High Concentrations Of Molybdate Research Articles

Presence of the molybdenum-cofactor in nitrate reductase-deficient mutant cell lines of Nicotiana tabacum

Nicotiana tabacum mutant cell cultures lacking nitrate reductase activity were assayed for the presence of the molybdenum-cofactor using its ability to restore NADPH-nitrate reductase activity in extracts of Neurospora crassa nit-1 mycelia. The molybdenum-cofactor of the tobacco wild-type line was shown to complement efficiently the N. crassa nit-1 mutant in vitro. The molybdenum-cofactor seems to exist in a bound form, as acid-treatment was required for release of cofactor activity. Molybdate (5–10 mM), ascorbic acid, and anaerobic conditions greatly increased the activity of the cofactor, demonstrating its high lability and sensitivity to oxygen. Similar results were obtained with two tobacco nia mutants, which are defective in the apoprotein of nitrate reductase. The four cnx mutants studied were shown to contain exclusively an inactive form of the molybdenum-cofactor. This inactive cofactor could be reactivated in vitro and in vivo by unphysiologically high concentrations of molybdate (1–10 mM), thereby converting the cnx cells into highly active cofactor sources in vitro, and restoring nitrate reductase and xanthine dehydrogenase in vivo to partial acitivity. Thus the defect of the cnx mutants resides in a lack of molybdenum as a catalytically active ligand metal for the cofactor, while the structural moiety of the cofactor seems not to be impaired by the mutation. The subunit assembly of the nitrate reductase was found to be independent of the molybdenum content of the cofactor.

Determination of serum-phosphate without deproteinization by ultraviolet spectrophotometry of the phosphomolybdic acid complex.

Phosphate may be determined in serum without deproteinization as the phosphomolybdic acid complex by UV-spectrophotometry. The rate of complex formation is dependent on the acidity and the concentration of molybdate. The photometric measurements should be performed at a longer wavelength than in maximum absorptivity of the phosphomolybdic acid complex to permit practical high concentrations of molybdate in the reagents. Citric acid will hinder the complex formation, but will not interfere with the stability of the phosphomolydbic acid. The phosphomolybdic complex is determined by adding citric acid to the samples before (blank) and after (test) the molybdate. The analytical procedure has been automated on a Greiner GSA II, Greiner Electronics, Switzerland.

Selenium requirement for active xanthine dehydrogenase from Clostridium acidiurici and Clostridium cylindrosporum.

The xanthine dehydrogenase of Clostridium acidiurici and C. cylindrosporum was assayed with methyl viologen as acceptor. In C. acidiurici the basal activity level was about 0.3 mumol/min x mg of protein. Cells grown on uric acid in the presence of 10(-7) M selenite showed a 14-fold increase in xanthine dehydrogenase activity, which decreased with higher selenite concentrations (10(-5) M). The supplementation with 10(-7) M molybdate or tungstate was without effect. High concentrations of tungstate decreased the xanthine dehydrogenase if selenite was also present. In comparison, high concentrations of molybdate affected only a small decrease in activity level at the optimal concentration for selenite and relieved to some degree the inhibitory effect of 10(-5) M selenite. With hypoxanthine and xanthine as substrates for growth again only the addition of selenite was necessary to show a similar increase in xanthine dehydrogenase activity. C. acidiurici could be grown in a mineral medium. Both xanthine dehydrogenase and formate dehydrogenase exhibited the highest level of activity if selenite and tungstate were present in that medium. In C. cyclindrosporum the basal activity level of xanthine dehydrogenase was about 0.95 mumol/min x mg of protein. The addition of 10(-7) M selenite to the growth medium increased the activity level about 3-fold, but the highest level (3.7 U/mg) was reached if 10(-7) M molybdate was also added. The presence of tungstate resulted in a decreased enzyme activity.

Mapping and characerization of mutants of Pseudomonas aeruginosa affected in nitrate respiration in aerobic or anaerobic growth.

SUMMARY: Genes which are involved in anaerobic growth with nitrate or nitrite as terminal electron acceptor in Pseudomonas aeruginosa were mapped by interrupted mating and linkage analysis after conjugation. Times of entry were mostly consistent with the linkage frequencies. Late markers were not located precisely. A gene order was obtained for the genes involved in dissimilatory nitrate (nar) and nitrite reduction (nir) and anaerobical growth (ana) and for some already known auxotrophic genes. A mutant unable to grow aerobically (aer) was found. The aer mutation appeared to be an early marker. The cytochrome spectra of nir mutants and part of the cytochrome oxidase activities were abnormal in that haem d was absent. Most of the nar mutants were pleiotropic. Nar A mutants were affected in assimilatory and dissimilatory nitrate reductase, and nar B, nar D and nar E mutants lacked assimilatory and dissimilatory nitrate reductase and xanthine dehydrogenase. In nar D mutants high concentrations of molybdate restored xanthine dehydrogenase and assimilatory and dissimilatory nitrate reductase and, consequently, anaerobic growth. Just as in the aer mutants, which had a normal respiratory system, the denitrifying system and the cytochrome system in ana mutants were normal. It is therefore unlikely that ana and aer mutants synthesize defective energy generating systems.

Phenotypic restoration by molybdate of nitrate reductase activity in chlD mutants of Escherichia coli.

ChlD mutants of Escherichia coli are pleiotropic, lacking formate-nitrate reductase activity as well as formate-hydrogenlyase activity. Whole-chain formate-nitrate reductase activity, assayed with formate as the electron donor and measuring the amount of nitrite produced, was restored to wild-type levels in the mutants by addition of 10(-4)m molybdate to the growth medium. Under these conditions, the activity of each of the components of the membrane-bound nitrate reductase chain increased after molybdate supplementation. In the absence of nitrate, the activities of the formate-hydrogenlyase system were also restored by molybdate. Strains deleted for the chlD gene responded in a similar way to molybdate supplementation. The concentration of molybdenum in the chlD mutant cells did not differ significantly from that in the wild-type cells at either low or high concentrations of molybdate in the medium. However, the distribution of molybdenum between the soluble protein and membrane fractions differed significantly from wild type. We conclude that the chlD gene product cannot be a structural component of the formate-hydrogenlyase pathway or the formate-nitrate reductase pathway, but that it must have an indirect role in processing molybdate to a form necessary for both electron transport systems.