Test for Bioenergetic Progress and Specific Energy Metabolism in Isopod Crustaceans (Isopoda) of Various Ecology

We studied energy metabolism of terrestrial and cavernicolous isopods and demonstrated much lower standard metabolism in the troglobionts as compared to other Isopoda representatives. The test for bioenergetic progress proved to be applicable for both aromorphosis and katamorphosis. Different patterns of the relationship between energy metabolism and temperature in stenothermal and eurythermal species have been proposed.     

Dynamics of body mass and oxygen consumption in the ontogeny of the Spanish ribbed newt (<Emphasis Type="Italic">Pleurodeles waltl</Emphasis>): 2. Larval stage

The correlation between characteristics of growth and energy metabolism during the larval stage of development of the Spanish ribbed newt (Pleurodeles waltl) has been studied. During this period, its body mass is found to increase 140 times and the oxygen consumption rate, 77 times. The highest rate of specific body mass increase and oxygen consumption rate are noted in the early larval stage. Later, these characteristics decrease except for a brief period before completion of metamorphosis when the rate specific body mass increase rises. Comparison of the studied characteristics allows us to note a similar pattern in changes of the specific growth rate and the oxygen consumption rate during the premetamorphic development of the Spanish ribbed newt.     

Effect of α-crystallin on thermal aggregation of glycogen phosphorylase <Emphasis Type="Italic">b</Emphasis> from rabbit skeletal muscle

Thermal aggregation of rabbit skeletal muscle glycogen phosphorylase b (Phb) has been investigated using dynamic light scattering under conditions of a constant rate of temperature increase (1 K/min). The linear behavior of the dependence of the hydrodynamic radius on temperature for Phb aggregation is consistent with the idea that thermal aggregation of proteins proceeds in the kinetic regime wherein the rate of aggregation is limited by diffusion of the interacting particles (the regime of "diffusion-limited cluster-cluster aggregation"). In the presence of alpha-crystallin, a protein exhibiting chaperone-like activity, the dependence of the hydrodynamic radius on temperature follows the exponential law; this suggests that the aggregation process proceeds in the kinetic regime where the sticking probability for colliding particles becomes lower than unity (the regime of "reaction-limited cluster-cluster aggregation"). Based on analysis of the ratio between the light scattering intensity and the hydrodynamic radius of Phb aggregates, it has been concluded that the addition of alpha-crystallin results in formation of smaller size starting aggregates. The data on differential scanning calorimetry indicate that alpha-crystallin interacts with the intermediates of the unfolding process of the Phb molecule. The proposed scheme of thermal denaturation and aggregation of Phb includes the stage of reversible dissociation of dimers of Phb into monomers, the stage of the formation of the starting aggregates from the denatured monomers of Phb, and the stage of the sticking of the starting aggregates and higher order aggregates. Dissociation of Phb dimer into monomers at elevated temperatures has been confirmed by analytical ultracentrifugation.     

Mechanism of thermal aggregation of rabbit muscle glyceraldehyde-3-phosphate dehydrogenase

Thermal denaturation and aggregation of rabbit muscle glyceraldehyde-3-phosphate dehydrogenase (GAPDH) have been studied using differential scanning calorimetry (DSC), dynamic light scattering (DLS), and analytical ultracentrifugation. The maximum of the protein thermal transition (T(m)) increased with increasing the protein concentration, suggesting that the denaturation process involves the stage of reversible dissociation of the enzyme tetramer into the oligomeric forms of lesser size. The dissociation of the enzyme tetramer was shown by sedimentation velocity at 45 degrees C. The DLS data support the mechanism of protein aggregation that involves a stage of the formation of the start aggregates followed by their sticking together. The hydrodynamic radius of the start aggregates remained constant in the temperature interval from 37 to 55 degrees C and was independent of the protein concentration (R(h,0) approximately 21 nm; 10 mM sodium phosphate, pH 7.5). A strict correlation between thermal aggregation of GAPDH registered by the increase in the light scattering intensity and protein denaturation characterized by DSC has been proved.     

The rate of oxygen consumption during embryogenesis of Lymnaea stagnalis (Gastropoda)

We studied the rate of oxygen consumption by the Lymnaea stagnalis embryos. The rate of oxygen consumption increased consistently during embryogenesis. The volume specific rate of oxygen consumption increased initially from the early cleavage stages until the gastrula stage and then decreased gradually to the eclosion of snails. There are three periods in embryogenesis of L. stagnalis, which differ in the coefficients of allometric dependence between the rate of oxygen consumption and volume of embryos: (1) early embryogenesis, when the increase in the rate of oxygen consumption is not accompanied by the growth of volume of the embryos; (2) larval period (trochophore and veliger stages; exponential coefficient k = 0.514), and (3) postlarval period (exponential coefficient k = 0.206).     

Mechanism of chaperone-like activity. Suppression of thermal aggregation of betaL-crystallin by alpha-crystallin

Thermal denaturation and aggregation of beta(L)-crystallin from bovine lens have been studied using differential scanning calorimetry (DSC) and dynamic light scattering (DLS). According to the DLS data, the distribution of the beta(L)-crystallin aggregates by their hydrodynamic radius (R(h)) remains monomodal to the point of precipitating aggregates (sodium phosphate, pH 6.8; 100 mM NaCl; 60 degrees C). The size of the start aggregates (R(h,0)) and duration of the latent stage (t(0)) leading to the formation of the start aggregates have been determined from the light scattering intensity versus the hydrodynamic radius plots and the dependences of R(h) on time. The R(h,0) value remains constant at variation of the beta(L)-crystallin concentration, whereas the t(0) value increases with diminishing beta(L)-crystallin concentration. The suppression of beta(L)-crystallin aggregation by alpha-crystallin is connected with the decrease in the R(h,0) value and increase in the t(0) value. In the presence of alpha-crystallin the aggregate population is split into two components. The first component is represented by stable aggregates whose size remains constant in time. The aggregates of the other kind grow until they reach the size characteristic of aggregates prone to precipitation. The DSC data show that alpha-crystallin has no appreciable influence on thermal denaturation of beta(L)-crystallin.     

Stabilization of plant formate dehydrogenase by rational design

Recombinant formate dehydrogenase (FDH, EC 1.2.1.2) from soy Glycine max (SoyFDH) has the lowest values of Michaelis constants for formate and NAD⁺ among all studied formate dehydrogenases from different sources. Nevertheless, it also has the lower thermal stability compared to enzymes from bacteria and yeasts. The alignment of full sequences of FDHs from different sources as well as structure of apo- and holo-forms of SoyFDH has been analyzed. Ten mutant forms of SoyFDH were obtained by site-directed mutagenesis. All of them were purified to homogeneity and their thermal stability and substrate specificity were studied. Thermal stability was investigated by studying the inactivation kinetics at different temperatures and by differential scanning calorimetry (DSC). As a result, single-point (Ala267Met) and double mutants (Ala267Met/Ile272Val) were found to be more stable than the wild-type enzyme at high temperatures. The stabilization effect depends on temperature, and at 52°C it was 3.6- and 11-fold, respectively. These mutants also showed higher melting temperatures in DSC experiments — the differences in maxima of the melting curves (T _m) for the single and double mutants were 2.7 and 4.6°C, respectively. For mutations Leu24Asp and Val127Arg, the thermal stability at 52°C decreased 5- and 2.5-fold, respectively, and the T _m decreased by 3.5 and 1.7°C, respectively. There were no differences in thermal stability of six mutant forms of SoyFDH — Gly18Ala, Lys23Thr, Lys109Pro, Asn247Glu, Val281Ile, and Ser354Pro. Analysis of kinetic data showed that for the enzymes with mutations Val127Arg and Ala267Met the catalytic efficiency increased 1.7- and 2.3-fold, respectively.     

Iron-dependent superoxide dismutase from novel thermoacidophilic crenarchaeon Acidilobus saccharovorans: from gene to active enzyme

A gene encoding superoxide dismutase was revealed in the genome of the thermoacidophilic crenarchaeon Acidilobus saccharovorans. A recombinant expression vector was constructed and transformed into E. coli cells. The novel recombinant superoxide dismutase was purified and characterized. The enzyme was shown to be an iron-dependent super-oxide dismutase able to bind various bivalent metals in the active site. According to differential scanning calorimetric data, the denaturation temperature of the enzyme is 107.3°C. The maximal activity of the Fe(II) reconstituted enzyme defined by xanthine oxidase assay is 1700 U/mg protein. Study of the thermal stability of the superoxide dismutase samples with various metal contents by tryptophan fluorescence indicated that the thermal stability and activity of the enzyme directly depend on the nature of the reconstituted metal and the degree of saturation of binding sites.     

A comparative study of the thermal stability of formate dehydrogenases from microorganisms and plants

A comparative study of the thermostability of NAD⁺-dependent formate dehydrogenases (FDHs; EC 1.2.1.2) from both methylotrophic bacteria Pseudomonas sp. 101 and Moraxella sp. C1, the methane-utilizing yeast Candida boidinii, and plants Arabidopsis thaliana and Glycine max (soybean) was performed. All the enzymes studied were produced by expression in E. coli cells. The enzymes were irreversibly inactivated in one stage according to first-order reaction kinetics. The FDH from Pseudomonas sp. 101 appeared as the most thermostable enzyme; its counterpart from Glycine max exhibited the lowest stability. The enzymes from Moraxella sp. C1, C. boidinii, and Arabidopsis thaliana showed similar thermostability profiles. The temperature dependence of the inactivation rate constant of A. thaliana FDH was studied. The data of differential scanning calorimetry was complied with the experimental results on the inactivation kinetics of these enzymes. Values of the melting heat were determined for all the enzymes studied.