Kinetic mechanism of Fo x F1 mitochondrial ATPase: Mg2+ requirement for Mg x ATP hydrolysis

The initial rates of ATP hydrolysis catalyzed by Fo x F1 (bovine heart submitochondrial particles) preincubated in the presence of Pi for complete activation of the oligomycin-sensitive ATPase were measured as a function of ATP, Mg2+, and Mg x ATP concentrations. The results suggest the mechanism in which Mg x ATP complex is the true substrate of the ATPase and the second Mg2+ bound at a specific pH-dependent site is needed for the catalysis. Simple hyperbolic Michaelis--Menten dependences of the reaction rate on the substrate (Mg x ATP) and activating Mg2+ were found. In contrast to the generally accepted view, no inhibition of ATPase by free Mg2+ was found. Inhibition of the reaction by free ATP is due to a decrease of free Mg2+ needed for the catalysis. In the presence of both Ca2+ and Mg2+ the kinetics of ATP hydrolysis suggest that the Ca x ATP complex is neither hydrolyzed nor competes with Mg x ATP, and free Ca2+ does not affect the hydrolysis of Mg x ATP complex. A crucial role of free Mg2+ in the time-dependent inhibition of ATPase by azide is shown. The dependence of apparent Km for Mg x ATP on saturation of the Mg2+-specific site suggests the formal ping-pong mechanism in which bound Mg2+ participates in the overall reaction after dissociation of one product (most likely Pi) thus promoting either release of ADP (catalytic turnover) or slow isomerization of the enzyme--product complex (formation of the dead-end ADP(Mg2+)-inhibited enzyme). The rate of Mg x ATP hydrolysis only slightly depends on pH at saturating Mg2+. In the presence of limited amounts of free Mg2+ the pH dependence of the initial rate corresponds to the titration of a single group with pKa = 7.5. The simple competition between H+ and activating Mg2+ was observed. The specific role of Mg2+ as a coupling cation for energy transduction in Fo x F1-ATPase is discussed.     

Comparative effectiveness of antioxidant melatonin and radioprotectors indralin and phenylephrine in local radiation injuries

In experiments on mice radioprotective properties of indraline, phenyleprine and melatonin were compared at topical application as an ointment at a place of local exposure of animal hind to a dose of 38.3 Gy of 60Co gamma-quanta. Factor of dose reduction was 1.27-1.32 for indraline (1-10% ointment) and 1.29 for phenyleprine (0.25% ointment). Antioxidants were low efficient at radiation skin burn. At later local radiation injuries, such as hind contracture, the efficiency of indraline was 1.33-1.5, that of phenyleprine was 1.28, and that of melatonine (2 and 5% ointment) was 1.23-1.47.     

Crystal structure of the YffB protein from <Emphasis Type="Italic">Pseudomonas aeruginosa</Emphasis> suggests a glutathione-dependent thiol reductase function

Background  

The yffB (PA3664) gene of Pseudomonas aeruginosa encodes an uncharacterized protein of 13 kDa molecular weight with a marginal sequence similarity to arsenate reductase from Escherichia coli. The crystal structure determination of YffB was undertaken as part of a structural genomics effort in order to assist with the functional assignment of the protein.     

Insertional inactivation of genes encoding eukaryotic type serine/threonine protein kinases in cyanobacterium Synechocystis sp. PCC 6803

Synechocystis sp. PCC 6803 mutants, in which one of the eukaryotic-type serine/threonine protein kinase genes pknD, pknE, pknG, and pknH was inactivated, were obtained by insertion mutagenesis. None of these mutants differed phenotypically from the wild-type strain, indicating that the pknD, pknE, pknG, and pknH genes are not of crucial importance for the photoautotrophically grown cyanobacterium. Mutant with the inactivated pknE gene was resistant to L-methionine-D,L-sulfoximine and especially to methylamine. The resistance was neither due to the impaired transport of these compounds nor to the inhibition of the production of toxic gamma-glutamylmethylamide from methylamine. The data presented suggest that resistance to methylamine may be associated with alterations in the regulation of the glutamine synthetase system and that the PknE protein kinase may be involved in the regulation of nitrogen metabolism in the cyanobacterium studied.     

Localization of cohesin complexes of polytene chromosomes of Drosophila melanogaster located on interbands

The distribution of cohesin complex in polytene chromosomes of Drosophila melanogaster was studied. Cohesin is a complicated protein complex which is regulated by the DRAD21 subunit. Using immunostaining for DRAD21p, the cohesins were shown to be preferentially located in the interband regions. This specificity was not characteristic for puffs, where uniform staining was observed. The presence of a few brightly fluorescent regions (five to ten per chromosome arm) enriched with cohesin complexes was shown. Some of these regions had permanent location, and the others, variable location. No antibody binding was detected in the chromocenter. Immunostaining of interphase nuclei of neuroblasts revealed large cohesin formations. On the polytene chromosomes of D. melanogaster, the Drad21 gene was mapped to the chromocentric region (81) of the L arm of chromosome 3.     

Molecular mechanisms of microheterogeneity-induced defect formation in ferritin crystallization

We apply in situ atomic force microscopy to the crystallization of ferritins from solutions containing approximately 5% (w/w) of their inherent molecular dimers. Molecular resolution imaging shows that the dimers consist of two bound monomers. The constituent monomers are likely partially denatured, resulting in increased hydrophobicity of the dimer surface. Correspondingly, the dimers strongly adsorb on the crystal surface. The adsorbed dimers hinder step growth and on incorporation by the crystal initiate stacks of up to 10 triple and single vacancies in the subsequent crystal layers. The molecules around the vacancies are shifted by approximately 0.1 molecular dimensions from their crystallographic positions. The shifts strain the lattice and, as a consequence, at crystal sizes > 200 microm, the accumulated strain is resolved by a plastic deformation whereupon the crystal breaks into mosaic blocks 20-50 microm in size. The critical size for the onset of mosaicity is similar for ferritin and apoferritin and close to the value for a third protein, lysozyme; it also agrees with theoretical predictions. Trapped microcrystals in ferritin and apoferritin induce strain with a characteristic length scale equal to that of a single point defect, and, as a consequence, trapping does not contribute to the mosaicity. The sequence of undesired phenomena that include heterogeneity generation, adsorption, incorporation, and the resulting lattice strain and mosaicity in this and other proteins systems, could be avoided by improved methods to separate similar proteins species (microheterogeneity) or by increasing the biochemical stability of the macromolecules against oligomerization.     

The HI0073/HI0074 protein pair from Haemophilus influenzae is a member of a new nucleotidyltransferase family: structure,sequence analyses,and solution studies

The crystal structure of HI0074 from Haemophilus influenzae, a protein of unknown function, has been determined at a resolution of 2.4 A. The molecules form an up-down, four-helix bundle, and associate into homodimers. The fold is most closely related to the substrate-binding domain of KNTase, yet the amino acid sequences of the two proteins exhibit no significant homology. Sequence analyses of completely and incompletely sequenced genomes reveal that the two adjacent genes, HI0074 and HI0073, and their close relatives comprise a new family of nucleotidyltransferases, with 15 members at the time of writing. The analyses also indicate that this is one of eight families of a large nucleotidyltransferase superfamily, whose members were identified based on the proximity of the nucleotide- and substrate-binding domains on the respective genomes. Both HI0073 and HI0074 were annotated "hypothetical" in the original genome sequencing publication. HI0073 was cloned, expressed, and purified, and was shown to form a complex with HI0074 by polyacrylamide gel electrophoresis under nondenaturing conditions, analytic size exclusion chromatography, and dynamic light scattering. Double- and single-stranded DNA binding assays showed no evidence of DNA binding to HI0074 or to HI0073/HI0074 complex despite the suggestive shape of the putative binding cleft formed by the HI0074 dimer.     

Fibrillar actin promotes assembly of dissociated 20S-proteasome complex

20S-proteasome was purified from rat liver cells by ultracentrifugation, gel-chromatography and ultrafiltration. The ability of 20S-proteasome to interact with fibrillar actin (F-actin) was examined by rapid cosidementation of these dissociated particles with F-actin in isokinetic sucrose gradient. Proteasomes, which were dissociated with Zn2+ ions, can be assembled on the fibrillar actin once again (with the exception of protein 27 kDa) at deleting ions Zn2+ from the solution with the help of EDTA.     

ND3, ND1 and 39 kDa subunits are more exposed in the de-active form of bovine mitochondrial complex I

An intriguing feature of mitochondrial complex I from several species is the so-called A/D transition, whereby the idle enzyme spontaneously converts from the active (A) form to the de-active (D) form. The A/D transition plays an important role in tissue response to the lack of oxygen and hypoxic deactivation of the enzyme is one of the key regulatory events that occur in mitochondria during ischaemia. We demonstrate for the first time that the A/D conformational change of complex I does not affect the macromolecular organisation of supercomplexes in vitro as revealed by two types of native electrophoresis. Cysteine 39 of the mitochondrially-encoded ND3 subunit is known to become exposed upon de-activation. Here we show that even if complex I is a constituent of the I + III₂ + IV (S₁) supercomplex, cysteine 39 is accessible for chemical modification in only the D-form. Using lysine-specific fluorescent labelling and a DIGE-like approach we further identified two new subunits involved in structural rearrangements during the A/D transition: ND1 (MT-ND1) and 39 kDa (NDUFA9). These results clearly show that structural rearrangements during de-activation of complex I include several subunits located at the junction between hydrophilic and hydrophobic domains, in the region of the quinone binding site. De-activation of mitochondrial complex I results in concerted structural rearrangement of membrane subunits which leads to the disruption of the sealed quinone chamber required for catalytic turnover.     

Cold shock domain proteins in the extremophyte <Emphasis Type="Italic">Thellungiella salsuginea</Emphasis> (salt cress): Gene structure and differential response to cold

Four genes encoding cold shock domain (CSD) proteins have been identified in salt cress Thellungiella salsuginea (halophila), an extremophyte currently recognized as a promising model for studying stress tolerance]. The deduced proteins prove highly homologous to those of Arabidopsis thaliana (up to 95% identity) and are accordingly enumerated TsCSDP1-TsCSDP4; after the N-proximal conserved CSD, they have respectively 6, 2, 7, and 2 zinc finger motifs evenly spaced by Gly-rich stretches. Much lower similarity (∼45%) is observed in the regions upstream of TATA-box promoters of TsCSDP1 vs. AtCSP1, with numerous distinctions in the sets of identifiable cis-regulatory elements. Plasmid expression of TsCSDP1 (like AtCSP1/3) rescues a cold-sensitive csp-lacking mutant of Escherichia coli, confirming that the protein is functional. In leaves of salt cress plants under normal conditions, the mRNA levels for the four TsCSDPs relate as 10: 27: 1: 31. Chilling to 4°C markedly alters the gene expression; the 4-day dynamics are different for all four genes and quite dissimilar from those reported for their Arabidopsis homologous under comparable conditions. Thus, the much greater cold hardiness of Thellungiella vs. Arabidopsis cannot be explained by structural distinctions of its CSDPs, but rather may be due to expedient regulation of their expression at low temperature.