Some newly characterized collagenases from procaryotes and lower eucaryotes

Keil, B.

doi:10.1007/bf00226230

Cited by 38 publications

(27 citation statements)

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“…The numerous bands of cross-reactivity shown by immunoelectrophoresis of strain CIP 82.01 collagenase with antibodies against strain 1.029 collagenase suggest the presence of some partially degraded forms of the enzyme, in which the major determinants are conserved. Previous studies have shown that the subunit of Achromobacter collagenase (strain 1.029) may be structurally similar to the neutral proteinases recovered from Bacillus thermoproteolyticus (thermolysin; EC 3.4.24.4) and B. subtilis (10). This association is based on a comparison of molecular weights, amino acid compositions (9,14,32), essential components of the enzyme active sites (7,33), and circular dichroism measurements (6).…”

Section: Discussionmentioning

confidence: 99%

“…In the presence of collagen or macromolecular fragments of collagen (15), a subculture of this organism (strain 1.029) produces a collagenase having a very high specific activity (12,20). The structural and functional properties of this Achromobacter collagenase (EC 3.4.24.8), as well as the metabolism of strain 1.029, have been described in a series of papers by Woods et al (21,26,27,29,35) and by workers in our laboratory (4, 10,11,13,14,19,30).…”

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confidence: 99%

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Assignment of "Achromobacter iophagus" Strain I.029 to Vibrio alginolyticus Chemovar iophagus

Emod

Soubigou

Tong

et al. 1983

International Journal of Systematic Bacteriology

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RICHARD^Strain CIP 82.01 of Vibrio alginolyticus and strain 1.029 (previously designated "Achromobacter iophagus") were compared. Strain 1.029 produces a collagenase of high specific activity (Achrornobacter collagenase; EC 3.4.24.8). Collagenase production is induced in strain CIP 82.01 by collagen or macromolecular fragments of collagen in a manner similar to collagenase induction in strain 1.029; caseinolytic proteinase is constitutive. In this study we demonstrate that both strains also produce a constitutive extracellular endonuclease. Collagenases from both strains cleave either native collagen in its helical region or a similar synthetic peptide; both enzymes are inhibited by ethylenediamine tetraacetate, but not by diisopropyl fluorophosphate. The collagenase subunit (molecular weight, 35,000) of strain CIP 82.01 is similar in amino acid composition to the subunit of the strain 1.029 enzyme, although some of the aspartic and threonine residues in strain CIP 82.01 are replaced by glutamic and serine residues in strain 1.029. Surface radioiodination followed by two-dimensional electrophoresis showed that there are quantitative differences in the major outer membrane proteins of the two strains. Strains CIP 82.01 and 1.029 differ qualitatively in resistance to ampicillin and carbenicillin, in cellobiose fermentation, in ornithine decarboxylase activity, and in halophilism. We propose that strain 1.029, which was originally designated A . iophagus, be included within the species V. alginolyticus, but that this organism be distinguished from other strains of this species by the designation Vibrio alginolyticus chemovar iophagus, with the corresponding collagenase designated "iophagus collagenase" (EC 3.4.24.8).In 1973, Welton and Woods isolated and described the gram-negative aerobic bacterium Achromobacter iophagus (34). In the presence of collagen or macromolecular fragments of collagen (15), a subculture of this organism (strain 1.029) produces a collagenase having a very high specific activity (12,20). The structural and functional properties of this Achromobacter collagenase (EC 3.4.24.8), as well as the metabolism of strain 1.029, have been described in a series of papers by Woods et al. (21,26,27,29,35) and by workers in our laboratory (4,10,11,13,14,19,30).In a recent study (27), which confirmed the original observation of collagenase induction by macromolecular collagen fragments (15), Achromobacter iophagus was reclassified as a strain of Vibrio alginolyticus; however, a direct experimental comparison of strain 1.029 with a welldefined wild-type strain of V . alginolyticus has been lacking. It is well established that the other strains of the Vibrionaceae (23, 31) can be induced (by collagen and fragments of collagen) to produce collagenolytic and gelatinolytic enzymes.We undertook a comparative study to determine the differences between strain 1.029 isolated from a random selection of cured hides in South Africa (34) and a wild-type strain (strain CIP 82.01) of V . alginolyticus from a different ge...

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Section: Discussionmentioning

confidence: 99%

mentioning

confidence: 99%

Assignment of "Achromobacter iophagus" Strain I.029 to Vibrio alginolyticus Chemovar iophagus

Emod

Soubigou

Tong

et al. 1983

International Journal of Systematic Bacteriology

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“…As part of a comparative study on collagenases from procaryotes and lower eucaryotes [17], we have re-examined in the present paper the chemical and enzymatic properties of the Hypoderma collagenase. A new method of preparation allowed us to obtain the enzyme in homogeneous state and in high yields.…”

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Chemical and Enzymatic Characterization of the Collagenase from the Insect Hypodearma lineatum

Boulard²,

1979

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The collagenase from the larvae Hypoderma lineaturn, with a molecular weight of 24000 and isoelectric point of 4.1, was obtained in homogeneous form by ion-exchange chromatography. It is stoichiometrically inhibited by diisopropylfluorophosphate. On the other hand it is unaffected by ethylenediaminetetraacetate, p-chloromercuribenzoate, dithiothreitol, N-tosyllysine chloromethyl ketone, N-tosylphenylalanine chloromethyl ketone and ovomucoid trypsin inhibitor. The enzyme which degrades native collagen in its helical parts, has a specific activity on thermally reconstituted collagen fibrils of 150 pg collagen degraded x min-' x (mg enzyme)-' at 37 "C. It hydrolyses casein but has no esterolytic activity characteristic of trypsin, chymotrypsin nor elastase. It has no action on the synthetic peptide 4-phenylazobenzyloxycarbonyl-~-prolyl-~-leucyl-glycyl-~-prolyl-~-arginine. The amino acid composition of Hypoderma collagenase indicates a distinct similarity with the serine proteinases of the trypsin family and with another athropode serine collagenase, that of the fiddler crab Uca pugilator. This suggests that eucaryotic collagenases with digestive rather than morphogenic function represent a new category of members of the trypsin family.Since the occurence of a collagenase from a eucaryote was first described in tadpole skin culture media [l], a number of collagenases from various animal and human tissues have been isolated and characterized [2-51. Little is known, however, about the collagenases from eucaryotes which do not play a role in the physiological remodelling of connective tissues. Such collagenases functioning as digestive enzymes have been demonstrated in the hepatopancreas from the fiddler crab, Ucapugilator [6,7], and in the pyloric caeca from a fish, Seriola quinquecadiata [8]. Many years ago, collagenolytic enzymes were also reported in parasites which enter through the skin of their host [9 -131. The demonstration of a collagenolytic enzyme in the midgut of the first instar migrating larvae from and migrate during eight months while digesting its connective tissues, helped by the enzymic secretion of their salivary glands. As they migrate, the larvae partially reabsorb their salivary enzymes together with the degradation products of the connective tissue of their host and stock them in their midgut which is closed in its hind end and can thus act as a reservoir In a previous biochemical study, the collagenase from H. lineatum was purified and partially characterized [16]. Its mode of attack on collagen was shown to be similar to that of vertebrate tissue collagenases in that it cleaves native collagen, at neutral pH and under non-denaturing conditions, at a site located three-quarters of the length of the molecule from the N terminus. H. lineatum collagenase differs however from vertebrate collagenases in regard to its behaviour to various inhibitors, particularily EDTA. Although this agent completely blocks the action of most tissues collagenases, which are metalloenzymes, it was without effect on t...

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“…Recent studies using purified bacterial collagenases have shown that the initial cleavage of native collagen occurs in the central helical region of the a-chains [17,18] and not progressively from the amino-terminal end. These enzymes are, therefore, endopeptidases and as such might be expected to be inhibited by serum components.…”

Section: Discussionmentioning

confidence: 99%

“…These enzymes are, therefore, endopeptidases and as such might be expected to be inhibited by serum components. The major cleavage sites of the clostridial enzymes, which are the source of the collagenolytic activity in both enzyme preparations we have used, would produce fragments with molecular weights in the range of 60,000, 55,000, 45,000, and 35,000 [17]. Because of the anomalous behavior of collagenous proteins on SDS-polyacrylamide, determination of accurate molecular weights for the fragments we obtained was not possible.…”

Section: Discussionmentioning

confidence: 99%

Uptake of collagenolytic enzymes by bone cells during isolation from embryonic rat calvaria

Sodek

Heersche

1981

Calcif Tissue Int

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Summary. Collagenolytic activity extracted from bone cells, isolated from calvaria with a proteolytic enzyme mixture, was analyzed using 14C-labeled collagen and a combination of gel electrophoresis and fluorography. The pattern of collagen degradation products obtained did not include 3/4-fragments, typical of vertebrate collagenases, but was essentially identical to the patterns generated by very dilute samples of the proteolytic enzyme mixture used to isolate the bone cells, and purified collagenase from Clostridium histolyticum (E.C. 3.4.24.3.). These and other results strongly suggest that the previously reported collagenolytic activity of bone cells [1] is exogenous in origin.Key words: Bacterial collagenase --Vertebrate collagenase --Sodium dodecyl sulfate polyacrylamide gel electrophoresis --Fluorography.Mixtures of proteolytic enzymes containing bacterial collagenases are routinely used to release bone cells from calvaria [1][2][3][4]. These bone cells can be further subdivided into distinct populations using sequential enzymic digestion [5,6]. Within these populations cells have been characterized as osteoblast-like and osteoclast-like, based on the response of the particular population to parathyroid hormone (PTH) and calcitonin (CT) [5][6][7]. It has been reported [1] that a collagenolytic enzyme activity can be extracted from freshly isolated bone cells from rat calvaria, and that the release of activity was increased when the calvaria were exposed to PTH prior to bone cell isolation [8]. Our initial interest was to determine if this collagenolytic activity could be ascribed to any particular bone cell population.Send offprint requests to Dr. J. Sodek at the above address.However, from preliminary results the origin of the collagenolytic activity became questionable. We report here results of experiments that we feel demonstrate that collagenases are taken up by bone cells during the isolation procedure. Materials and Methods Isolation of Bone CellsBone cells were isolated from 21-day fetal rat calvaria using the method of Rao et al. [6], which is a modification of the original method of Wong and Cohn [5]. Enzyme mixture (1 ml ofa 3 mg/ml solution) containing bacterial collagenase [6] was added to minced calvaria (7 calvafia per ml enzyme) in magnetically stirred spinner flasks and incubation carried out at 37~ Released cells were collected after 10 min (population 1), 20 min (population 2), 30 rain (population 3), 50 min (population 4), and 70 min (population 5), fresh enzyme being added after each collection. Immediately after harvesting, an equal volume (1.0 ml) of fetal bovine serum (FBS) at 0~ was added to stop the enzyme activity. The cells were then pelleted by centrifugation at 1000 g (10 min), resuspended in 10 ml c~-minimal essential medium (c~-MEM) containing 15% FBS, and recentrifuged. This procedure was repeated five times. Half the number of cells in each population were plated in 16 mm culture dishes (approximately 1 x 10" cells per dish) and cultured in c~-MEM containing 15% heat-in...

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Some newly characterized collagenases from procaryotes and lower eucaryotes

Cited by 38 publications

References 54 publications

Assignment of "Achromobacter iophagus" Strain I.029 to Vibrio alginolyticus Chemovar iophagus

Assignment of "Achromobacter iophagus" Strain I.029 to Vibrio alginolyticus Chemovar iophagus

Chemical and Enzymatic Characterization of the Collagenase from the Insect Hypodearma lineatum

Uptake of collagenolytic enzymes by bone cells during isolation from embryonic rat calvaria

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