Istvan Emod

Chemical characterization of the homogeneous collagenase from Clostridium histolyticum

Pure collagenase (clostridiopeptidase A, EC 3.4.24.3) having a molecular weight of 70 000 was obtained from the culture medium of Clostridium histolyticym by a combination of ultrafiltrations, molecular sieve, affinity and hydrophobic chromatography. The value of its specific activity is the highest of those described previously but 6-times lower than that of the collagenase from Achromobacter iophagus (EC 3.4.24.8). Its amino acid composition differs from previous data, namely by the presence of cysteine, methionine, tryptophan and O-phosphoserine residues. In contrast to Achromobacter collagenase it does not dissociate in subunits during the deactivation by EDTA or LiCl/glycine buffer at pH 10.5. Existence of multiple forms of Clostridium collagenase previously described is discussed as being due to autolysis of a single molecular species or to a different degree of phosphorylation.

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

Strain CIP 82.01 of Vibrio alginolyticus and strain I.029 (previously designated “Achromobacter iophagus”) were compared. Strain I.029 produces a collagenase of high specific activity (Achromobacter 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 I.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 I.029 enzyme, although some of the aspartic and threonine residues in strain CIP 82.01 are replaced by glutamic and serine residues in strain I.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 I.029 differ qualitatively in resistance to ampicillin and carbenicillin, in cellobiose fermentation, in ornithine decarboxylase activity, and in halophilism. We propose that strain I.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).

Cell-surface collagen-binding protein in the procaryote Achromobacter iophagus

Collagen and its high-molecular-weight fragments specifically induce an extracellular collagenase (EC 3.4.24.8) in the Gram-negative Achromobacter iophagus. During the induction process the inducer is concentrated on the bacterial outer membrane. Two-dimensional electrophoresis of 125I-labelled outer membrane proteins has shown that, in particular, the amount of one protein which is already present on the surface of non-induced bacteria increases quantitatively when the inducer is added. After 125I-labelling of the cell membrane and its solubilization, the same protein is retained selectively on a gelatin-Sepharose column. It has isoelectric point of 4.9-5.1 and molecular weight of 40000. This molecular weight is close to that of the 35000 of the collagenase subunit. However, their non-identity was proved in three independent ways: upon two-dimensional electrophoresis, only those proteins in the range corresponding to the collagenase dimer (Mr 70000-80000) react with fluorescent anticollagenase antibody system, whereas the spot of the collagen-binding protein (mr 40000) is negative; the solubilized collagen-binding protein is not retained by anticollagenase-Sepharose affinity chromatography; in vivo, it is not protected by anti-collagenase antibodies against lactoperoxidase iodination. A hypothesis for the possible role of the collagen-binding protein in the induction of collagenase is proposed.

SEPARATION OF PROTEINASES AND IN PARTICULAR OF CLOSTRIPAIN AND COLLAGENASE BY AFFINITY CHROMATOGRAPHY

ABSTRACT Selective separations of proteinases can be achieved by affinity chromatography using different Sepharose-bound amino acids. Trypsin, chymotrypsin, papain, clostripain, pepsin, collagenases of CI. histolyticum and A. iophagus , carboxypeptidase A and B were assayed on columns of Sepharose-bound arginine, histidine and phenylalanine. Highly active preparations of clostripain and colla-genase were isolated from crude collagenase of C1. histolyticum using five different Sepharose-bound ligands.