No abstract

The stability of halophilic malate dehydrogenase increases with increasing salt concentration and with decrease in temperature. Stabilization by various salts, at high salt concentrations, follows the Hofmeister series. The enzyme inactivation rates closely match dissociation of the dimeric enzymes into monomeric subunits and unfolding of the polypeptide chains, as followed by velocity sedimentation, light. scattering and circular dichroism measurements. The a-helix content goes to zero upon denaturation. Unusual water and salt binding properties of the native enzyme (cf. preceding paper, in this journal) are believed to be largely lost upon enzyme dissociation and unfolding. These properties thus seem to be associated with the intact structure of the enzyme.In our previous work [l] we have shown that, although malate dehydrogenase (h) becomes unstable at NaCl concentrations lower than about 2.5 M, enzyme activity and structure is maintained for short times at NaCl concentrations as low as 1 M. It was therefore possible to provide a description of the properties of the active enzyme over the NaCl concentration range 1-5 M, by performing a series of diffusion, sedimentation and circular dichroism experiments. These essentially could be considered as being performed at 'zero time', before any significant changes in enzymatic activity or conformation arises.The recent striking observation resulting from the above work is the fact that malate dehydrogenase (h), as well as glutamate dehydrogenase (h), complex in striking fashion with large amounts of both water and salt. Concepts relating to 'binding' of low-molecular-weight components to large particles in solution are largely operational and different methods may yield different results. We have used an approach based on volume exclusion, as manifested by density increments at specified compositions and thermodynamic potentials of water and salt [2,3]. This concept has previously yielded reasonable values for hydration and interaction with salts of DNA and of non-halophilic proteins [l]. The behavior of the halophilic enzymes is distinctly different from that of the non-halophilic macromolecules examined. We shall present evidence in the present work to suggest that these unusual hydration and salt binding characteristics are largely lost when malate dehydrogenase (h) dissociates and unfolds. An indication is thus provided for a mechanism of adaptation to extreme conditions of high salt concentration which is dependent on the intactness of the active enzyme structure.Ahbreviaiions. Malate dehydrogenase (h), halophilic malate dchydrogenase; glutamate dehydrogenase (h), halophilic glutamate dehydrogenase.Enzymes. Malate dehydrogenase (EC 1.1.1.37); glutamate dehydrogenase [NAD(P)+] (EC 1.4.1.3).In an earlier investigation Mevarech and Neumann [4] described the reversible inactivation of malate dehydrogenase (h) at low NaCl concentration. They suggested that inactivation of the dimeric enzyme involved dissociation into two subunits, since the kinetics of reactivation...

A long-standing question in bacterial chemotaxis is whether repellents are sensed by receptors or whether they change a general membrane property such as the membrane fluidity and this change, in turn, is sensed by the chemotaxis system. This study addressed this question. The effects of common repellents on the membrane fluidity of Escherichia coli were measured by the fluorescence polarization of the probe 1,6-diphenyl-1,3,5-hexatriene in liposomes made of lipids extracted from the bacteria and in membrane vesicles. Glycerol, indole, and L-leucine had no significant effect on the membrane fluidity. NiSO4 decreased the membrane fluidity but only at concentrations much higher than those which elicit a repellent response in intact bacteria. This indicated that these repellents are not sensed by modulating the membrane fluidity. Aliphatic alcohols, on the other hand, fluidized the membrane, but the concentrations that elicited a repellent response were not equally effective in fluidizing the membrane. The response of intact bacteria to alcohols was monitored in various chemotaxis mutants and found to be missing in mutants lacking all the four methyl-accepting chemotaxis proteins (MCPs) or the cytoplasmic che gene products. The presence of any single MCP was sufficient for the expression of a repellent response. It is concluded (i) that the repellent response to aliphatic alcohols can be mediated by any MCP and (ii) that although an increase in membrane fluidity may take part in a repellent response, it is not the only mechanism by which aliphatic alcohols, or at least some of them, are effective as repellents. To determine whether any of the E. coli repellents are sensed by periplasmic receptors, the effects of repellents from various classes on periplasm-void cells were examined. The responses to all the repellents tested (sodium benzoate, indole, L-leucine, and NiSO4) were retained in these cells. In a control experiment, the response of the attractant maltose, whose receptor is periplasmic, was lost. This indicates that these repellents are not sensed by periplasmic receptors. In view of this finding and the involvement of the MCPs in repellent sensing, it is proposed that the MCPs themselves are low-affinity receptors for the repellents.The nonstimulated swimming of peritrichously flagellated bacteria is a random walk consisting of swimming in rather straight lines interrupted occasionally by brief episodes of tumbling. Smooth swimming results from counterclockwise rotation of the flagella, and tumbling results from clockwise rotation and from nonsynchronized pauses in the rotation.