Immunochemistry of bacterial ornithine carbamoyltransferase and bacterial arginase

Soru, E; Zaharia, O

doi:10.1016/0019-2791(70)90113-8

Cited by 3 publications

(4 citation statements)

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“…C. histolyticum collagenases are metalloproteinases, which require Zn 2+ and Ca 2+ for their proteolytic activity (). C. histolyticum collagenase activities are inhibited by chelation of Ca 2+ , and their activity is temperature and pH dependent ( , ). Our data confirm the Ca 2+ - and pH-dependent properties of C. histolyticum collagenases (Figures , , and 8).…”

Section: Discussionmentioning

confidence: 99%

Imaging Real-Time Proteolysis of Single Collagen I Molecules with an Atomic Force Microscope

1999

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The dynamic process of synthesis and degradation of extracellular matrix molecules, including various collagens, is important in normal physiological functions and pathological conditions. Existing models of collagen enzymatic degradation reactions are derived from bulk biochemical assays. In this study, we have imaged in real-time individual collagen I molecules and their proteolysis by Clostridium histolyticum collagenases in phosphate-buffered saline (PBS) with atomic force microscopy (AFM). We have also imaged the likely binding and unbinding of collagenase molecules to single triple-helical collagen I molecules and subsequent proteolysis of subsets of the collagen molecules. The proteolysis of collagen molecules was inhibited by reduced calcium and acidification. Results from AFM study of collagen proteolysis are consistent with SDS-PAGE biochemical assays. The real-time proteolysis of single collagen I molecules followed simple Michaelis-Menton kinetics previously derived from bulk biochemical assays. This is the first report of imaging real-time proteolysis of single macromolecules and its inhibition on a molecular scale. A strong correspondence between the kinetics of proteolysis of single collagen molecules and the kinetics of proteolysis derived from bulk biochemical assays will have a wide applicability in examining real-time enzymatic reactions and their regulation at single molecule structural level. Such real-time study of single molecule proteolysis could provide a better understanding of the interactions between proteases and target proteins as well as proteases and protease inhibitors.

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Section: Discussionmentioning

confidence: 99%

Imaging Real-Time Proteolysis of Single Collagen I Molecules with an Atomic Force Microscope

1999

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“…Over the years, many different molecular weights have been reported for Clostridial collagenases. Without specifying the designation given to the collagenase or the method used to determine the molecular weight, values that have been reported include 57 400 (Harper et al, 1965), 66000 (Keil, 1979), 70000 (Emod et al, 1981), 72000 (Lwebuga-Mukasa et al, 1976), 79000 (Yoshida & Noda, 1965), 81000 (Lwebuga et al, 1976), 95 000 (Yoshida & Noda, 1965), 105000 (Harper et al, 1965), 109 000 (Seifter et al, 1959;Strauch & Grassman, 1966), 112000 (Mandl et al, 1964), and 147000 (Soru & Zaharia, 1972). There is little doubt that some of the quoted values differ due to the fact that investigators have simply isolated different individual or subsets of the individual collagenases.…”

Section: Discussionmentioning

confidence: 99%

“…Moreover, there seems to be little agreement concerning those basic properties that have been examined. For example, the molecular weights of these collagenases have variously been reported to be as low as 66 000 (Keil, 1979) and as high as 147 000 (Soru & Zaharia, 1972).…”

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confidence: 99%

Characterization of the individual collagenases from Clostridium histolyticum

Bond¹,

Wart²

1984

Biochemistry

212

135

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The six collagenases (alpha, beta, gamma, delta, epsilon, and zeta) from Clostridium histolyticum isolated in the preceding paper [Bond, M. D., & Van Wart, H. E. (1984) Biochemistry (first paper of three in this issue)] have been characterized in detail. The molecular weights determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis range from 68 000 to 125 000. Isoelectric focusing experiments demonstrate that the isoelectric points of the collagenases are in the 5.35-6.20 range. These experiments also reveal that the subspecies of alpha- and gamma-collagenases (alpha1 vs. alpha 2 and gamma 1 vs. gamma 2) have different isoelectric points but the same molecular weights. Microheterogeneity is also observed for the beta- and epsilon-collagenases. The amino acid compositions of all six collagenases have been determined, and analysis for neutral sugars and hexosamines shows that none of the enzymes have a significant carbohydrate content. Zinc and calcium are the only metals that copurify with the collagenases. The purified enzymes contain approximately 1 mol of zinc/mol of protein and a calcium content that varies from about 2 mol/mol for alpha-collagenase to about 7 mol/mol for beta-collagenase. All of the collagenases are 5-10 times more active against gelatin than collagen. The alpha-, beta-, and gamma-collagenases are significantly less active toward the synthetic peptide substrates examined than the delta-, epsilon, and zeta-collagenases. This property, taken together with data on the stabilities and amino acid compositions of these enzymes, strongly supports their assignment to two distinct classes. This establishes clearly that C. histolyticum does, indeed, produce more than one different type of collagenase.

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“…This harmful effect acts in a time‐dependent manner and can be due to the direct effect of crude collagenase on cells and not some degraded components produced during the enzymatic digestion of connective tissue because the same pattern of cell death was observed in in vitro established dermal fibroblasts, which revealed that extracellular matrix is not only the target of crude collagenase but may also directly influence some molecules such as membranous collagen present on the cell surface, thus leading to a higher rate of death among cells with a longer time of incubation with this enzyme. Also, regarding the presence of other components in crude collagenase (nonspecific proteases, clostripain, neutral protease, aminopeptidase activities, and trypsin), it is possible that some of them, such as proteases, have a detrimental effect on cell survival 24–29. However, as protease accelerates tissue dissociation, it should be considered as a double‐edged sword that can damage cells subsequent to prolonged exposure to crude collagenase 30.…”

Section: Discussionmentioning

confidence: 99%

Short exposure to collagenase and coculture with mouse embryonic pancreas improve human dermal fibroblast culture

Pandamooz

Hadipour

Akhavan‐Niaki

et al. 2012

Biotech and App Biochem

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The dermal fibroblast as a major component of connective tissue has attracted much attention in the past few years, and application of these very fast growing cells in several fields has been intensively studied. Isolating dermal fibroblasts is an appropriate way to expand these fast growing cells in vitro. Although using a dissociated fibroblast culture method is more convenient than skin explant culture, its enzymatic digestion is critical because a large number of cells can be lost over prolonged exposure to collagenase. This study was performed to increase the number of viable cells after digestion of fresh human foreskin of donors aged from 1 to 3 months with collagenase and also by to design a coculture system for resuscitation of the injured fibroblast. Our results demonstrate that we can maximize cell yield while maintaining cell viability by cutting the specimens into very small pieces (1-2 mm³) after removing the epidermal layer with dispase II and also by collecting released cells every 20 Min subsequent to digesting the dermal layer with collagenase. Moreover, our data strongly indicate that coculturing of isolated fibroblasts with embryonic pancreas explants can enhance the rate of proliferation in cultured fibroblasts.

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Immunochemistry of bacterial ornithine carbamoyltransferase and bacterial arginase

Cited by 3 publications

References 0 publications

Imaging Real-Time Proteolysis of Single Collagen I Molecules with an Atomic Force Microscope

Imaging Real-Time Proteolysis of Single Collagen I Molecules with an Atomic Force Microscope

Characterization of the individual collagenases from Clostridium histolyticum

Short exposure to collagenase and coculture with mouse embryonic pancreas improve human dermal fibroblast culture

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