Kinetic studies of a soluble alpha beta complex of nitrate reductase A from Escherichia coli. Use of various alpha beta mutants with altered beta subunits

A soluble alpha beta complex of nitrate reductase can be obtained from a strain of Escherichia coli that lacks the narI gene and expresses only the alpha and beta subunits. The beta subunit contains four Fe-S centres and the alpha subunit contains the molybdenum cofactor, which is the site at which nitrate is reduced. Despite the lack of the gamma subunit of the complete enzyme, this complex can still catalyse the reduction of nitrate with artificial electron donors such as benzyl viologen, so that it is suitable for studying the transfer of electrons between these two types of redox centre. To examine whether the electrons from reduced benzyl viologen are initially delivered to the Fe-S centres, or directly to the molybdenum cofactor, or both, we have studied the steady-state kinetics and the binding of benzyl viologen to the alpha beta complex and mutants alpha beta* with altered beta subunits. Reduction of the enzyme by reduced benzyl viologen in the absence of nitrate showed that all four Fe-S centres and the molybdenum cofactor could be reduced. Two classes of site with different equilibrium constants could be distinguished. The kinetic results suggest that benzyl viologen supplies its electrons directly to the molybdenum cofactor, at a rate showing a hyperbolic dependence on the square of the concentration of the electron donor. A reaction mechanism is proposed for the reduction of nitrate catalysed by the alpha beta complex of nitrate reductase with artificial electron donors.     

Enhanced security protocol engineering through a unified multidimensional framework

Multidimensional security protocol engineering is effective for creating cryptographic protocols since it encompasses a variety of design, analysis, and deployment techniques, thereby providing a higher level of confidence than individual approaches offer. SPEAR II, the Security Protocol Engineering and Analysis Resource II, is a protocol engineering tool built on the foundation of previous experience garnered during the SPEAR I project in 1997. The goal of the SPEAR II tool is to facilitate cryptographic protocol engineering and to aid users in distilling the critical issues during an engineering session by presenting them with an appropriate level of detail and guiding them as much as possible during design, analysis and implementation. The SPEAR II tool currently consists of four components that have been integrated into one consistent and unified graphical interface: a protocol specification environment (GYPSIE), a GNY statement construction interface (visual GNY), a Prolog-based GNY analysis engine (GYNGER), and a message rounds calculator. The multidimensional approach realized by SPEAR II is combined with a graphical interface that focuses on making specification of a protocol and its associated conditions for formal analysis as straight forward and painless as possible. Experiments that we have conducted confirm that the analysis engine is able to generate accurate proofs for achievable GNY-based goals, while preliminary usability experiments have indicated that the interface utilized by SPEAR II is both expressive and simple to use for specifying cryptographic protocols and constructing logic statements pertaining to these protocols.     

Isoproterenol evokes extracellular Ca2+ spikes due to secretory events in salivary gland cells

Secretory cells should in principle export substantial amounts of calcium via exocytosis since Ca2+ is sequestered in secretory granules. Based on a new technique for measurements of the extracellular calcium concentration in the vicinity of the cell membrane and on the droplet technique, we have monitored the rate of calcium extrusion from salivary gland acinar cells. Isoproterenol (ISP), a beta-adrenergic agonist and powerful secretogogue, evoked no change in the cytosolic free Ca2+ concentration ([Ca2+]i) but induced vigorous extracellular Ca2+ concentration ([Ca2+]i) spiking. The absence of [Ca2+]i elevation and the pulsatile nature of the changes in [Ca2+]i indicate that these spikes are most likely due to calcium release from secretory granules. The cholinergic agonist acetylcholine (ACh), which induces moderate secretion, evoked a marked rise in [Ca2+]i and a smooth rise in [Ca2+]i, most likely induced by plasma membrane calcium pumps, on which shortlasting [Ca2+]i spikes were superimposed. The rate of ISP-induced calcium efflux was very substantial. The calculated calcium loss during the first 100 s of supramaximal stimulation corresponded to a reduction of the total cellular calcium concentration of approximately 0.4 mM. We conclude that in salivary glands, calcium release via exocytosis is one of the main mechanisms extruding calcium from cells to the extracellular milieu.     

Sugar transport by the marine chitinolytic bacterium Vibrio furnissii. Molecular cloning and analysis of the glucose and N-acetylglucosamine permeases

Chitin catabolism by the marine bacterium Vibrio furnissii involves chemotaxis to and transport of N-acetyl-D-glucosamine (GlcNAc) and D-glucose. We report the properties of the respective permeases that complemented E. coli Glc- Man- mutants. Although the V. furnissii Glc-specific permease (55,941 Da) shares 38% identity with E. coli IIGlc (ptsG), it is 67% identical to MalX of the E. coli maltose operon (Reidl, J., and Boos, W. (1991) J. Bacteriol. 173, 4862-4876). An adjacent open reading frame encodes a protein with 52% identity to E. coli MalY. Glc phosphorylation requires only V. furnissii MalX and the accessory phosphoenolpyruvate:glycose phosphotransferase system proteins. The V. furnissii equivalent of IIGlc was not found in the 25,000 transformants screened. The GlcNAc/Glc-specific permease (52,894 Da) shares 47% identity with the N-terminal, hydrophobic domain of E. coli IINag, but is unique among IINag proteins in that it lacks the C-terminal domain and thus requires IIIGlc for sugar fermentation in vivo and phosphorylation in vitro. While there are similarities between the phosphoenolpyruvate:glycose phosphotransferase system of V. furnissii and enteric bacteria, the differences may be important for survival of V. furnissii in the marine environment.     

Separations of derivatized amino acid enantiomers by cyclodextrin-modified capillary electrophoresis: mechanistic and molecular modeling studies

The enantiomers of 5-dimethylamino-1-naphthalene sulfonyl (DNS)-derivatives of selected amino acids were successfully separated using capillary electrophoresis (CE) employing cyclodextrins (CD) as enantio-selective running buffer additives. A previously described model for retention and chiral recognition in CD-modified CE is shown to adapt well in this application. Resolution of the isomers is strongly influenced by the type and concentration of cyclodextrin employed, as predicted by the model. Although data indicates differences in the electrophoretic mobilities for some of the completely complexed enantiomer pairs, selectivity generally requires exploiting differences in the amino acid-CD complexation constants for enantiomer pairs. In this work, the D-enantiomers exhibit larger formation constants and are complexed to a greater degree (elute first) at moderate CD concentration. When mixtures of amino acids are analyzed, the effects of separation conditions on general elution behavior must be considered or separated enantiomer pairs will co-elute with other enantiomers. Preliminary results aimed at predicting the strength of DNS-amino acid enantiomer-CD interactions based on molecular modeling studies are presented. A statistical mechanical approach to treating computationally derived enantiomer-CD interaction energies is shown to provide reasonable correlation with separation performance.