Sialidase ( Neuraminidase ) of Corynebacterium Diphtheriae Leonard Warren

WARREN, LEONARD (National Institute of Arthritis and Metabolic Diseases, Bethesda, Md.) AND C. W. SPEARING. Sialidase (neuraminidase) of Corynebacterium diphtheriae. J. Bacteriol. 86:950-955. 1963.-The characteristics of a sialidase produced by Corynebacterium diphtheriae were studied. The enzyme was partially purified from preparations of diphtheria toxin on a column of Sephadex G-75. By this means the lethal factor of diphtheria toxin was separated, in part, from the sialidase activity. There appeared to be a close immunological relationship between the sialidases of C. diphtheriae and clostridia, since a preparation of diphtheria antitoxin was as effective an inhibitor of diphtheria sialidase as of the sialidase of three species of clostridia. Conversely, antitoxin to clostridia inhibited diphtheria sialidase. Diphtheria antitoxin was essentially inactive toward influenza virus sialidase, and was completely inactive against purified sialidase of Vibrio cholerae. Removal of sialic acid from the proteins in a preparation of diphtheria antitoxin did not alter the inhibitory activity of the antitoxin against diphtheria sialidase. The enzyme operated optimally at pH 5.5 and did not require calcium ions for activity. The substrate specificity of diphtheria sialidase appears to be the same as that of other previously described sialidases. The glycosidase called neuraminidase or sialidase has been found in a variety of bacteria and myxovirus preparations (Gottschalk, 1960; Rafelson, 1963). In addition, this enzyme has been observed in preparations of bovine and human plasma proteins (Warren and Spearing, 1960), in the developing chick egg (Ada and Lind, 1961), and in several tissues of the rat (Carubelli, Trucco, and Caputto, 1962). In this paper, the characteristics of a sialidase formed by Corynebacterium diphtheriae, and partially purified from the toxin, will be described. The presence of a sialidase in diphtheria toxin was suspected when it was observed that the changes in electrophoretic mobility of human transferrin produced by the enzyme sialidase (Parker and Bearn, 1961; Blumberg and Warren, 1961) were in many respects the same as changes in an earlier study on the effects of diphtheria toxin on proteins (Poulik, 1956). Preliminary enzymatic tests by the present authors showed that diphtheria toxin did, in fact, contain a sialidase (Blumberg and Warren, 1961; Poulik, 1961). While this work was in progress, Heide and Haupt (1962) and Jamieson and Poulik (1962) briefly reported their work on diphtheria sialidase. MATERIALS AND METHODS Human plasma proteins, used for routine assays, were purchased from Pentex, Inc., Kankakee, Ill. Crystalline human transferrin was the gift of A. L. Schade. Orosomucoid was the gift of E. A. Popenoe. Several preparations of diphtheria toxin were employed: oneand five-times recrystallized diphtheria toxin obtained from C. G. Pope, Wellcome Research Laboratories; Eli Lilly Toxin (lot no. 778765) from F. E. Kamplain; and Parke, Davis & Company Toxin from G. D. Brigham. A sample of toxin was also provided by M. D. Poulik. Eli Lilly commercial diphtheria antitoxin (equine) was used (2000 units per ml). The following preparations were obtained from D. R. Seligman of the Division of Biologics Standards, National Institutes of Health: Standard Schick diphtheria toxin, and toxin and antitoxins of Clostridium perfringens, C. septicum, and C. sordelli. These materials had been stored in a lyophilized state for general medical distribution. Diphtheria toxoid was obtained from the National Drug Co., Philadelphia, Pa. Cholera sialidase was purified approximately 700-fold by the method of Ada and French (1959). Crystalline N-acetylneuraminic acid, isolated from human plasma proteins (Svennerholm, 1956), was used as a standard.