Investigation of common and specific components of Cl. septicum and Cl. histolyticum toxins

Interrelations between the common and specific components in the toxins of several strains of Cl. septicum and Cl. histolyticum were investigated. The method of tissue culture, which yields more stable results than biological tests on animals, was used. It has been demonstrated that native toxins of Cl. seticum (7 strains) and Cl. histolyticum (7 strains) cause cytotoxic changes in chick embryo fibroblasts. These changes are similar to each other and identical with changes occurring under the effect of concentrated toxins of the mentioned microorganisms. Cross reactions of neutralization with antitoxic and species-specific sera against Cl. septicum and Cl. histolyticum have shown that the strains of Cl. septicum and Cl. histolyticum synthesize toxins with components possessing common antigenic properties. The strains of Cl. histolyticum synthesize a greater amount of components common with Cl. septicum than the strains of Cl. septicum in which the amount of heterologous antigens varies.     

An extracellular aminopeptidase from Clostridium histolyticum.

An aminopeptidase was isolated from the culture filtrate of Clostridium histolyticum and purified to homogeneity. Absence of endopeptidase activity in the purified preparation was demonstrated. Gel filtration on a calibrated column indicates an apparent molecular weight of 340000 for the native enzyme. Gel electrophoresis of the denatured enzyme in the presence of dodecylsulfate in constant acrylamide concentration and in a concentration gradient, resulted in the appearance of a single component for which a molecular weight of 51000 and 59000 respectively, was calculated. From mobilities of crosslinked and denatured protein species a molecular weight of 56000 was obtained for the monomer. Specificity studies show that the enzyme cleaves all types of N-terminel amino acid residues including proline and hydroxyproline from small peptides and from polypeptides. The peptide bond formed between an N-terminal amino acid residue and proline is not cleaved by the enzyme. The combined action of aminopeptidase-P and clostridal aminopeptidase leads to complete hydrolysis of the proline-rich nonapeptide bradykinin. Low rates of hydrolysis was observed for charged residues, and amides of amino acids. Kinetic studies with five tripeptides of the general structure X-Gly-Gly, where X stands for Leu, Phe, Val, Ala, or Pro, show a decrease in Km with the increasing size of the hydrophobic side chain of X. The highest Kcat values are observed with proline and alanine. In the series Pro-Gly, Pro-Gly-Pro, Pro-Gly-Pro-Pro, the last peptide is the best substrate, indicating an active site complementary to at least four amino acid residues. The enzymatic activity is dependent on the presence of divalent cations, maximal activation being reached with Mn2+ and Co2+. The optimal pH for the Mn2+ and Co2+- activated enzyme is 8.6 and 8.2 respectively. The optimal temperature is 40 degrees C. Inhibition of the aminopeptidase was achieved with Zn2+, Cu2+ and p-mercuribenzoate, but not with diisopropylphosphofluoridate.     

Toxigenicity of Clostridium histolyticum

Nishida, Shoki (Kanazawa University, Kanazawa, Japan), and Masaaki Imaizumi. Toxigenicity of Clostridium histolyticum. J. Bacteriol. 91:477-483. 1966.-From 234 soil samples, 21 strains of Clostridium histolyticum of different levels of alpha-toxigenicity were isolated by a new method specially designed for the isolation of this species. The alpha-toxigenicity of freshly isolated strains and of stock strains was closely associated with the potentiality for sporulation, growth, and smooth-colony formation. The presence of sugars, particularly xylose and arabinose, was inhibitory for growth. A few controversies on the biological properties of this species seem to be due to disregard for the growth-inhibiting effects of these sugars.     

Immunoenzymology of clostridium histolyticum collagenase

Summary Rabbits inoculated with purifiedClostridium histolyticum collagenase (Clostridiopeptidase A (E.C. 3.44.19) formed precipitating and enzyme activity inhibiting antibodies.The inhibiting effect of immune -globulin and of the immune Fab fragment depends on the antibody to enzyme ratio, on the time of preincubation between these two components, and on the presence of Ca²⁺ during preincubation.Ca²⁺ appears to be necessary to the formation of the collagenase-anticollagenase antibody complex and implicitly to the inhibition of collagenase activity by the antibody.Neither the high molecular substrate collagen, nor the small molecular substrate CBC-hexapeptide protect the collagenase activity against the inhibiting effect of the anticollagenase antibody.     

Preparation of crude Clostridium histolyticum collagenase for therapeutic purposes.

The production of a freeze-dried enzymatic preparation from the category of crude collagenases has been described. The method is based on the utilization of a highly proteolytic Clostridium histolyticum strain whose products have more advantageous properties for therapeutic purposes than the products of the strain commonly used as yet.     

Glycine fermentation by Clostridium histolyticum

Clostridium histolyticum grew on glycine, arginine, or threonine as sole substrate. Arginine degradation preceded that of glycine and partially inhibited that of threonine when two amino acids were present. Each amino acid seemed to be individually catabolized, not by a Stickland type of reaction. Glycine fermentation required the presence of complex ingredients. Therefore, an effect of selenite on glycine catabolism could only be demonstrated after scavenging selenium contamination by preculturing Peptostreptococcus glycinophilus in that medium. C. acidiurici was not suited as selenium accumulating organism as C. histolyticum was inhibited by the residual uric acid. Arginine catabolism was unaffected by seleniuum depriviation. The labelling pattern obtained in acetate after incubation of C. histolyticum with [1-¹⁴C]- or [2-¹⁴C]glycine strongly indicated the metabolism of glycine via the glycine reductase pathway.     

Consecutive use of omega-aminoalkylagaroses. Resolution and purification of clostripain and collagenase from Clostridium histolyticum.

Clostripain and collagenase A were resolved and purified from the culture filtrate of $\text{Clostridium histolyticum}$ by consecutive use of two columns from the homologous series of ω-aminoalkylagaroses. Clostripain was extracted by passing the culture filtrate through ω-aminobutylagarose and purified ～9-fold with 70% overall yield. Collagenase A was then extracted by passing the excluded mixture of proteins through ω-amino-heptylagarose and was purified about 7-fold with an overall yield of 90%. This procedure yields two useful proteolytic enzymes with narrow specificity and also illustrates the potential of homologous series of hydrocarbon-coated agaroses for the development of consecutive fractionators. Such fractionators would separate several proteins out of the same crude extract, thus leading to maximal utilization of expensive or scarcely-available tissues.     

Effect of divalent metal ions on collagenase from Clostridium histolyticum.

The collagenase from Clostridium histolyticum (EC 3.4.24.3) degrades type IV collagen with Km 32 nM, indicating a high affinity for this substrate. Ferrous and ferric ions can inhibit Clostridium collagenase. Inhibition by Fe++ was of the mixed, non-competitive type, with Ki 90 microM. The inhibitory effect of Fe++ may be due to Zn++ displacement from the intrinsic functional center of this metalloprotease, since in the presence of excess amounts of Zn++ enzyme activity is retained. This inhibitory effect of Fe++ may be common for all types of collagenases, since this ion can also inhibit type IV collagenase purified from Walker 256 carcinoma, with IC50 80 microM. Cu++ can only partially inhibit Clostridium collagenase, while other divalent metal ions such as Cd++, Co++, Hg++, Mg++, Ni++ or Zn++ are devoid of any inhibitory effect on the enzyme.