THE RECEPTOR‐DESTROYING ENZYME OF V. CHOLERAE

Abstract

In previous papers we have described (Burnet, McCrea and Stone, 1946) the activity of filtrates from Y. cholerae in removing "virus receptors" from red cells. Cells so treated also develop the condition of panagglutinability described by Thomson (1926) and Friedenrich (1928) and evidence has been given that both effects are due to the action of an enzyme which for convenience we refer to as receptor-destroying enzyme, RDE. In the second paper (Stone, 1947) of the series, it was shown that except for minor details the action of viruses of tlie influenza group on the red-cell surface was essentially the same as that of RDE.The present paper reports further work which has been directed mainly, toward determining the optimal conditions for the production of enzyme by the vibrio, and for the preparation of purified material by elution from red cells. EXPERIMENTAL. Tlie Production of BDE.In the work reported previously, EDE was prepared by the growth of a suitable V. cholerae strain in nutrient broth for 16-20 hours. Filtrates from such cultures gave titres of the order 3.60-240. Growth in simple peptone water, though producing similar degrees of turbidity, gave only very small yields (<30).Much higher titre filtrates could be obtained by the method used by one o-f us in 1930 for the production of staphylococcal toxin (Burnet, 1930). Plates of weak (1 p.c.) nutrient agar wero inoculated with a few drops of broth culture of the desired strain and incubated for 16 hours at 37° 0. The surface growth was scraped ofE with a glass rod and the agar removed into a gauze bag inside the top of a Buchner funnel. Usually a batch of 20 Petri dishes waa used. The gau^e bag waa tied and the rfluid pressed out by a weighted cylindrical jar fitting closely into the top of the Buchner funnel. The expressed fiuid was centrifuged if necessary and paBsed through a Seitz filter. These "agar filtrates" had a pH between 7-5 and 8-5. Their RDE titres with suitable strains ranged from 320 to 2,000, and the method has been used as a routine both for the production of EDE and also for the desquamating enzyme described elsewhere.A second effective method is growth of a suitable strain in the allantoic cavity of 12-day chick embryos. Death occurs with a small inoculum in approxima.tely 24 hours and fluid harvested at this time and centrifuged gave titres of almost exactly the same range, 400-2,000, as those obtained with agar filtrates.

Regeneration of Virus Receptors in Mouse Lungs After Artificial Destruction

“…For further details of the properties and preparation of EDE see Stone (1947b) and Burnet and Stone (1947).…”

“…The effect of standard preparations {Stone, 1947a;Burnet and Stone, 1947) is identical on red cells (McCrea, 1947; Stone, 1947b}, the respiratory lining of excised lungs (Pazekas de St.Groth, 1948) and glycoproteins of various origin which act as Francis inhibitors (Anderson aud McCrea, 1948); the preparations render all these biological and chemical materials incapable of adsorbing viruses of the influenza group or release these viruses artificially from the adsorbed state. The "receptors" of virus adsorption are destroyed-at least in the biological senseby their action.…”

DESTRUCTION OF INFLUENZA VIRUS RECEPTORS IN THE MOUSE LUNG BY AN ENZYME FROM V. CHOLERAE

“…Readings were made by estimating "-|-" agglutination from the pattern of settled cells. (For full details sec Burnet and Stone, 1947).…”

“…(For further details of BDE preparation see Burnet, McCrea and Stone, 1946;Burnet and Stone, 1947. ) MisccUavcovs Preparations.…”

Studies on Host-Virus Interactions in the Chick Embryo-Influenza Virus System

“…One batch of RDE was kindly supplied by Dr. John Spizizen of Sharp & Dohme, Inc. The preparations were standardized according to the technic of Buruet and Stone (23). They contained between 640 and 2560 units per ml.…”