Title: INTERACTIONS OF ATABRINE, THIAMINE, AND COCARBOXYLASE
Abstract: The observation that salts of the alkaline earth metals reverse the inhibitory effects of atabrine in the growth of Escherichia coli (1) suggested, as one possibility, that the inhibitor acts by forming complexes with metal-catalyzed enzyme systems.The effect of atabrine on yeast carboxylase was therefore investigated, but the results show no evidence for complex formation involving atabrine and this metallo-enzyme.Instead, the investigation has disclosed that atabrine competitively inhibits the dephosphorylation of cocarboxylase by a yeast phosphatase and, further, that the drug inhibits the synthesis of thiamine by yeast cultures. MethodsA batch of brewers' yeast which had been washed extensively in water, then dried and stored at room temperature, was used as the source of carboxylase.Atiozymase was prepared daily from this lot of yeast by rapidly washing 1 gm.with six successive 45 ml.portions of 0.1 M K2HPOI.A final washing was carried out with 25 ml. of 0.067 M KH2POI.The temperature of the wash solutions was maintained at 10".Cocarboxylase concentrations were determined manometrically by the customary Warburg procedures.The components of the assay system and the order of the additions to the vessels were as follows: (1) Mn++, 4.4 x lo4 M; (2) standard cocarboxylase solution or sample; (3) thiamine, 104, or atabrine, 10" M (see the text); (4) phosphate buffer, pH 6.1, 2.7 X 10m2 M; (5) atiozymase, 50 mg.in solution (4); (6) sodium pyruvate, 2.7 X 1O-2 M; total volume 2.5 ml.; temperature 30".The pyruvate was added after temperature equilibrium was attained and the CO2 evolved was measured after 15 minutes.Thiamine concentrations were determined by a thiochrome procedure of Papageorge and Lamar (2).Thiamine was extracted from cell suspensions by boiling for 1 minute in 0.05 N HCl.To&a utilis, ATCC 9255, was grown in the mineral salts-glucose medium of Gray and Tatum (3).The inoculum employed was the growth from 10 ml. of 1 per cent Bacto-peptone incubated at 30" for 24 hours.