104 ^- MAMELAK, J. H. QUASTEL VOL. 12 (1953) 



A variety of substances undergoes aerobic oxidation in presence of CI. sporogencs 

 e.g. pyruvate (Nisman^), (though Rosenberg and Nisman^ report no aerobic oxidation 

 of pyruvate by this organism), and amino acids such as alanine, leucine, threonine, 

 serine, methionine, phenylalanine (Nisman and Vinet^*'), the oxidation of amino acids 

 being accompanied by ammonia liberation. The presence of arsenite exerts a much 

 larger inhibitory effect on the amino acid reductases'^- ^^ than upon the amino acid 

 oxidative enzymes. Nisman and Vinet'^ conclude, from the kinetics of arsenite- 

 inhibited reactions, that the enzymes in CI. sporogenes responsible for the aerobic oxi- 

 dation of amino acids are identical with those involved in the anaerobic amino acid inter- 

 actions. Extracts of CI. sporogenes in presence of pyruvate and phosphate give rise to 

 acetyl phosphate'^ and, in presence of alanine, bring about the reduction of diphospho- 

 pyridine nucleotide'^. Moreover such extracts will oxidise reduced diphosphopyridine 

 nucleotide in presence of pyruvate and ammonium ions. 



It is the purpose of this preliminary communication to throw further light on the 

 mechanisms of amino acid interactions in CI. sporogenes and to indicate how these 

 mechanisms are linked with the phenomenon of oxygen toxicity to the strict anaerobes. 



materials and methods 



Manometric technique. The conventional Warburg apparatus was used. 



Nutrient media 



Stock cultures of CI. sporogenes were maintained on Brewer's meat with bi-weekly transfers to 

 fresh media. A newly inoculated tube of media was heated at 80° C for 10 min prior to incubation 

 at 38° C. 



One ml of a suspension of organisms from the Brewer's meat was added to i litre of medium 

 of the following composition. 



15 g Bacto-casitone 



I g Sodium thioglycollate 



1.25 g Sodium chloride 



1.5 g Potassium chloride 



0.75 g Cysteine hydrochloride 



I mg D-Biotin 



I litre water 



The medium was adjusted to pH 7.0 before autoclaving. 



Preparation of bacterial suspensions 



One litre of a 14-17 hours culture (grown at 38°) was centrifuged and washed twice in 200 ml 

 0.15 M KCl. The cells were suspended in 8-16 ml 0.15 M KCl. One ml of a thick bacterial suspension, 

 whose dry weight varied from 7. 7-1 1.8 mg, was used in each experiment. 



Preparation of bacterial extract 



A bacterial crusher described by Hughes^^ was used in these experiments and found to be 

 very efficient. Two litres of bacteria were grown for 14-17 hours at 38°, washed once in 200 ml i % 

 sodium thioglycollate and a second time in 10 ml of the same solution and the washed cells were 

 frozen for 5-10 minutes in dry ice. The frozen cells were inserted into the crushing machine which 

 had been cooled to — 20° C. After crushing, the material was extracted with 7-9 ml 1% sodium 

 thioglycollate and centrifuged in the cold at 20,000 g for 2 minutes. One ml of the supernatant was 

 used in each experiment. In order to get good activity in the extract, the cells and the crushing 

 machine must be kept very cold ( — 20° C). If this precaution is not observed and the cells thaw, the 

 activity of the extract is very low. Sodium thioglycollate was used for washing and extraction to 

 prevent the deleterious effects of exposure to oxygen. 



Preparation of lyophylized bacteria 



Cells of CI. sporogenes were washed with 1% sodium thioglycollate and suspended in 20 ml 

 0.15 M KCl. The cell .suspension was frozen in a mixture of dry ice and acetone and lyophilized for 

 5 hours. The dry cells were stored in a vacuum dessicator at o°-io° C. 



References p. 120. 



