VOL. 12 (1953) AMINO ACID INTERACTIONS IN STRICT ANAEROBES IO9 



Amino Acid , j)pj^jj ^ Reduced -pp^ 



Hydrogen Acceptor ^ Amino Acid 



As, however, a-ketonic acids do not accumulate in the anaerobic amino acid inter- 

 actions, it follows that these, too, must undergo oxidation, presumably through the DPN 

 system, the following reaction taking place: 



a-Ketonic acid + DPN + HgO = Lower fatty acid + COg + DPNHg 



Pyruvate, an intermediate in alanine-proline interaction in CI. sporogenes 



So far there has been no satisfactory evidence that pyruvate is an intermediate in 

 the alanine-proline interaction, though the indirect evidence is in favour of this con- 

 clusion. Proof that pyruvate is an intermediate in this interaction is given in the typical 

 results quoted in Table V. 



TABLE V 



LACTIC ACID FORMATION FROM ALANINE-PROLINE IN PRESENCE OF AN EXTRACT OF CI. SpOrOgenCS 



AND LACTIC DEHYDROGENASE OF BRAIN 



Warburg manometer vessels contained i ml cell free extract of CI. sporogenes (in i % sodium 

 thioglycollate and 0.002 71/ phosphate) and, where indicated, 0.5 ml of rat brain homogenate (i g 

 brain homogenized in 3 ml 0.002 M phosphate -f i % nicotinamide solution) as source of lactic de- 

 hydrogenase. DPN present = 3 mg per vessel. 0.028 M NaHCOg present. Gas = 93 % Ng -|- 7 % COj. 

 Temp. 37". Time 70 min. Total vol 3.2 ml. Amino acid 0.02 M L-form. Alanine tipped in. 



Contents of vessel fj,M COi fj,M Lactate formed 



Bacterial extract 



Bacterial Extract -|- Brain homogenate 



Alanine + Proline -|- Bacterial extract 



Alanine 4- Proline -1- Bacterial extract -1- Brain homogenate 



Alanine -|- Proline -|- Brain homogenate 



The addition of a preparation of lactic dehydrogenase, in the form of a brain 

 homogenate, to a cell free extract of CI. sporogenes, containing alanine, proline and 

 DPN, gives rise to high rate of lactate formation. This could only occur if the pyruvate 

 formed normally as an intermediate is diverted by DPN and lactic dehydrogenase into 

 lactate. It is noteworthy that in spite of the diversion of pyruvate into lactate by the 

 added lactic dehydrogenase, the rate of evolution of carbon dioxide in the amino acid 

 interaction is not diminished (Table V). Since the carbon dioxide is presumably wholly 

 derived from the oxidation of pyruvate, it follows that pyruvate must be formed in 

 sufficient amount from alanine to saturate the pyruvic oxidase-DPN system even in 

 presence of lactic dehydrogenase. 



Effect of DPN on pyruvic acid hreakdonni in presence of CI. sporogenes 



Sodium pyruvate undergoes anaerobic breakdown in presence of an extract of CI. 

 sporogenes giving rise to carbon dioxide but with little or no lactate formation. The 

 speed of this process is markedly accelerated by the addition of DPN, so that there is 

 little doubt that the initial anaerobic oxidation of pyruvate is DPN-linked (see Table VI) . 

 If a source of lactic dehydrogenase, in the form of a brain homogenate, is added to the 

 pyruvate-DPN-bacterial extract system, lactate formation takes place (Table VI, 

 Expt. 2). This result is to be expected if the reduced DPN formed by the oxidation of 



References p. 120. 



