126 THE BIOCHEMISTRY OF B VITAMINS 



protein can inactivate an active undialyzed preparation (presumably by 

 competing with the active enzyme for the coenzyme) ; but a combination 

 of the dialyzed protein and the dialyzable coenzyme does not exhibit the 

 inhibitory effect upon an undialyzed preparation. Furthermore, no inacti- 

 vation of the protein component occurs if the enzyme is dialyzed against 

 a solution containing the thermostable coenzyme. 



In view of these findings a study using inositol inhibitors was carried 

 out to demonstrate a possible relationship of the coenzyme to inositol. 

 The y-isomer of 1,2,3,4,5,6-hexachlorohexane, a compound whose inhibi- 

 tory action on the growth of yeast and molds can be prevented by meso- 

 inositol, was found to inactivate purified a-amylase preparations. 3 The 

 presence of inositol, however, counteracted this inhibition. To demonstrate 

 the inactivation of the enzyme, it was necessary that the inhibitor be 

 incubated with the enzyme for at least fifteen hours. The amount of 

 inactivation produced by the inhibitor depended on the ratio of inhibitor 

 to inositol. When the molar ratio of hexachlorohexane to inositol was 10, 

 only a 10 per cent inactivation occurred. Increasing this ratio to 50 com- 

 pletely inactivated the enzyme. Thus, the authors concluded that inositol 

 is an active constituent of a-amylase. 



Although the results obtained with the inositol inhibitor are highly 

 indicative, they do not conclusively answer a pertinent question: Is "free" 

 inositol the coenzyme of a-amylase? The only logical explanation for the 

 inhibition by hexachlorohexane and its reversal by inositol in the highly 

 purified system used is that inositol itself is a dissociable component of 

 the system, or that it can effectively replace some dissociable component. 

 It would be interesting to know whether inositol by itself would behave 

 in a manner identical to the thermostable dialyzable component obtained 

 by dialysis of the enzyme. If the coenzyme is simply inositol, a-amylase 

 could be dialyzed against a solution of inositol with no loss in activity; 

 also, the inactivation of undialyzed preparations by dialyzed protein 

 fractions could be prevented by the addition of inositol along with the 

 dialyzed protein. 



In considering possible mechanisms by which inositol, which contains 

 no salt-forming groups, could combine with the apoenzyme, the relation- 

 ship of the chemical structure of inositol to that of the glucosidic units in 

 the amylase substrate should not be overlooked. 



A crystalline preparation from hog pancreas was used for the dial- 

 ysis experiments which demonstrated the existence of a coenzyme for 

 a-amylase. Since that time, a-amylases have been isolated in a crystalline 

 state from human pancreas, 4 human saliva, 5 and Bacillus subtilis. G The 

 hog pancreatic amylase is not the same protein as the enzyme isolated 

 from human pancreas, but apparently the human salivary amylase is 



