Culture of Clostridium pasteurianum in Defined Medium and Growth as a Function of Sulfate Concentration

Clostridium pasteurianum strain W-5 was selected as an anaerobe which may be grown from large inocula in defined media with sulfate as its primary sulfur source. Since it is important to keep inocula small in minimizing transfer of sulfur sources, culture conditions were optimized. The medium devised decreased lag period and generation time when compared with other media, but growth could not be induced consistently with 6 x 10(6) cells per ml or less. Addition of trace elements, chelating agents, reducing agents, metabolites, and spent medium from various stages of growth did not stimulate growth from small inocula. Generation time was 85 min on inoculation with 10(7) or more cells per ml taken from young stocks, but the lag period decreased somewhat with larger inocula. On the other hand, generation time and lag period increased with age of the inoculum. The total yield of cells increased when buffer capacity was increased. Growth of C. pasteurianum W-5 was dependent upon sulfate at relatively low sulfate concentrations, and the organism is thus suitable for study of sulfur metabolism. No evidence of a maintenance requirement for sulfate was detected.     

The interaction of folic acid derivatives in the methylation of homocysteine

1. The cobalamin-independent synthesis of methionine from serine and homocysteine by ultrasonic extracts of E. coli with tetrahydropteroyltriglutamate as cofactor was inhibited competitively by tetrahydropteroylmonoglutamate and derivatives which were readily converted into this compound. 2. The potency of these inhibitors was directly related to their ability to function as cofactors or substrates in the alternative, cobalamin- dependent mechanism for homocysteine methylation. 3. The cobalamin-dependent and -independent mechanisms of homocysteine methylation were both inhibited by reduced derivatives of aminopterin in a similar manner. 4. It was tentatively concluded that the inhibition was due to a competitive interaction between the folates for N(5)N(10)-methylenetetrahydrofolate reductase.     

A new method for the assay of glutaminase activity: direct measurement of product formation by an ammonia electrode

A simple, reliable procedure is described for the quantitative assay of glutaminase reaction by measuring product formation using an ammonia electrode. The ammonia electrode is a gas-detecting electrode, sensing the level of dissolved ammonia in aqueous solutions. Ammonia concentration can be read from calibration curves after converting ammonium ion to ammonia by adding sufficient base. Sample color and turbidity do not affect measurements, and samples need not be distilled. The concentrations of the three glutaminase isoenzymes from rat tissues measured by this method are strictly comparable to those measured by other methods.     

Activation and inhibition of rabbit muscle pyruvate kinase by transition-metal ions.

The paper reports a comparative study of the effects of Mn2+, Ni2+ and Co2+ on the reaction of ADP with phosphoenolypyruvate when catalysed by K+-activated rabbit muscle pyruvate kinase. The activation and subsequent inhibition that occurs as the bivalent ion concentration is increased is taken as evidence that the substrates of the enzyme are phosphoenolypyruvate, uncomplexed ADP and free bivalent cation. Kinetic constants for the binding of the bivalent cation to the enzyme are reported.