242 E. S. GUZMAN BARRON, F. GHIRETTI VOL. 12 (1953) 



TABLE II 



OXIDATION OF INTERMEDIATES OF THE CITRIC ACID CYCLE 



BY Pseudonionas fluorescens 

 Buffer, o.o5j1/ phosphate, pH. 5.5; substrate, o.oiM. Figures give jul per mg dry weight per hour. 



oxidizes acetate readily and, according to Seaman^^- accumulates succinate in the pres- 

 ence of malonate. (The succinate figures given by him were too small to be considered 

 reliable). Tetrahymena geleii obtained from 24-hour cultures oxidized acetate, citrate, 

 and succinate. Although pyruvate did not increase the O.2 uptake, there was definite 

 utilization. Malonate inhibited acetate oxidation, as found by Seaman (Table III). 



Citric acid synthesis and isocitric acid oxidation. Neither kinetic nor isotope experi- 

 ments are reliable tools to determine whether acetate is oxidized by the dicarboxylic 

 acid pathway. Since these two pathways differ only by addition to the latter of a syn- 

 thesis reaction and two more oxidative steps, it was necessary to look for these steps in 

 the cells we were studying by preparing cell-free extracts and measuring citric acid 

 synthesis and isocitric acid oxidation under optimum conditions for these enzymatic 

 reactions. Cell-free extracts of M. tremellosus, M. niveus, C. creatinovorans, Rhyzopus 

 nigricans, and /I. aerogenes contained Ochoa's condensing enzyme for the synthesis of 

 citric acid on addition of acetyl phosphate, oxalacetate, and coenzyme A (Table IV). 

 The cell-free extracts of Tetrahymena geleii contained so much citric acid that search for 

 the enzyme was considered unnecessary. All these cell-free extracts, including those of 

 Tetrahymena, contained isocitric dehydrogenase, as shown by the reduction of TPN on 

 addition of zsocitrate (Table V). OcHOA found the condensing enzyme in cell-free extracts 

 of Pseudomonas fluorescences ^^. 



Additional evidence that oxidation of acetate by the moulds M. niveus, and M. 

 tremellosus occurs via the citric acid cycle was obtained by the determination of the 

 citric acid cycle intermediate-oxidation products. For these experiments the moulds were 

 grown at 30° with continuous shaking for 7 days. The filtered media were reduced in 

 volume under vacuum, and determinations were made in aliquots of these solutions. 

 In all the specimens were found by chemical analysis citric, a-ketoglutaric, malic, gly- 

 colic, and oxalic acids. By paper chromatography succinic and fumaric acids were 

 identified (Table VI). The a-keto acid, which was determined as the coloured complex 

 of the phenylhydrazone derivative, was assigned to a-ketoglutaric acid because the 

 absorption spectrum was identical to that of the phenylhydrazone complex of a sample 

 of a-ketoglutaric acid. 



The effect of malonate on the oxidation of acetate. Malonate inhibition experiments 

 with bacterial cells are sometimes difficult to interpret because of the possible lack of 

 penetration through the cell membrane. In some cases this difficulty is surmounted by 



References p. 24^. 



