240 



E. S. GUZMAN BARRON, F. GHIRETTI 



VOL. 12 (1953) 



adenine dinucleotide (FAD). This enzyme, we believe, is responsible for the formation 

 of the oxalacetic acid necessary to initiate the citric acid cycle. 



600 



600 



^00 



200 



EXPERIMENTAL 



Methods. Aerobacter aerogenes and Corynebacterium creatinovorans were obtained from the 

 American Type Culture collection. Merulius niveus and M. tremellosus were kindly provided by 

 Dr. William J. Robbins of the New York Botanical Garden. Tetrahymena geleii was kindly provided 

 by Dr. G. M. Kidder. Pseudomonas fluorescens and Aspergillus fumigaius were isolated at the labora- 

 tory. All bacteria were grown in the medium described by Barron et al.^ with acetate as the main 

 source of carbon. The moulds were grown in the medium used by Nord and Vitucci^* with acetate as 

 the main source of carbon. Tetrahymena was grown in Kidder's yeast-peptone medium containing ace- 

 tate and glucose. The bacteria were grown generally at 38 ".Moulds were grown at 30° in flat-bottomed 

 bottles with continuous shaking. Protozoa were grown at 30° in a dark room and in flat-bottomed 

 bottles. The bacteria were washed three times by centrifugation. The moulds were filtered through 

 gauze and washed several times with distilled water. Fleischmann baker's yeast was washed three 

 times, and oxygen was bubbled through the suspension for six hours. It was then centrifuged again 

 and suspended in water. Oxygen uptake and COj formation were measured manometrically with the 

 usual Warburg technique in vessels of different size. Cell-free extracts of micro-organisms were prepared 

 by grinding the cells in a mechanical mortar at 3° for 3 to 4 hours in the presence of alumina, A-303. 

 During the grinding process the paste was kept moist by addition of drops of 0.154^/ KCl. The 

 mortar was washed with the KCl solution, and the suspension was centrifuged in a Sorvall angle 

 centrifuge at 8,000 r.p.m. at 3°. The supernatant fluid was a clear golden yellow or green yellow 

 solution. Yeast cells were broken in the press described by Hughes^^. Citric acid was determined by 

 the method of Ettinger et al.^^] pyruvic and a-ketoglutaric acids, according to Friedemann and 

 Haugen^''; malic acid, according to Hummel^*; glycolic acid, according to Link et al.*; oxalic acid, 

 by titration with permanganate of the precipitated calcium oxalate. For the detection of organic 

 acids by paper chromatography the method of Lugg and Overell^" was employed after several 

 uni- and bi-dimensional methods were tested; unidimensional descending chromatography with 



Whatman No. i filter paper and redistilled mesityl 

 ox.ide-^N formic acid (1:1) as solvent was used. 

 The chromatograms were run for 8 hours at 18°. 

 The papers were dried overnight in a hood and 

 were developed with a bromocresol green and thymol 

 blue solution. The activity of isocitric dehydrogenase 

 in cell-free extracts was determined spectrophotometri- 

 cally by measuring at 340 m/i the rate of reduction of 

 triphosphopyridine nucleotide (TPN). In these experi- 

 ments the quartz cells contained i ml of cell-free 

 extract; 0.7 ml of o.iM phosphate, pH 7.46; o.i ml of 

 0.2M MgClj; 0.5 ml of TPN (200 micrograms); 1.6 ml 

 of HjO. At time zero, o.i ml of o.iM isocitrate was ad- 

 ded. Reactions were run at room temperature (±26°). 

 For citric acid synthesis, the following system was em- 

 ployed: I ml of cell-free extract; 0.2 ml of o.iM K cy- 

 steine; 0.2 ml of 0.04M of MgClj; 0.2 ml of 0.2M K 

 oxalacetate; 0.2 ml of o.iM acetyl phosphate; and 0.1 

 ml of coenzyme A containing 150 units. The vessels were 

 incubated at 26° for two hours, at the end of which 2 

 ml of 20% CCI3COOH was added, and citric acid was 

 determined in the filtrate. 



Coenzyme A (150 units per mg) was kindly provided 

 by Dr. F. Lipmann; TPN and isocitric acid, by Dr. S. 



OCHOA. 



Oxidation of intermediates of the di- and tri- 

 carboxylic acid cycles. It is known that most 

 Fig. I. Rate of Oxidation of Citrate, organic acids do not penetrate the cell mem- 

 a-ketoglutarate, succinate, and acetate by brane except as undissociated acids^° and that 



A.^rogenes^ Abcissa, time in mmutes; ^^ ^ consequence it is necessary in many cases to 



Ordmate, Oj uptake m //I. i. buccmate; ^ j j 



1. a-ketoglutarate; 3. Acetate; 4. Citrate, lower the pH value of the solution to increase 



References p. 249. 



