IV. BIOCHEMICAL SYSTEMS 581 



of normal rat liver homogenate or within 21 lioiu's uft<M- inicdion of 200 

 7 of biotin into deficient animals.^' 



The synthesis of citruUinc from ornithine, glutamate, carbon dioxide, 

 and ammonia by washed residue of rat liver homogenate'*- '^ is greatly 

 reduced when the liver enzyme preparation is obtained from biot in-de- 

 ficient rats.'*' Inasmuch as it is carbamyl-L-glutamate rather than glutamate 

 which acts as actual catalytic intermediate in citrulline synthesis from orni- 

 thine,"' '* it would be interesting to learn whether the effect of biotin on 

 citrulline synthesis may be placed prior to or subsequent to the reactions 

 in which carbamyl-L-glutamate participates. It has been shown by means 

 of enzymatic preparations obtained from normal and biotin-deficient rats 

 that replacement of glutamate by carbamyl-L-glutamate has compensated 

 for the lack of the specific "biotin-enzyme preparation" and resulted in 

 equal rates of citrulline synthesis by the biotin-deficient and control prep- 

 arations.*^ Futhermore, inhibition of citrulline formation by the biotin- 

 deficient liver enzyme preparation in the presence of glutamate was affected 

 by fumarate, oxalacetate, and aspartate. These observations*^ are in good 

 accord with the assumption that "the influence of biotin on CO2 fixation 

 into citrulline is at a step prior to that at which carbamyl-L-glutamate 

 functions in the conversion of ornithine to citrulline.*^ 



The incorporation of C^^Oo from bicarbonate into the carboxyl group of 

 acetoacetate formed from crotonate, butyrate, capronate, caprylate, iso- 

 valerate, heptylate, nonylate, or p3a-uvate by rat liver homogenates^" is 

 markedly decreased when the enzyme is prepared from biotin-deficient 

 animals.^^ However, the quantity of acetoacetate as such is not affected 

 by the state of biotin nutrition of the animals. The incorporation of C^'^O-j 

 into the carboxyl group of acetoacetate metabolically formed from p.yru- 

 vate, caproate, caprylate, or isovalerate was increased ten- to one hundred- 

 fold when biotin-deficient rats received biotin either in their diet or by 

 injection.^- These observations offer further proof for the important role 

 of biotin in the intermediate carbon dioxide fixation mechanism. 



In a cell-free enzyme system prepared from Micrococcus lysodeikticus the 

 addition of avidin will prevent the fixation of carbon dioxide in oxalacetate. 

 The addition of biotin in excess results in a return of normal fixation.^* 



" P. P. Cohen and M. Hayano, J. Biol. Chem. 170, 687 (1947). 



3« P. P. Cohen and M. Hayano, ./. Biol. Chem. 172, 405 (1948). 



" P. P. Cohen and S. Grisolia, J. Biol. Chrm. 182, 747 (1950). 



38 S. Grisolia and P. P. Cohen, J. Biol. Chem. 191, 189 (1951). 



" G. Feldott and H. A. Lardy, J. Biol. Chem. 192, 447 (1951). 



^» G. W. E. Plaut and II. .\. Lardy, ./. Biol. Chem. 192, 435 (1951). 



« G. W. E. Plaut and II. .\. Lardy, ./. Biol. Chem. 186. 705 (19.50). 



« G. W. E. Plaut, Proc. Soc. Exptl .Biol. Med. 78, 769 (1951). 



" G. E. Wessnian and C. H. Werkman, Arch. Biochcw. 26, 214 (1950). 



