400 4. ALLOXAN 



inactivation of coenzyme A could be reversed, but the inhibition directly 

 on the acetylating enzyme could not. The inactivation of rat liver hexo- 

 kinase could not be reversed at all with cysteine and it was concluded 

 that something more than simple oxidation must be involved (Bhatta- 

 charya, 1959). Siliprandi and Daghetta (1950) noted that alloxan inhibits 

 urease more strongly in oxygen than in nitrogen, and thought that this 

 pointed to an oxidation mechanism. 



The inhibition is usually noncompetitive — as with renal phosphatase 

 (Burgen and Lorch, 1947), xanthine oxidase (Bruns, 1954), and urease 

 (Gray et al., 1959) — but in some cases one finds a kinetic competition, 

 in that protection is exerted by substrates or coenzymes — for example, 

 fructose-6-P and ATP protect phosphofructokinase (Engelhardt and Sa- 

 kov, 1943), glucose protects pancreatic hexokinase (Villar-Palasi et al., 

 1957), and NAD protects a yeast apozymase system (Kensler et al., 1942 b). 

 The inhibition of urease by alloxan is perhaps partially competitive, due 

 to the urea moiety, but this is complicated by the fact that alloxanate is 

 a more potent inhibitor than alloxan and may be responsible for the inhi- 

 bition when alloxan is used. 



Another type of effect of alloxan on dehydrogenases must be considered. 

 Dixon and Zerfas (1940) showed that alloxan could act as a hydrogen ac- 

 ceptor in several dehydrogenase systems when used at a low concentra- 

 tion. Unlike methylene blue or quinone, it does not require NAD to be 

 reduced to dialurate. Keleti (1958) determined the equilibrium constant 



A' = (dialurate) (acetaldehyde)/(alloxan) (alcohol) = 1.24 X 10 ^^ 



using the alcohol dehydrogenase system, and felt that alloxan might com- 

 bine with the Zn++ ions on the enzyme in the same manner as NAD. What- 

 ever the mechanism, it is clear that alloxan can function in certain oxida- 

 tion-reduction sequences and thereby possibly stimulate oxygen uptake. 



Succinate dehydrogenase is inhibited more potently by alloxan the lower 

 the pH (Table 4-2) (Klebanoff, 1955). It is not known whether this is 

 a real pH effect on the reaction of alloxan with the enzyme, or merely an 

 expression of the fact that alloxan is more stable at lower pH. The inhi- 

 bition is found to be progressive over 30 min when 20 mM alloxan is used, 

 but this is due to the fall in pH during the first 1-2 min; if this fall is pre- 

 vented, the inhibition does not increase following the rapid initial depression, 

 showing that it is an acid effect. It must be emphasized again that pH 

 control is especially important in work with alloxan. 



We come now to the important question: Have any enzymes been found 

 sufficiently sensitive to alloxan to be seriously considered as involved in 

 the diabetogenic action? Lazarow (1954 b) has estimated that in the rat 

 a diabetogenic dose of 40 mg/kg intravenously would give a maximal al- 

 loxan concentration of 0.5 milf . Other species require higher doses, so that 



