The bacterial stringent response is induced by nutrient deprivation and is mediated by enzymes of the RSH (RelA/SpoT homologue; RelA, (p)ppGpp synthetase I; SpoT, (p)ppGpp synthetase II) superfamily that control concentrations of the ‘alarmones’ (p)ppGpp (guanosine penta- or tetra-phosphate). This regulatory pathway is present in the vast majority of pathogens and has been proposed as a potential anti-bacterial target. Current understanding of RelA-mediated responses is based on biochemical studies using Escherichia coli as a model. In comparison, the Francisella tularensis RelA sequence contains a truncated regulatory C-terminal region and an unusual synthetase motif (EXSD). Biochemical analysis of F. tularensis RelA showed the similarities and differences of this enzyme compared with the model RelA from Escherichia coli . Purification of the enzyme yielded a stable dimer capable of reaching concentrations of 10 mg/ml. In contrast with other enzymes from the RelA/SpoT homologue superfamily, activity assays with F. tularensis RelA demonstrate a high degree of specificity for GTP as a pyrophosphate acceptor, with no measurable turnover for GDP. Steady state kinetic analysis of F. tularensis RelA gave saturation activity curves that best fitted a sigmoidal function. This kinetic profile can result from allosteric regulation and further measurements with potential allosteric regulators demonstrated activation by ppGpp (5′,3′-dibisphosphate guanosine) with an EC 50 of 60±1.9 μM. Activation of F. tularensis RelA by stalled ribosomal complexes formed with ribosomes purified from E. coli MRE600 was observed, but interestingly, significantly weaker activation with ribosomes isolated from Francisella philomiragia .

Glutathionylation plays a central role in cellular redox regulation and anti-oxidative defence. Grx (Glutaredoxins) are primarily responsible for reversing glutathionylation and their activity therefore affects a range of cellular processes, making them prime candidates for computational systems biology studies. However, two distinct kinetic mechanisms involving either one (monothiol) or both (dithiol) active-site cysteines have been proposed for their deglutathionylation activity and initial studies predicted that computational models based on either of these mechanisms will have different structural and kinetic properties. Further, a number of other discrepancies including the relative activity of active-site mutants and contrasting reciprocal plot kinetics have also been reported for these redoxins. Using kinetic modelling, we show that the dithiol and monothiol mechanisms are identical and, we were also able to explain much of the discrepant data found within the literature on Grx activity and kinetics. Moreover, our results have revealed how an apparently futile side-reaction in the monothiol mechanism may play a significant role in regulating Grx activity in vivo .

2′- O ,4′- C -methylene-linked ribonucleotide derivatives, named LNA (locked nucleic acid) and BNA (bridged nucleic acid) are nucleic acid analogoues that have shown high-affinity recognition of DNA and RNA, and the employment of LNA oligomers for antisense activity, gene regulation and nucleic acid diagnostics seems promising. Here we show kinetic and thermodynamic results on the interaction of a series of 10 bases long LNA–DNA mixmers, gabmers as well as full length LNA's with the complementary DNA, RNA and LNA oligonucleotides in the presence and absence of 10 mM Mg 2+ - ions. Our results show no significant differences in the reaction thermodynamics and kinetics between the LNA species, only a tendency to stronger duplex formation with the gabmer and mixmer. Introduction of a few LNA's thus may be a better strategy, than using full length LNA's to obtain an oligonucleotide that markedly increases the strength of duplexes formed with the complementary DNA and RNA.

The transport system of folic acid (Pte-Glu) by brush-border membrane vesicles (BBMV) isolated from prawn ( Penaeus japonicm ) hepatopancreas, was studied by measuring the uptake of Pte-Glu. This uptake was found to have two components, intravesicular transport and membrane binding. Membrane binding was not affected by the presence of a transmembrane pH-gradient at a short incubation period. However, a transmembrane pH-gradient increased membrane binding at 60 min. The transport of Pte-Glu appeared to be carrier-mediated, was stimulated by an inwardly proton gradient (pH 5.5 outside, 7.4 inside) and was unaffected by a sodium-gradient. The relationship between pH gradient-driven Pte-Glu uptake and medium Pte-Glu concentration followed saturating Michaelis–Menten kinetics. Eadie–Hofstee representation of the pH gradient-driven Pte-Glu uptake indicated a single transport system with a Km of 0.37 μM and Vmax of 1.06 pmol/mg protein/15 s. These findings indicate that BBMV isolated from prawn hepatopancreas possesses a Pte-Glu transport system similar to that described in mammalian intestine.