derived from the literature [9][10][11][12][13][14][15][16][17][18][19][20][21][22][23][24][25][26][27][28] on the substrate specificity of the three enzymes.…”

Section: Methodsmentioning

confidence: 99%

T‐Helper‐Cell Determinants in Protein Antigens are Preferentially Located in Cysteine‐Rich Antigen Segments Resistant to Proteolytic Cleavage by Cathepsin B, L, and D

et al. 1991

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We report on a computer algorithm capable of predicting the location of T-helper-cell epitopes in protein antigen (Ag) by analysing the Ag amino acid sequence. The algorithm was constructed with the aim of identifying segments in Ag which are resistant to proteolytic degradation by the enzymes cathepsin B, L, and D. These are prominent enzymes in the endocytic pathway through which soluble protein Ag enter APC, and resistant segments in Ag may, therefore, be expected to contain more T-cell determinants than susceptible segments. From information available in the literature on the substrate specificity of the three enzymes, it is clear that a cysteine is not accepted in any of the S2, S1, S1', and S2' subsites of cathepsin B and L, and not in the S1 and S1' subsites of cathepsin D. Moreover, we have noticed that cysteine-containing T-cell determinants in a number of protein Ag are particularly rich in the amino acids alanine, glycine, lysine, leucine, serine, threonine, and valine. By searching protein Ag for clusters of amino acids containing cysteine and two of the other amino acids we were able to predict 17 out of 23 empirically known T-cell determinants in the Ag with a relatively low number of false (positive) predictions. Furthermore, we present a new principle for searching Ag for potential amphipatic alpha-helical protein segments. Such segments accord well with empirically known T-cell determinants and our algorithm produces a lower number of false positive predictions than the principle based on discrete Fourier transformations previously described.

show abstract

The kinetics of hydrolysis of some synthetic substrates containing neutral hydrophilic groups by pig pepsin and chicken liver cathepsin D.

Cited by 6 publications

References 21 publications

Cathepsin D-mediated processing of procollagen: lysosomal enzyme involvement in secretory processing of procollagen.

Cathepsin D-mediated processing of procollagen: lysosomal enzyme involvement in secretory processing of procollagen.

Digestion Assays in Allergenicity Assessment of Transgenic Proteins

T‐Helper‐Cell Determinants in Protein Antigens are Preferentially Located in Cysteine‐Rich Antigen Segments Resistant to Proteolytic Cleavage by Cathepsin B, L, and D

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