The steady-state kinetic mechanism of ATP hydrolysis catalyzed by membrane-bound (Na+ + K+)-ATPase from ox brain IV. Rate constant determination

The expressions for the kinetic constants corresponding to the steady state model for hydrolysis of ATP catalyzed by (Na⁺ + K⁺)-ATPase proposed recently are analyzed with the object of determining the rate constants. The theoretical background for the necessary procedures is described. The results of this analysis are: (1) A small class (four) of rate constants are determined directly by the previously published values of the kinetic constants. (2) For a somewhat larger class of rate constants upper and lower bounds may be established. For several rate constants the upper and lower bounds differ by less than a factor 1.6 (for the ‘(Na⁺ + K⁺)-enzyme’, i.e. the enzyme activity with K⁺ and millimolar substrate concentration) and 1.2 (for the ‘Na⁺-enzyme’, i.e. the activity at micromolar substrate concentrations). (3) Experiments on inhibition by K⁺ of the Na⁺-enzyme at various Mg²⁺ concentrations are reported and analyzed. With the additional assumption that the rate constants governing the addition to ATP of Mg²⁺ is independent of whether or not ATP is bound to an enzyme molecule, a set of consistent values for all the 23 rate constants in the mechanism may be obtained. (4) The values of some rate constants lend further support to the contention discussed in a previous paper that the enzyme hydrolyzes ATP along two kinetically distinct pathways, depending on the presence of K⁺ and on the concentration of substrate, without the necessity of having more than one active substrate site per enzyme unit at any time. (5) The results show that while the two enzyme forms, the ‘Na⁺-enzyme’ E₁ and the “K⁺-enzyme” E₂K, add substrate with (second order) rate constants of the same order of magnitude (differing only by a factor of four in favor of the former), the rate constants for the reverse processes differ by a factor of 100, being largest for the K⁺-enzyme. This is the main reason for the large difference in the Michaelis constants for the two forms reported previously. (6) Compatibility of the model with the well-known rapid dephosphorylation of the phosphorylated enzyme in the presence of K⁺ requires the presence, at non-zero steady state concentration, of an enzyme-potassium-phosphate intermediate, which is acid labile and is therefore not detected as a phosphorylated enzyme using conventional methods.     

NODULE ROOT and COCHLEATA Maintain Nodule Development and Are Legume Orthologs of Arabidopsis BLADE-ON-PETIOLE Genes

During their symbiotic interaction with rhizobia, legume plants develop symbiosis-specific organs on their roots, called nodules, that house nitrogen-fixing bacteria. The molecular mechanisms governing the identity and maintenance of these organs are unknown. Using Medicago truncatula nodule root (noot) mutants and pea (Pisum sativum) cochleata (coch) mutants, which are characterized by the abnormal development of roots from the nodule, we identified the NOOT and COCH genes as being necessary for the robust maintenance of nodule identity throughout the nodule developmental program. NOOT and COCH are Arabidopsis thaliana BLADE-ON-PETIOLE orthologs, and we have shown that their functions in leaf and flower development are conserved in M. truncatula and pea. The identification of these two genes defines a clade in the BTB/POZ-ankyrin domain proteins that shares conserved functions in eudicot organ development and suggests that NOOT and COCH were recruited to repress root identity in the legume symbiotic organ.     

Structural properties of plant and mammalian lipoxygenases. Temperature-dependent conformational alterations and membrane binding ability

Lipoxygenases form a heterogeneous family of lipid peroxidizing enzymes, which have been implicated in the synthesis of inflammatory mediators, in cell development and in the pathogenesis of various diseases with major health and political relevance (atherosclerosis, osteoporosis). The crystal structures of various lipoxygenase-isoforms have been reported, and X-ray coordinates for enzyme-ligand complexes are also available. Although the 3D-structures of plant and animal lipoxygenase-isoforms are very similar, recent small-angle X-ray scattering data suggested a higher degree of motional flexibility of mammalian isozymes in aqueous solutions. To explore the molecular basis for these differences we performed dynamic fluorescence measurements that allowed us to study temperature-induced conformational changes arising from three-dimensional fluctuations of the protein matrix. For this purpose, we first investigated the impact of elevated temperature on activity, secondary structure, tertiary structure dynamics and conformational alterations. Applying fluorescence resonance energy transfer we also tested the membrane binding properties of the two lipoxygenase-isoforms, and compared their binding parameters. Taken together, our results indicate that the rabbit 12/15-lipoxygenase is more susceptible to temperature-induced structural alterations than the soybean enzyme. Moreover, the rabbit enzyme exhibits a higher degree of conformational flexibility of the entire protein molecule (global flexibility) and offers the possibility of augmented substrate movement at the catalytic center (local flexibility).     

Fluorescent in situ Hybridization on Mitotic Chromosomes of Mosquitoes

Fluorescent in situ hybridization (FISH) is a technique routinely used by many laboratories to determine the chromosomal position of DNA and RNA probes. One important application of this method is the development of high-quality physical maps useful for improving the genome assemblies for various organisms. The natural banding pattern of polytene and mitotic chromosomes provides guidance for the precise ordering and orientation of the genomic supercontigs. Among the three mosquito genera, namely Anopheles, Aedes, and Culex, a well-established chromosome-based mapping technique has been developed only for Anopheles, whose members possess readable polytene chromosomes ¹. As a result of genome mapping efforts, 88% of the An. gambiae genome has been placed to precise chromosome positions ^2,3 . Two other mosquito genera, Aedes and Culex, have poorly polytenized chromosomes because of significant overrepresentation of transposable elements in their genomes ^{4, 5, 6}. Only 31 and 9% of the genomic supercontings have been assigned without order or orientation to chromosomes of Ae. aegypti ⁷ and Cx. quinquefasciatus ⁸, respectively. Mitotic chromosome preparation for these two species had previously been limited to brain ganglia and cell lines. However, chromosome slides prepared from the brain ganglia of mosquitoes usually contain low numbers of metaphase plates ⁹. Also, although a FISH technique has been developed for mitotic chromosomes from a cell line of Ae. aegypti ¹⁰, the accumulation of multiple chromosomal rearrangements in cell line chromosomes ¹¹ makes them useless for genome mapping. Here we describe a simple, robust technique for obtaining high-quality mitotic chromosome preparations from imaginal discs (IDs) of 4^th instar larvae which can be used for all three genera of mosquitoes. A standard FISH protocol ¹² is optimized for using BAC clones of genomic DNA as a probe on mitotic chromosomes of Ae. aegypti and Cx. quinquefasciatus, and for utilizing an intergenic spacer (IGS) region of ribosomal DNA (rDNA) as a probe on An. gambiae chromosomes. In addition to physical mapping, the developed technique can be applied to population cytogenetics and chromosome taxonomy/systematics of mosquitoes and other insect groups.     

DNA damage response in peripheral mouse blood leukocytes in vivo after variable,low-dose rate exposure

Environmental contamination and ingestion of the radionuclide Cesium-137 (137Cs) is a large concern in fallout from a nuclear reactor accident or improvised nuclear device, and highlights the need to develop biological assays for low-dose rate, internal emitter radiation. To mimic low-dose rates attributable to fallout, we have developed a VAriable Dose-rate External 137Cs irradiatoR (VADER), which can provide arbitrarily varying and progressive low-dose rate irradiations in the range of 0.1–1.2 Gy/day, while circumventing the complexities of dealing with radioactively contaminated biomaterials. We investigated the kinetics of mouse peripheral leukocytes DNA damage response in vivo after variable, low-dose rate 137Cs exposure. C57BL/6 mice were placed in the VADER over 7 days with total accumulated dose up to 2.7 Gy. Peripheral blood response including the leukocyte depletion, apoptosis as well as its signal protein p53 and DNA repair biomarker γ-H2AX was measured. The results illustrated that blood leukocyte numbers had significantly dropped by day 7. P53 levels peaked at day 2 (total dose = 0.91 Gy) and then declined; whereas, γ-H2AX fluorescence intensity (MFI) and foci number generally increased with accumulated dose and peaked at day 5 (total dose = 2.08 Gy). ROC curve analysis for γ-H2AX provided a good discrimination of accumulated dose < 2 Gy and ≥ 2 Gy, highlighting the potential of γ-H2AX MFI as a biomarker for dosimetry in a protracted, environmental exposure scenario.     

A method for allelic replacement in Francisella tularensis

A vector for mutagenesis of Francisella tularensis was constructed based on the pUC19 plasmid. By inserting the sacB gene of Bacillus subtilis, oriT of plasmid RP4, and a chloramphenicol resistance gene of Shigella flexneri, a vector, pPV, was obtained that allowed specific mutagenesis. A protocol was developed that allowed introduction of the vector into the live vaccine strain, LVS, of F. tularensis by conjugation. As a proof of principle, we aimed to develop a specific mutant defective in expression of a 23-kDa protein (iglC) that we previously have shown to be prominently upregulated during intracellular growth of F. tularensis. A plasmid designated pPV-DeltaiglC was developed that contained only the regions flanking the encoding gene, iglC. By a double crossover event, the chromosomal iglC gene was deleted. However, the resulting strain, denoted DeltaiglC1, still had an intact iglC gene. Southern blot analysis verified that LVS harbors two copies for the iglC gene. The mutagenesis was therefore repeated and a mutant defective in both iglC alleles, designated DeltaiglC1+2, was obtained. The DeltaiglC1+2 strain, in contrast to DeltaiglC1, was shown to display impaired intracellular macrophage growth and to be attenuated for virulence in mice. The developed genetic system has the potential to provide a tool to elucidate virulence mechanisms of F. tularensis and the specific F. tularensis mutant illustrates the critical role of the 23-kDa protein, iglC, for the virulence of F. tularensis LVS.