D-AMINO ACID OXIDASE 349 



ylation in liver mitocliondria is inhibited 55% by 10 mM benzoate (Wein- 

 bacli, 1961). One can conclude from this limited material that benzoate is 

 certainly a weak inhibitor of cycle oxidations and phosphorylations. Bosund 

 (1959, 1960 a, b) has investigated the effects of benzoate on the metabolism 

 of glucose and pyruvate in Proteus vulgaris in attempting to elucidate 

 the mechanisms for the bacteriostatic activity. There is no interference 

 with glucose metabolism to the acetate level, and acetate was found to 

 accumulate. The respiratory quotient is increased from 1.24 to 1.82 by 

 benzoate during the oxidation of pyruvate and the Oa/pyruvate ratio is 

 decreased. The oxidation of pyruvate in yeast is quite strongly inhibited 

 by benzoate (50% at 0.4 mM), especially at low pH's where penetration is 

 better, but acetate oxidation is less sensitive. It is quite possible that these 

 inhibitions play a role in the suppression of growth, which for yeast requires 

 5 mM benzoate at pH 5.1 and 60 mM at pH 6. It is clear that much more 

 work must be done before the mechanisms of respiratory inhibition are 

 understood. 



Benzoate can also interfere in lipid metabolism, as demonstrated many 

 years ago by Jowett and Quastel (1935 a, b), but the mechanism is still 

 unknown. In liver slices it was claimed that benzoate at around 0.5-2 

 mM inhibits specifically the oxidation of fatty acids, and the oxidation of 

 crotonate 63% at 1 mM. There is progressively less effect on the higher 

 fatty acids, little inhibition of decanoate being observed. It is possible 

 that the benzoate ring simulates the aliphatic chains of butyrate or croton- 

 ate enabling it to compete with these substrates for some enzyme; it would 

 be interesting to know if benzoate can participate in any of these reactions 

 (e. g., if benzoyl-CoA is formed) and deplete the systems of some cofactor. 

 Benzoate is a weak inhibitor of tyrosinase (Ludwig and Nelson, 1939), 

 chymotrypsin (Foster and Niemann, 1955 b), p-aminobenzoate acetylation 

 (Koivusalo and Luukkainen, 1959), and NADPH dehydrogenase (Kasa- 

 maki et al., 1963); it does not effect shikimate dehydrogenase (Balinsky and 

 Davies, 1961 b) or D-glutamate oxidase (Mizushima and Izaki, 1958) at 

 1 mM, or a-ketoisocaproate decarboxylase at 4 mM (Sasaki, 1962). 



Kojic Acid 



The potent inhibition of D-amino acid oxidase by kojic acid is interesting 

 in light of the central nervous system effects observed in dogs, rabbits, and 

 rats, namely, ataxia, excitement, and convulsions (Friedemann, 1934). 

 Kojic acid was first isolated by Saito in 1907 from Aspergillus oryzae and 

 has since been found in many species of Aspergillus. It is a weak antibiotic, 

 inhibiting growth of most bacteria at 2-15 mM, but is particularly active 

 against Leptospira, complete growth inhibition being observed at 0.007 m.N 

 (Morton et al., 1945). Toxic effects are produced in dogs by 150 mg/kg and 

 in mice by 250 mg/kg when injected parenterally; the LD50 for mice is 



