Peptide substrates for a proteinase of Clostridium histolyticum

McQuade, A.B.; Crewther, W.G.

doi:10.1016/0005-2744(68)90055-7

Cited by 17 publications

(6 citation statements)

References 8 publications

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“…NP seems to prefer Gly residue at P1 site, and some polar amino acids, like Tyr, Lys, and Gln, were acceptable. Our results are consistent with those previously reported (McQuade and Crewther 1968). McQuade and Crewther (1968) demonstrated that NP could digest GlyXaa-amide, where Xaa may be Leu or Phe, but could not digest Gly-Phe-amide, benzyloxycarbonyl-glycine-L-phenylalanine (Z-Gly-Phe), and benzyloxycarbonyl-glycine-glycine (Z-Gly-Gly).…”

Section: Discussionsupporting

confidence: 95%

“…Our results are consistent with those previously reported (McQuade and Crewther 1968). McQuade and Crewther (1968) demonstrated that NP could digest GlyXaa-amide, where Xaa may be Leu or Phe, but could not digest Gly-Phe-amide, benzyloxycarbonyl-glycine-L-phenylalanine (Z-Gly-Phe), and benzyloxycarbonyl-glycine-glycine (Z-Gly-Gly). At P2′, Arg was suitable, but a Gly at the P2 position negates the effect of Arg, as revealed in our pentapeptide analysis.…”

Section: Discussionsupporting

confidence: 95%

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Cloning a neutral protease of Clostridium histolyticum, determining its substrate specificity, and designing a specific substrate

Maeda

Nakagawa

Murayama

et al. 2015

Appl Microbiol Biotechnol

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Islet transplantation is a prospective treatment for restoring normoglycemia in patients with type 1 diabetes. Islet isolation from pancreases by decomposition with proteolytic enzymes is necessary for transplantation. Two collagenases, collagenase class I (ColG) and collagenase class II (ColH), from Clostridium histolyticum have been used for islet isolation. Neutral proteases have been added to the collagenases for human islet isolation. A neutral protease from C. histolyticum (NP) and thermolysin from Bacillus thermoproteolyicus has been used for the purpose. Thermolysin is an extensively studied enzyme, but NP is not well known. We therefore cloned the gene encoding NP and constructed a Bacillus subtilis overexpression strain. The expressed enzyme was purified, and its substrate specificity was examined. We observed that the substrate specificity of NP was higher than that of thermolysin, and that the protein digestion activities of NP, as determined by colorimetric methods, were lower than those of thermolysin. It seems that decomposition using NP does not negatively affect islets during islet preparation from pancreases. Furthermore, we designed a novel substrate that allows the measurement of NP activity specifically in the enzyme mixture for islet preparation and the culture broth of C. histolyticum. The activity of NP can also be monitored during islet isolation. We hope the purified enzyme and this specific substrate contribute to the optimization of islet isolation from pancreases and that it leads to the success of islet transplantation and the improvement of the quality of life (QOL) for diabetic patients.

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Section: Discussionsupporting

confidence: 95%

Section: Discussionsupporting

confidence: 95%

Cloning a neutral protease of Clostridium histolyticum, determining its substrate specificity, and designing a specific substrate

Maeda

Nakagawa

Murayama

et al. 2015

Appl Microbiol Biotechnol

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“…There is also likely to be considerable nonspecific enzymatic attack on cell membrane constituents of any of the cells, causing breaks in the membranes. Crude bacterial collagenase contains at least three noncollagenase proteases [21] and perhaps other as yet unidentified hydrolytic enzymes. A similar decrease in cell potassium and increase in cell sodium has been observed in freshly isolated liver cells [10].…”

Section: Discussionmentioning

confidence: 99%

Potassium, sodium, and the intracellular fluid space of cells from bone

1979

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Cells enzymatically dispersed from fetal rat calvaria were analyzed for sodium and potassium content and intracellular fluid space (ICF). Even when obtained in comparatively high yield, the cells are damaged by the isolation procedure as evidenced by high sodium and low potassium content immediately after isolation. During a post-incubation period potassium is accumulated and sodium extruded to steady-state levels. Although electrolyte content of cells after recovery did not vary as a function of cell yield, ICF was increased in cells obtained in lower yield, suggesting cell swelling as a result of membrane damage. The weighted mean values obtained for the best cell preparations were 117 mM K+ and 27 mM Na+. Based on DNA assay of isolated cells and the whole tissue, 20- to 21-day calvaria were found to have an average of 8.1 x 10(6) cells/calvarium. Combining cell data with analysis of total tissue sodium, potassium, and water, it was concluded that the tissue extracellular sodium is in equilibrium with blood but that the potassium concentraiton is approximately 5-fold higher than blood levels.

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“…This implies that the extracellular matrix (ECM), a complex network of collagen, proteoglycans including glycosaminoglycans, glycoproteins and elastin [15], should be degraded without affecting the structural and functional integrity of the islets. The usual approach applies digestion of the tissue with commercial Clostridium histolyticum collagenase, which is a mixture of six collagenases with distinct substrate specificities [16,17] and various other enzymes such as aminopeptidase [18], clostripain [19], phospholipase C [3] and neutral protease [20]. The composition of C. histolyticum collagenase is an important source of variability in islet isolation [2,3].…”

mentioning

confidence: 99%

An analysis of the role of collagenase and protease in the enzymatic dissociation of the rat pancreas for islet isolation

et al. 1992

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Crude Clostridium histolyticum collagenase is widely used for the enzymatic degradation of pancreatic extracellular matrix in order to isolate the islets of Langerhans. The variable enzymatic composition of crude collagenases is a critical issue which contributes to the poor reproducibility of islet isolation procedures. In this study, the separate contributions of collagenase and protease to the islet isolation process were analysed by testing various combinations of purified collagenase and purified protease in rat pancreas dissociations under conditions which eliminated all other proteolytic activity. Under these conditions, complete tissue dissociation by purified collagenase required 99 +/- 10 min, whereas increasing amounts of protease progressively reduced this time to a minimum of 36 +/- 1 min. Histochemical analysis of the dissociation process showed that protease enhanced the degradation of all four major components of the extracellular matrix: collagen was degraded more completely, while proteoglycans, glycoproteins and elastin were degraded at a higher rate. Pancreas dissociation under the present, strictly controlled conditions resulted in a high yield of viable islets: 4.2-5.0 microliters islet tissue volume (3,300-3,800 islets) were isolated per g pancreas in the presence of a high or low protease concentration, respectively. Prolonged dissociation in the presence of protease resulted in a dramatic decrease in islet yield which correlated with the observation that the enzyme accelerated islet disintegration. It is concluded that the collagenase-induced dissociation of the extracellular matrix is facilitated by protease. Our study shows that high yields of viable islets can be obtained under controlled enzymatic conditions, provided that the exposure of islets to protease is limited.

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Peptide substrates for a proteinase of Clostridium histolyticum

Cited by 17 publications

References 8 publications

Cloning a neutral protease of Clostridium histolyticum, determining its substrate specificity, and designing a specific substrate

Cloning a neutral protease of Clostridium histolyticum, determining its substrate specificity, and designing a specific substrate

Potassium, sodium, and the intracellular fluid space of cells from bone

An analysis of the role of collagenase and protease in the enzymatic dissociation of the rat pancreas for islet isolation

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