164 PTEROYLGLUTAMIC ACID 



logical test, it was oV)served that the activity for microorganisms could be 

 increased by appropriate enzymatic digestion. The name "vitamin B,. 

 conjugase^' was given by Bird et al}^ to this enzyme because it was capable 

 of splitting vitamin Be conjugate (pteroylheptaglutamic acid). 



Two main types of conjugase have been described, one present in hog 

 liver and having a pH optimum of 4.5,^^- ^- the other in chicken pancreas 

 with a pH optimum of 7 to 8.^" Chicken pancreas conjugase is inactivated 

 at temperatures above 45° and is relatively stable to the action of crystalline 

 trypsin. Autolysis of chicken pancreas tissue increases the amount of ex- 

 tractable enzyme activity a thousandfold. CrystaUine chicken pancreas 

 conjugase has not been obtained, but concentrates 2800 times as active as 

 the crude starting material have been prepared by Mims and Laskowski^^ 

 using adsorption on tricalcium phosphate gel, precipitation with alcohol, 

 and fractional precipitation with sodium sulfate. Calcium ions were found 

 to be a component of the enzyme system, which accounted for the apparent 

 loss of activity which sometimes occurred during dialysis and the increase 

 in activity observed on purification by adsorption on calcium phosphate. 



Hog kidney conjugase has a pH optimum of 4.5. This enzyme does not 

 attack the methyl ester of pteroylheptaglutamate, a fact which led to its 

 classification as a carboxypeptidase. However, neither of the conjugases is 

 identical with carboxypeptidase, since the purified carboxypeptidase has no 

 conjugase activity.^^ 



Studies on the specificity of chicken pancreas conjugase by Dabrowska 

 et al}^ show that 7-peptides of pteroylglutamic acid are attacked much 

 more readily than a-peptides. In the action of the enzyme on pteroyl-7- 

 glutamyl-7-glutamylglutamic acid only one mole of glutamic acid is re- 

 moved. This presumably is the terminal glutamic acid group, since pteroyl- 

 7-glutamylglutamic acid is not attacked by chicken pancreas conjugase. 

 Thus it appears that the final product resulting from the cleavage of pteroyl- 

 heptaglutamic acid by chicken pancreas conjugase is pteroyl-7 -glutamyl- 

 glutamic acid rather than the monoglutamate. 



Hog kidney conjugase is capable of splitting pteroylheptaglutamate all 

 the way down to the monoglutamate. This is shown by the fact that pteroyl- 

 glutamic acid has been isolated from a hog kidney conjugase digest of the 



" O. D. Bird, S. B. Binkley, E. S. Bloom, A. D. Emmett, and J. J. Pfiffner, /. Biol. 

 Chem. 157, 413 (1945). 



12 O. D. Bird, M. Robbiiis, J. M. Vandenbelt, and J. J. Pfiffner, /. Biol. Chem. 163, 

 649 (1946). 



13 M. Laskowski, V. Mims, and P. L. Day, /. Biol. Chan. 157, 731 (1945). 

 " V. Mims and M. Laskowski, /. Biol. Chem. 160, 493 (1945). 



16 M. Laskowski, Ann. Rev. Biochem. XIX, 21 (1950). 



1^ W. Dabrowska, A. Kazenko, and M. Laskowski, Science 110, 95 (1949). 



