Sandra W. Clark

2,4,6-Trinitrotoluene Reduction by an Fe-Only Hydrogenase in Clostridium acetobutylicum

ABSTRACT The role of hydrogenase on the reduction of 2,4,6-trinitrotoluene (TNT) in Clostridium acetobutylicum was evaluated. An Fe-only hydrogenase was isolated and identified by using TNT reduction activity as the selection basis. The formation of hydroxylamino intermediates by the purified enzyme corresponded to expected products for this reaction, and saturation kinetics were determined with a Km of 152 μM. Comparisons between the wild type and a mutant strain lacking the region encoding an alternative Fe-Ni hydrogenase determined that Fe-Ni hydrogenase activity did not significantly contribute to TNT reduction. Hydrogenase expression levels were altered in various strains, allowing study of the role of the enzyme in TNT reduction rates. The level of hydrogenase activity in a cell system correlated (R2 = 0.89) with the organisms ability to reduce TNT. A strain that overexpressed the hydrogenase activity resulted in maintained TNT reduction during late growth phases, which it is not typically observed in wild type strains. Strains exhibiting underexpression of hydrogenase produced slower TNT rates of reduction correlating with the determined level of expression. The isolated Fe-only hydrogenase is the primary catalyst for reducing TNT nitro substituents to the corresponding hydroxylamines in C. acetobutylicum in whole-cell systems. A mechanism for the reaction is proposed. Due to the prevalence of hydrogenase in soil microbes, this research may enhance the understanding of nitroaromatic compound transformation by common microbial communities.

Regulation of purine metabolism. Adenylosuccinate synthetase from Novikoff ascites tumor cells.

Adenylosuccinate synthetase has been partially purified from Novikoff ascites tumor cells. The properties of the protein are quite different from the enzyme from rat liver in that the Km for asparate is higher and the K1 for the feedback inhibitor AMP is also higher. The antibiotic hadacidin has a preferential inhibitory effect on the tumor enzyme. These results suggest that the Novikoff ascites tumor enzyme is less sensitive to normal feedback controls but may be more sensitive to specific antitumor drugs.

Effects of rifampicin and chloramphenicol on product and enzyme levels of the acid- and solvent-producing pathways of Clostridium acetobutylicum (ATCC 824)

Abstract As part of an ongoing study of regulation of acid and solvent production in Clostridium acetobutylicum , the effects of rifampicin and chloramphenicol were determined on product formation, enzyme activity, and enzyme stability in vivo of enzymes and metabolites associated with those pathways. The in vitro activities of phosphotransbutyrylase, butyrate kinase, CoA transferase, butyraldehyde dehydrogenase, and butanol dehydrogenase were assayed over the entire fermentation. The enzymes in the solvent-producing pathways were more affected by the addition of rifampicin and chloramphenicol than those in the acid-producing pathways. All of the enzymes measured appeared to be stable in vivo , with the important exception of butyraldehyde dehydrogenase. The position of butyraldehyde dehydrogenase as a branchpoint enzyme in the use of butyryl-CoA, coupled with the short half-life for butyraldehyde dehydrogenase in vivo , indicates that butyraldehyde dehydrogenase may be a key enzyme in controlling the switch from the production of butyrate to the production of butanol.

Purine biosynthesis in Helix aspersa: Metabolic fate of labelled precursors

1. The hepatopancreas of terrestial snails actively synthesizes purines from labelled formic acid. 2. The guanine nucleotides are initially labelled to the highest specific activity followed by adenine and hypoxanthine. 3. The labelling patterns suggest that uric acid synthesis is the primary catabolic process for nitrogen excretion while guanine excretion is due to an inability to reutilize the base.

Regulation of purine metabolism: A comparative study of the kinetic properties of adenylosuccinate synthetases from various sources

1. Adenylosuccinate synthetase has been partially purified from rat liver, fetal rat liver, Novikoff ascites cells, Walker carcinoma 256 solid tumors, chicken liver and muscle, rabbit muscle and pig brain. 2. Considerable differences exist in Michaelis constants among the various species and the changes possibly reflect differences in regulation. 3. The kinetic properties of the enzyme are generally consistent with proposed metabolic roles in various tissues.

High-performance liquid chromatography of proteins: Purification of the acidic isozyme of adenylosuccinate synthetase from rat liver☆

Abstract High-performance ion-exchange liquid chromatography was utilized for the purification of the acidic isozyme of adenylosuccinate synthetase from rat liver. Initial steps in the purification included ammonium sulfate fractionation and DEAE-cellulose and agarose-GTP affinity columns. The final steps were done on a SynChropak AX-300 anion-exchange support. The enzyme was purified 3000-fold with an overall yield of 10%. The enzyme preparation exhibited only one protein band on gel electrophoresis.

Enzymes of the purine nucleotide cycle in muscles from normal and dystrophic chickens.

Summary The tissue levels of the enzymes involved in the proposed purine nucleotide cycle in muscle, adenylosuccinate synthetase and lyase and adenylate deaminase, have been determined in the sartorius and breast muscles of normal and genetically dystrophic chickens. The deaminase and synthetase levels in the breast decrease while the lyase levels rise.