VI. KSTIMATION 163 



1. EXTUACTK)N' KliOM TlSSUKS AND 1 1 V DIM IIASIS OF ( '( )XJlI(rATKS 



A nuniluM' of oxpiM'inn'nts Ikia'c .shown that inicrol)iolof5i('ally inactive 

 ('onju<>;al(\s (>\ist in natural protlucts. Vitamin He conjugate (pteroylhoxa- 

 glutamylglutaniif acid) found in yeast is inactive both for *S'. faccalis and 

 L. casei. Pteroyltriglutamic acid isolated from a fermentation material is 

 actix'c for L. casei but only slightly active for S.faecalis. Pteroyl-7-glutamyl- 

 glutamic acid, which has been prepared synthetically but not yet ol)served 

 in natural products, is active for both S. faecalis and L. casei. It is thus 

 apparent that L. casei is capable of responding to larger conjugates than 

 S. faecalis. This offers a possible explanation for the fact that many natural 

 products give a higher result by L. casei assay than by *S'. faecalis assay. 

 Rhizopterin and pteroic ac^id are active for S. faecalis but inactive for L. 

 casei. Thus the presence of a high activity for *S. faccalis compared to L. 

 casei might indicate the presence of rhizopterin or pteroic acid in the sample. 



A number of methods have been proposed for the release of pteroylglu- 

 tamic acid from tissues. Cheldelin e( al.^ investigated the effect of a lumiber 

 of commercially available enzymes on the liberation of folic acid from tis- 

 sues. This hydrolysis presumablj^ consisted in two parts: first, the release 

 of pteroj^glutamic acid from the tissue to give a soluble form; second, the 

 hydrolysis of the soluble but inactive conjugates to yield active compounds. 

 These workers found taka-diastase to be more effective than papain, pepsin, 

 trypsin, pancreatin, malt diastase, or pancreatic amylase. Digestion of hog 

 heart with taka-diastase gave about ten times as much pteroylglutamic 

 acid activity as autolysis alone. However, subsequent work^' ^^ indicated 

 that, although taka-diastase may be effective in releasing the nutrilite from 

 the tissues, it does not hydrolyze all the conjugates present. Mims et al.^ 

 demonstrated the existence of an enzyme capable of activating a microbio- 

 logically inactive conjugate in yeast. Cell-free extracts of plants and tissues 

 which had been previously digested with taka-diastase could be further 

 activated by this enzyme which was obtained from li\'er. This distinguished 

 the action of the conjugate-splitting enzyme from that of taka-diastase. 



a. Conjugases 



Shortly after the observation that certain natural matei'ials possessed 

 more activity by animal assay than could be accounted for by microbio- 



ical Assay of Lactobacillus casei Factor (Vitamin Be , Folic Acid), Volume XII, 



p. 339. Jaques Cattell Press, Lancaster, Pa., 1947. 

 ^ E. E. Snell in Vitamin Methods, Microbiological Methods in Vitjimiii Hosearch, 



p. 327. Academic Press, New York, 1950. 

 8V. H. Cheldelin, M. A. Eppright, E. E. Snell, and B. .M. Guirard, Univ. Texas 



Pnhl. No. 4237, 15 (1942). 

 " V. Mims, J. R. Totter and P. L. Daj-, J. Biol. Chcm. 155, 401 (1944). 

 '« P. R. Burkholder, I. McVeigh and K. Wilson, Arch. Biochem. 7, 287 (1945). 



