32 S. G. ANDERSON 



D. Enzymes with Receptor-Destroying Properties 



In view of the work of Gottschalk and others (see Chapter IV) in char- 

 acterizing the substrate and mode of action of the enzyme (a neuraminidase) 

 of the influenza virus, only a brief account will be given of some implications 

 of these enzymes for hemagglutinin reactions. 



The first enzyme of bacterial origin to be studied was obtained from 

 Clostridium wdchii by McCrea (1947), but he did not investigate this or- 

 ganism further after the receptor-destroying enzyme was recognized as a 

 product of the growth of V. cholerae (Burnet et at., 1946). This has since 

 become a standard reagent for a wide variety of experimental studies with 

 influenza viruses. 



1. The Receptor-Destroying Enzyme (RDE) of Vibrio cholerae 



a. Biological Properties of the Enzyme. The methods of production and 

 purification of this enzyme are still based on those described by Burnet and 

 Stone (1947), but Ada and French (1957) have recently recommended an 

 improved method involving growth in a medium containing neuraminic acid 

 compounds derived from bovine colostrum. In an earlier paper, they de- 

 scribed a useful method for its partial purification (Ada and French, 1950). 



The enzyme is completely destroyed in 30 minutes at 52.5°C. in the 

 absence of calcium. In the presence of M/100 calcium at pH 5.6 to 8.5, the 

 thermal death point is about 60°C. RDE is destroyed by crystalline trypsin 

 at pH 7.2 to 8.5 and it is possible to produce a specific antibody to it (Stone, 

 1949a; Burnet, 1949a). 



RDE adsorbs to red cells, alters receptors to a greater degree than any 

 influenza virus, and elutes. These actions require calcium and occur best at 

 pH 6.8 (Burnet, 1948b, Briody, 1948). 



The enzyme does not agglutinate red cells, but when cells are saturated 

 with RDE in the cold, the enzyme will block the adsorption of influenza virus, 

 presumably independently of its enzymatic action, which is minimal at that 

 temperature (Stone, 1947). 



The role of calcium is interesting. At 37°C. in the presence of calcium, 

 nearly 97 % of RDE in solution adsorbed to red cells and eluted rapidly. 

 Receptor destruction covered the whole range of the receptor gradient. At 

 0°C. in the absence of calcium, equilibrium was reached with only about 55 % 

 of RDE on red cells. This system was then brought to 37°C. and receptor 

 destruction proceeded to a point at which only receptors for NDV had been 

 destroyed; at that point all RDE eluted from the cells (Edney, 1949). The 

 amount of calcium which allows the enzyme to develop full titer is in the range 

 0.2 toM to 0.5 vaM but depends on the anion concentration of the medium 

 (Edney, 1949; Porterfield, 1952). 



