Chemical characterization of the homogeneous collagenase from Clostridium histolyticum

Emod, Istvan; Tong, Nguyen Thanh; Keil, B.

doi:10.1016/0005-2744(81)90054-1

Cited by 12 publications

(8 citation statements)

References 31 publications

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“…Moreover, collagenase II has a composition that is intermediate between those of the two classes of enzymes, suggesting that it could have been a mixture. The analyses for collagenases A and B and the enzyme isolated by Emod et al (1981), although similar in many ways, do not agree well with any of the collagenases isolated here.…”

Section: Resultscontrasting

confidence: 56%

“…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%

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Characterization of the individual collagenases from Clostridium histolyticum

Bond¹,

Wart²

1984

Biochemistry

210

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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Section: Resultscontrasting

confidence: 56%

Section: Discussionmentioning

confidence: 99%

Characterization of the individual collagenases from Clostridium histolyticum

Bond¹,

Wart²

1984

Biochemistry

210

135

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“…The best means of comparing the activities of collagenases from different studies is by their action against PZ-Pro-Leu-Gly-Pro-D-Arg (Wunsch & Heidrich, 1963), since all such assays have been carried out under a single set of conditions. The activities of the -, 6-, and ("-collagenases toward this substrate, which are reported in the accompanying paper (Bond & Van Wart, 1984a), are significantly higher than reported (Kono, 1968;Emod et al, 1981) for any Clostridial collagenase.…”

Section: Discussionmentioning

confidence: 57%

“…In fact, the number of enzymes detected generally correlates with how extensively the starting preparation was subjected to ion-exchange chromatography and the extent to which the various chromatographic fractions obtained were assayed. Of the recent studies in the literature, only that by Emod et al (1981) has claimed the presence of a single collagenase in the culture filtrate. However, these authors made no attempt to purify all the collagenases present in their starting preparation.…”

Section: Discussionmentioning

confidence: 99%

Purification and separation of individual collagenases of Clostridium histolyticum using red dye ligand chromatography

Bond¹,

Wart²

1984

Biochemistry

160

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Six collagenases present in the culture filtrate of Clostridium histolyticum have been purified to homogeneity. Chromatography over hydroxylapatite, Sephacryl S-200, and L-arginine-Affi-Gel 202 removes the brown pigment and the great majority of the contaminating proteinases active against casein, benzoyl-L-arginine ethyl ester, and elastin. Reactive Red 120 dye ligand chromatography subdivides the collagenases, which have very similar physicochemical properties, among four fractions. The final purification is achieved by chromatography over DEAE-cellulose and SP-Sephadex. All six collagenases, designated alpha, beta, gamma, delta, epsilon and zeta by the order of their purification, are highly active against collagen and devoid of other proteolytic activities. Each exhibits a single band on sodium dodecyl sulfate-polyacrylamide gels. Two distinct subspecies of the alpha and gamma enzymes have been isolated, which have the same molecular weight and activity but different isoelectric points. There is some less pronounced microheterogeneity for the other collagenases. On the basis of their activities toward native collagen and the synthetic peptide 2-furanacryloyl-L-leucylglycyl-L-prolyl-L-alanine (FALGPA), the six collagenases are divided into two classes. Class I collagenases (alpha, beta, and gamma) have high collagenase activity and moderate FALGPA activity while the class II collagenases (sigma, epsilon, and sigma) have moderate collagenase and high FALGPA activities. The relationship between these six collagenases and other reported to have been isolated in the literature has also been examined.

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“…C. histolyticum collagenases are Ca 2+ -and pH-dependent (Figure 1; 23,24). The inhibitory effects of Ca 2+ -free/EDTA and acidic pH on collagenase reactions were imaged in realtime.…”

Section: Resultsmentioning

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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Chemical characterization of the homogeneous collagenase from Clostridium histolyticum

Cited by 12 publications

References 31 publications

Characterization of the individual collagenases from Clostridium histolyticum

Characterization of the individual collagenases from Clostridium histolyticum

Purification and separation of individual collagenases of Clostridium histolyticum using red dye ligand chromatography

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

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