TASTE FUNCTIONS IN FISH 225 



protein, mucin and other ethanol insoluble products were separated from 

 the ethanol soluble fraction. The ethanol soluble and insoluble fractions 

 were then dried by evaporation and distilled water was added to the 

 original volume of the saliva. The aqueous solution of the ethanol soluble 

 fraction was still as active a gustatory stimulant as the natural saliva, while 

 the ethanol insoluble fraction had a very weak activity (Fig. 9). Renewed 

 ethanol treatment yielded a precipitate which shows a weak Molisch's 

 test, and the ninhydrin test was always negative. The aqueous solution of 

 these precipitates produced only a weak response. Similarly, methanol 

 soluble fraction stimulated very strongly, although somewhat weaker 

 than the ethanol soluble fraction. Similar treatment with acetone, how- 

 ever, showed that the powerful stimulating agent in saliva is insoluble in 

 acetone. The active substance seems to be hardly destroyed by these 

 treatments and it is also resistant to heating. Repeated treatments with 

 ethanol followed by acetone demonstrated that the gustatory active sub- 

 stance always occurs in the ethanol soluble and acetone insoluble fraction. 

 While amino acids, primary amines and proteins were not detected in 

 either the gustatory active fraction or the inactive fraction, traces of car- 

 bohydrates were demonstrated in both fractions by Molisch's test. We first 

 guessed that the gustatory active substance in saliva might be a substance 

 chemically related to the phospholipid group from the following facts : 



(1) organic phosphorus was detected in the acetone insoluble fraction, and 



(2) the addition of CdCI.^ yielded a small amount of precipitate from the 

 alcoholic solution of the acetone insoluble fraction but not from the 

 acetone soluble fraction. The treatment with ether following ethanol, 

 however, revealed that the active substance appears in the elher insoluble 

 fraction (Fig. 9). This clearly indicates that the active substance is at 

 least not related to a glycerophospholipid. Since blood group substances 

 in saliva are alcohol-insoluble carbohydrates (Kabat, 1956), it seems un- 

 likely that they are involved in the gustatory excitation. 



The observation that silkworm pupa, earthworm and milk had strong 

 stimulating effects similar to the saliva, indicates that there may be gusta- 

 tory active substances in these sapid materials. In order to investigate the 

 problem, similar fractionation with organic solvents was performed (Fig. 9). 

 The results clearly showed that all active extracts of these sapid substances 

 occur in the ethanol-soluble and acetone-insoluble fractions as was the case 

 with saliva. The question whether the active agent in these sapid substances 

 would actually stimulate the receptors which are stimulated by saliva was 

 then investigated. For that purpose the technique of selective adaptation 

 was introduced. Application of saliva to the receptive field of the palatal 

 organ which was adapted to milk or the pupa fluid produced only a small 

 response, while the saliva response was not markedly changed by previous 

 adaptation to the solutions of salt, quinine and acid (Fig. 11 — 1). A similar 



