22 P-AMINOBENZOIC ACID 



by MacLeod^^ in a survey of a wide variety of substances of animal and 

 bacterial origin. "Sulfonamide inhibitor" is found in many tissue extracts. 

 The amount found is greatly increased by prior autolysis or acid hydrolysis. 

 In certain species of bacteria the inhibitor is found in the cells only and is 

 not demonstrable in the culture medium, whereas in other species the in- 

 hibitor is found in the culture supei'natant, and the cells themselves are 

 relatively free. Sulfonamide resistance (fastness) in a strain of pneumococ- 

 cus is accompanied by greatly increased production of "sulfonamide in- 

 hibitor." 



MacLeod was c^uite aware of the limitations of a microbiological assay 

 that depends upon the reversal of sulfonamides for the determination of 

 PABA (see section on sulfonamides). He found that the "sulfonamide in- 

 hibitor" present in some natural materials is not extractable b}^ ether at 

 an acid pH as would be expected if it were PABA. 



The first essentially specific microbiological method for the determina- 

 tion of PABA was proposed by Landy and Dicken.^^ Their method was 

 based on the primary observations of Underkofler et al.^^ and Lampen et al}- 

 that Acetobader suboxydans recjuires PABA as a growth factor. Their origi- 

 nal basal medium contained a purified acid hydrolyzate of casein, plus 

 tryptophan and cystine as nitrogen sources, glycerol as an energy source, 

 inorganic salts, and the only other growth factors required by the or- 

 ganism, namely, pantothenic and nicotinic acids. With this basal medium 

 the organism responds linearly to PABA over a concentration range of 

 to 0.03 7 of PABA per 10 ml. of medium. A variety of compounds with some 

 relationship to PABA in structure were examined for acti^'ity, but no com- 

 pound examined had more than 2 % of the activity of PABA. Water extracts 

 or solutions of natural materials were examined for apparent PABA con- 

 tent. Yeast and yeast extract were by far the best sources of PABA studied. 



Landy and Streightoff^^ subsequently have shown that the incorporation 

 of purines (adenine alone in relatively large amounts or a mixture of ade- 

 nine, guanine, and xanthine) in the original basal medium greatly increases 

 the sensitivity of response of Acetobader suboxydans to PABA. In the ab- 

 sence of purines (original medium) a response to PABA could be detected 

 at a level of 0.01 y per 10 ml. of culture. The inclusion of purines permits 

 a detectable response to PABA at a level of 0.001 y per 10 ml. Cheldelin 

 and Bennett^* have modified the Landy-Streightoff medium by the further 

 inclusion of glucose, Norit-treated pe})tono, Norit-treated liver, and addi- 



5«M. Landy and D. M. Dioken, ,/. Biol. Choi. 146, 109 (1942). 



31 L. A. Underkofler, A. C. Bantz, and W. H. Peterson, J. Bacterial. 45, 183 (1943). 



" J. O. .Lampen, L. A. Underkofler, and W. H. Peterson, /. Biol. Chcm. 146, 277 



(1942). 

 33 M. Landy and F. Strcightofi", Proc. Sac. Expti. Bial. Med. 52, 127 (1943). 

 =" V. H. Cheldelin and M. J. Bennett, /. Biol. Chem. 161, 751 (1945). 



