Gerard A. O'Donovan

Linear one-step assay for the determination of orthophosphate

A rapid one-step spectrophotometric assay for orthophosphate that requires a single stable reagent solution is presented. The reagent solution, an aqueous mixture of ammonium molybdate and zinc acetate at pH 5.0, produces a stable complex with orthophosphate that absorbs strongly in the near-visible region of the light spectrum. Response to concentration of phosphate was linear up to 300 microM phosphate with a molar absorptivity of 7200 M-1 cm-1 at 350 nm. The mild conditions for phosphate determination employed in this method are unique, making it particularly suitable for the assay of orthophosphate in the presence of labile organophosphates.

New assay for enzymatic phosphate release: Application to aspartate transcarbamylase and other enzymes

The colorimetric method for phosphate determination described in the preceding paper is adapted for the assay of orthophosphate liberated in the aspartate transcarbamylase reaction. The method provides for simple, accurate, and sensitive measurement of enzyme activity. The assay uses ammonium molybdate and zinc acetate to form a colored complex with the enzymatically released phosphate; mild conditions which minimize the nonenzymatic background degradation of the substrate, carbamoyl phosphate, are used. Since the assay procedure is relatively rapid, it is especially attractive in situations where results are desired immediately. The method can be used for the assay of any enzyme which releases inorganic phosphate, even in the presence of labile organophosphate compounds.

Regulatory divergence of aspartate transcarbamoylases within the enterobacteriaceae.

Abstract The regulatory characteristics of aspartate transcarbamoylases (ATCases, EC 2.1.3.2) isolated from various representatives of the Enterobacteriaceae have been compared. All of the enzymes had similar molecular weights (300,000 ± 15,000) and appeared to be “class B” bacterial ATCases like the native enzyme of Escherichia coli. Despite the apparent similarities in structures of these enzymes dramatic differences in the regulatory nature of the enzymes were observed. Cytidine 5′-triphosphate was a negative effector of ATCase in E. coli and closely related genera, but was a positive effector of ATCase activity from other enteric bacteria and had little or no effect on the enzymes from Yersinia and Erwinia species. Furthermore, adenosine 5′-triphosphate activation was observed for most of the ATCases isolated from the enterics but had no effect on some enzymes. Finally, these enzymes displayed dramatic variability in apparent [S]0.5 values ranging from 3 to 35 m m aspartate. These variations were used to define five classes of regulatory divergent ATCases in the Enterobacteriaceae. It was satisfying to observe that these class groupings compared favorably with the tribal classification of the Enterobacteriaceae in general. Thus, the regulatory character of ATCase appeared to be conserved in a specific manner within tribes of physiologically related bacteria. Based on these correlations we propose a model for the molecular evolution of ATCases in bacteria.

Improved colorimetric procedure for quantitating N-carbamoyl-β-alanine with minimum dihydrouracil interference☆

Abstract After modifying the Prescott-Jones colorimetric method for carbamoyl compound determination, it was possible to measure the concentration of N -carbamoyl-β-alanine (β-ureidopropionate) with little interference from its metabolic precursor, dihydrouracil. Color formation at 70°C was linear with respect to N -carbamoyl-β-alanine up to a concentration of 0.07 m m .

Regulatory variance of aspartate transcarbamoylase among strains ofYersinia enterocolitica andYersinia enterocolitica-like organisms

Aspartate transcarbamoylase (ATCase) has been isolated and characterized from 20 different strains ofYersinia enterocolitica andY. enterocolitica-like organisms. A variety of regulatory properties have emerged for the ATCases from the different strains. These regulatory properties may be used as a taxonomic tool to divideY. enterocolitica andY. enterocolitica-like organisms into separate groups. Results are in accord with the recent assignment ofY. enterocolitica andY. enterocolitica-like organisms to four DNA-relatedness groups and four correspondingYersinia species.

Repression of Cytidine Triphosphate Synthetase in Salmonella typhimurium by Pyrimidines during Uridine Nucleotide Depletion

Regulation of the synthesis of cytidine triphosphate (CTP) synthetase (EC 6.3.4.2) was investigated in Salmonella typhimurium. CTP synthetase appeared to be repressed only when intracellular concentrations of uridine nucleotides were significantly lowered. Under such nucleotide pool conditions, a cytidine compound and, to a lesser degree, a thymidine compound appeared as putative repressing metabolites of enzyme synthesis.

Location of the argR gene on the chromosome of Salmonella typhimurium

SummaryThe regulatory gene (argR) for the arginine biosynthetic pathway has been located at 106 min on the chromosome of S. typhimurium. In addition, the location of the gene specifying cytosine deaminase (cod) has been more precisely determined.