148 PTEROYLGLUTAMIC ACID 



xanthopterin, salts, cyanide, degree of dispersion of the tissue, pH of the 

 tissue, and the addition of taka-diastase. This observation was confirmed 

 by Totter et al.,^^ and it was found that the addition of xanthopterin in- 

 creased the content of microbiologically active PGA during incubation of 

 Uver from vitamin M-deficient monkeys but not of hver tissue from normal 

 chickens. This species difference in response of liver tissue to xanthopterin 

 is further emphasized by the fact that addition of this pterin to hog liver 

 produces tenfold increases of PGA activity''' in contrast with the earlier 

 reports of two- to threefold increases with rat liver. 



As an explanation of this phenomenon, it was suggested that liver con- 

 tains a precursor of pteroylheptaglutamic acid which is susceptible to attack 

 competitively by two types of enzymes.'*^ One reaction at pH 7.0 leads to 

 the formation of a compound, presumably pteroylheptaglutamic acid, 

 which is susceptible to further enzymic conversion at pH 4.7 to give PGA. 

 A second reaction at pH 7.0 leads to inactivation of this precursor and may 

 be inhibited by xanthopterin. This hypothesis rests on the following facts: 



1. Incubation of pig liver with xanthopterin at pH 7.0 increases the PGA 

 from an original of 0.25 to a final value of 3.0 7 per gram. 



2. Incubation at pH 4.7 gives 0.65 7 with or without xanthopterin. 



3. Incubation for 4 hours at pH 7.0 followed by 14 hours at pH 4.7 gives 

 18.5 7 with xanthopterin. 



4. At pH 7.0 pteroylheptaglutamic acid is only partiall}^ converted to 

 PGA; at pH 4.7 the conversion is nearly complete. 



If this explanation of the effect of xanthopterin is correct, then the true 

 amount of PGA derivatives in liver is much higher than that observed with 

 any combination of autolysis and digestion on unheated liver tissue. 



If one accepts this explanation of the effect of xanthopterin, one might 

 account for the transient and partial responses observed with xanthopterin 

 in rats. It may be that in PGA-deficient animals there are small amounts of 

 PGA conjugates in the liver whose major pathway of metabolism would 

 normally cross that of inactivation. Xanthopterin, by inhibiting this in- 

 activation, increases the utilizable quantity of the vitamin, and as soon 

 as the PGA precursor is completely exhausted, no further response can be 

 elicited by xanthopterin. 



The possibility that xanthopterin may be converted to PGA has been 

 entertained, but recent work with xanthopterin labeled with C'^ in the (> 

 and 7 positions makes such a view unlikely.^" Administration of this pterin 

 to a PGA-deficient pig did give a small hematological response, but no 

 radioactivity could be detected in PGA isolated from the liver by use of 

 the carrier vitamin. In addition, incubation of the liver in vitro with isotopic 



BO J. R. Totter, V. Mims, and P. L. Day, Science 100, 223 (1944). 



" A. D. WeloJi, i:. M. NelsiMi, and M. F. Wilson, Federation Proc. 8, 346 (1949). 



