[application of the analytical transmission electron microscopy techniques for detection, identification and visualization of localization of nanoparticles of titanium and cerium oxides in mammalian cells].

This work represents the results of the study on applicability of the modern methods of analytical transmission electron microscopy for detection, identification and visualization of localization of nanoparticles of titanium and cerium oxides in A549 cell, human lung adenocarcinoma cell line. A comparative analysis of images of the nanoparticles in the cells obtained in the bright field mode of transmission electron microscopy, under dark-field scanning transmission electron microscopy and high-angle annular dark field scanning transmission electron was performed. For identification of nanoparticles in the cells the analytical techniques, energy-dispersive X-ray spectroscopy and electron energy loss spectroscopy, were compared when used in the mode of obtaining energy spectrum from different particles and element mapping. It was shown that the method for electron tomography is applicable to confirm that nanoparticles are localized in the sample but not coated by contamination. The possibilities and fields of utilizing different techniques for analytical transmission electron microscopy for detection, visualization and identification of nanoparticles in the biological samples are discussed.

[The non-functioning chaperonin GroEL stimulates protein aggregation].

To clarify the role of chaperones in the development of amyloid diseases, the interaction of the chaperonin GroEL with misfolded proteins and recombinant prions has been studied. The efficiency of the chaperonin-assisted folding of denatured glyceraldehyde-3-phosphate dehydrogenase (GAPDH) was shown to decrease in the presence of prions. Prions are capable of binding to GroEL immobilized on Sepharose, but this does not prevent the interaction between GroEL and other denatured proteins. The sizes of individual proteins (GroEL, GAPDH, and the recombinant prion), as well as aggregates formed after their mixing, were determined by the dynamic light scattering method. It was shown that at 25 degrees C the non-functioning chaperonin (equimolar mixture of GroEL and GroES in the absence of Mg-ATP) bound prion yielding large aggregates (greater than 400 nm). The addition of Mg-ATP decreased significantly the aggregate size to 70-80 nm. On the blocking of one of the chaperonin centers by oxidized denatured GAPDH, the aggregate size increased to 1200 nm, and the addition of Mg-ATP did not prevent the aggregation. These data indicate the significant role of chaperonins in the formation of amyloid structures and demonstrate the acceleration of aggregation in the presence of functionally inactive chaperonins. The suggested model can be used for the analysis of the efficiency of antiaggregants in the system containing chaperonins.

[Coimmobilization of lactate dehydrogenase and low molecular weight thiols on sepharose]. / Sovmestnaia immobilizatsiia laktatdegidrogenazy i nizkomolekuliarnykh tiolov na sefaroze.

Lactate dehydrogenase (EC 1.1.1.27) and dithiothreitol (DTT) were coimmobilized on Sepharose activated with cyanogen bromide. It was demonstrated that addition of 10 mM DTT (but not 2-mercaptoethanol) during immobilization increased the enzyme specific activity 1.5-5-fold, depending on the initial extent of Sepharose activation by cyanogen bromide. The total activity increased two- to threefold. The lactate dehydrogenase preparations were rich in matrix-immobilized sulfhydryl groups (1.8-13.0 nmol per ml gel). The presence of DTT increased the stability of immobilized lactate dehydrogenase.

[Effect of erythrocyte membranes and tubulin on the activity of NAD-dependent dehydrogenases]. / Vliianie membran éritrotsitov i tubulina na aktivnost' NAD-zavisimykh degidrogenaz.

Interactions of NAD-dependent dehydrogenases (glyceraldehyde-3-phosphate dehydrogenase, GAPDH, and lactate dehydrogenase, LDH) with band 3 erythrocyte membrane protein and tubulin were characterized. At low ionic strength and un-saturating substrate concentrations, LDH tightly binds to tubulin and is thus inactivated. The Kd of the LDH-tubulin complex was calculated in inhibition and direct binding experiments (15.0 and 13.6 nM, respectively); the stoichiometry of the complex was 1.66 moles of tubulin dimer bound per mole of LDH tetramer. In the presence of 0.15 M NaCl, LDH does not bind to tubulin and tubulin-dependent inhibition of LDH activity is not detected. At low ionic strength, erythrocyte membranes affect both dehydrogenases similarly. GAPDH activity is completely inhibited by excess of erythrocyte membranes (or by excess of cytoplasmic fragment of band 3 protein). Under similar conditions, LDH activity was inhibited by 70% by erythrocyte membranes. In these experiments, 14.8.10(6) GAPDH tetramers or 25.6.10(6) LDH tetramers bound to one erythrocyte ghost (Kd is 0.13 and 0.6 microM, respectively). Increase in ionic strength (0.15 m NaCl) completely abolished the membrane-dependent inhibition of dehydrogenases; however, membranes still bound GAPDH and LDH. Under these conditions, the Kd for GAPDH was increased (up to 4.43 microM), whereas the number of membrane-bound enzyme molecules has not been significantly affected (0.75 nmoles of tetramer per 100 micrograms membrane protein). The Kd for LDH was not changed (0.76 microM), whereas the number of membrane-bound enzyme molecules was decreased (down to 0.48 nmoles of tetramer per 100 micrograms membrane protein). It is suggested that at low ionic strength, the "acidic tails" of band 3 protein and tubulin can interact with positively charged NAD-binding domains of both dehydrogenases thus inhibiting their activity. Increase in ionic strength reduces these interactions, decreasing the binding and inhibition of enzyme activities. At "physiological" ionic strength, catalytically active GAPDH and LDH can possibly bind to various sites of the erythrocyte membrane. This can be important in regulation of the transfer of the common cofactor (NAD/NADH) between their active sites.

[Glycolytic enzymes in human erythrocytes: association of glyceraldehyde-3-phosphate dehydrogenase with 3-phosphoglycerate kinase]. / Glikoliticheskie fermenty v éritrotsitakh cheloveka: assotsiatsiia glitseral'degid-3-fosfatdegidrogenazy s 3-fosfoglitseratkinazoi.

The ability of glyceraldehyde-3-phosphate dehydrogenase (GAPD) to associate with 3-phosphoglycerate kinase (3-PGK) in human erythrocytes has been studied. It was found that a stable GAPD-3-PGK complex can be isolated from human erythrocyte hemolysates using immobilized monoclonal antibodies that are specific for GAPD. The complex does not dissociate at high ionic strength (up to 0.3 M NaCl) but is decomposed in the presence of specific ligands interacting with GAPD and 3-PGK, e.g., 1,3-diphosphoglycerate. The interaction between GAPD and 3-PGK isolated from human erythrocytes was investigated. To assess the binding parameters, immobilized GAPD and soluble 3-PGK from erythrocytes were used. About 2.3 moles of monomeric 3-PGK (Kd = 2.4 microM) were bound per mole of the immobilized tetramer of GAPD. Under these conditions the rabbit muscle enzymes form more weak (Kd = 3.8 microM), whereas the yeast enzyme--more stable complexes (Kd = 1.5 microM). No such complexes were detected when the enzyme pairs were isolated from phylogenetically distant sources, such as yeast and mammalian tissues. The species specificity of binding of the two enzymes and possible causes of formation of such stable complexes in erythrocyte lysate are discussed.

[Phosphorylation of glyceraldehyde-3-phosphate dehydrogenase]. / Fosforilirovanie glitseral'degid-3-fosfat degidrogenazy.

Phosphorylation of D-glyceraldehyde-3-phosphate dehydrogenase (GPDH) by Ca2+/phospholipid- and Ca2+/calmodulin-dependent protein kinases was shown to take place in rabbit skeletal muscle and brain extracts. The kinases could be "picked up" from the extract, using GPDH immobilized on CNBr-activated Sepharose 4B as an affinity adsorbent. Washing of the column with GPDH solutions resulted in elution of the protein kinases; the same effect was observed when anti-GPDH antibodies were used. The most effective elution took place under the conditions favouring the dissociation of the immobilized GPDH into dimers. Based on these findings, a method for purification of Ca2+/calmodulin-dependent protein kinase has been elaborated, which includes chromatography on phenyl-Sepharose to separate the kinase from GPDH. The susceptibility of GPDH to phosphorylation by tissue protein kinases was confirmed by analyses of GPDH preparations purified from rabbit muscle for endogenous phosphate content: 0.7-1.5 moles of covalently bound phosphate were found per mole of the enzyme.

[The D-glyceraldehyde-3-phosphate dehydrogenase and 3-phosphoglycerate kinase complex from rabbit skeletal muscles]. / Kompleks mezhdu D-glitseral'degid-3-fosfatdegidrogenazoi i 3-fosfoglitseratkinazoi iz skeletnykh myshts krolika.

The experimental conditions favouring the association of Sepharose-bound D-glyceraldehyde-3-phosphate dehydrogenase with soluble 3-phosphoglycerate kinase were studied. Acylation of D-glyceraldehyde-3-phosphate dehydrogenase by 1.3-bisphosphoglycerate was found to be a prerequisite for the complex formation.

[Immobilized active monomers of D-glyceraldehyde-3-phosphate dehydrogenase from rabbit skeletal muscles and their coenzyme-binding properties]. / Immobilizovannye aktivnye monomery D-glitseral'degid-3-fosfatdegidrogenazy iz skeletnykh myshts krolika i ikh koénzimsviazyvaiushchie svoistva.

Experimental conditions favouring the dissociation of tetrameric rabbit muscle D-glyceraldehyde-3-phosphate dehydrogenase into active monomers were elaborated. The urea-induced dissociation of the tetramer was shown to be a stepwise process (in 2 M urea only dimers are formed; an increase in urea concentration up to 3 M causes the splitting of the dimers into monomers). The specific activity of immobilized monomers in the glyceraldehyde-3-phosphate oxidation reaction does not differ from that of the parent immobilized tetrameric form. The tetrameric enzyme molecule binds the coenzyme with a negative cooperativity (the first two NAD+ molecules bind with KD below 0.1 microM; for the third and fourth molecules the dissociation constant was determined to be equal to 5.5 +/- 1.5 microM (50 mM medinal buffer, 10 mM sodium phosphate, pH 8.2). The cooperativity of NAD+ binding is preserved in the immobilized preparation of tetrameric dehydrogenase. The immobilized monomers bind NAD+ with KD of 1.6 +/- 1.0 microM. The experimental results are consistent with the hypothesis according to which the association of catalytically active subunits into a tetramer changes their coenzyme-binding properties in such a way that the first two NAD+ molecules bind more firmly to a tetramer than to a monomer, whereas the third and the fourth NAD+ molecules bind less firmly.

[Use of immobilization in the study of glyceraldehyde 3-phosphate dehydrogenase. Immobilized monomers]. / Ispol'zovanie immobilizatsii dlia izucheniia glitseral'degid-3-fostfat-degidrogenazy. Immobilizirovannye monomery.

Active immobilized monomers of glyceraldehyde 3-phosphate dehydrogenase were prepared by means of dissociation of the tetrameric enzyme molecule covalently bound to Sepharose via a single subunit. The conditions were elaborated to achieve the inactivation and solubilization of the non-covalently bound subunits leaving the monomer coupled to the matrix intact. This procedure differs from the previously developed method of matrix-bound oligomeric enzymes dissociation in a detail which was found to be essentially important. The widely used method includes complete denaturation of all subunits during treatment with urea followed by reactivation of the immobilized one, whereas only the non-covalently bound subunits suffer denaturation under the conditions developed in the present work. The immobilized monomers of glyceraldehyde 3-phosphate dehydrogenase exhibit Vmax and Km (for NAD and substrate) values similar to those found for the immobilized tetramer. Reassociation of the immobilized monomers with soluble enzyme subunits obtained in the presence of urea produces matrix-bound tetrameric species. Immobilized trimers ae formed upon incubation of matrix-bound monomers in a diluted apoenzyme solution. The immobilized monomeric, trimeric and tetrameric enzyme species were used to study the role of subunit interactions in cooperative phenomena exhibited by the dehydrogenase.

[Effect of specific antibodies on association of glyceraldehyde 3-phosphate dehydrogenase subunits]. / Vliianie spetsificheskikh antitel na assotsistsiiu sub"edinits glitseral'degid-3-fosfatdegidrogenazy.

The rabbit antibodies against glyceraldehyde 3-phosphate dehydrogenase (EC 1.2.1.12) from baker's yeast or rat muscle are strictly specific to the corresponding antigens and are not involved in the cross reaction. The interaction of specific antibodies with Fab-fragments does not affect the activity of the yeast enzyme which is in agreement with the previous data on the rat muscle enzyme. The antibodies against yeast dehydrogenase immobilized on BrCN-activated Sepharose were used for the preparation of the enzyme linked to the matrix by the antigen--antibody complex. The tetrameric enzyme molecule thus immobilized completely retains its activity and the ability to dissociate into dimers in the cold in the presence of ATP. The dimer which remains bound within the antigen--antibody complex retains its activity in the presence of agents causing inactivation and release of the second dimer into solution. The tetramer of yeast dehydrogenase covalently bound to the BrCN-activated Sepharose by one subunit is completely stable under conditions causing dissociation, when it exists in the complex with three molecules of specific antibodies. The binding of four molecules of Fab-fragments of specific antibodies per molecule of tetramer protects the rat muscle apoenzyme against anion-dependent cold inactivation and thermal inactivation in solution. The binding of two antibody molecules specific to rat muscle glyceraldehyde 3-phosphate dehydrogenase to the hybrid tetramer made up of yeast dehydrogenase dimer covalently linked to Sepharose and of rat muscle dehydrogenase dimer, prevents the dissociation of the tetramer (i.e. release of the "muscle" type dimer into solution). Gel filtration through Sepharose 6B of the apo-glyceraldehyde phosphate dehydrogenase complex with the Fab-fragments of specific antibodies demonstrated that the binding of the Fab-fragments shifts the tetramer in equilibrium or formed from dimers equilibrium in the apoenzyme solution towards the tetramer. It is concluded that the specific antibodies increase the intersubunit interactions in the oligomer by stabilizing the native tertiary structure of individual subunits.

[Use of immobilization for the study of glyceraldehyde 3-phosphate dehydrogenase. Immobilized dimers of the enzyme]. / Ispol'zovanie immobilizatsii dlia izucheniia glitseral'degid-3-fosfatdegidrogenazy. Immobilizovannye dimery fermenta.

The immobilized dimers of glyceraldehyde 3-phosphate dehydrogenase have been obtained after dissociation of the tetrameric enzyme molecule linked by one of the subunits with Sepharose 4B. The catalytic parameters (V, Km and pH-dependence of activity) of the immobilized dimers and tetramers of the enzyme are identical. The immobilized dimers are capable of reassociating with the enzyme subunits from solution and possess a higher stability a compared to soluble dehydrogenase. The type of the dimer interaction with the reagents causing the "half-of-the-site reactivity" effect suggests that the immobilized dimers of the yeast and rat skeletal muscle enzymes possess the catalytic activity and, besides, are capable to express cooperative interactions between the active centers of the both subunits. The immobilized dimers of yeast dehydrogenase are able to form hybrids with soluble modified dimers of the yeast enzyme and with those from other sources. A procedure for the enzyme isolation from tissue extracts based on hybridization between the immobilized and soluble dimers of homologous dehydrogenases have been developed.

[Use of immobilization for investigation of glyceraldehyde 3-phosphate dehydrogenase. Immobilized tetramers]. / Ispol'zovanie immobilizatsii dlia izucheniia glitseral'degid-3-fosfatdegidrogenazy. Immobilizovannye tetramery.

Glyceraldehyde-3-phosphate dehydrogenase from yeast and rat skeletal muscle was covalently linked to CNBr-activated Sepharose 4B. When the activation was as high as 4-5 mg of CNBr per ml of Sepharose, the enzymes had their maximal activity and were linked to the carrier only by one of the four subunits. The specific activity of immobilized dehydrogenases makes up to 50-60% of that of soluble preparations, since the rate of the substrate diffusion into Sepharose granules is too low. The Km values for NAD and substrate and the pH dependence of the immobilized enzymes were determined. It was found that the enzymes used as adsorbents for isolation of specific antibodies reveal their maximal activity when CNBr concentration reaches 150-200 mg per ml of gel. No inhibition of activity of the immobilized dehydrogenase from yeast or stabilizing effect of antibodies on the enzyme structure were observed.

[Immobilization of glyceraldehyde-3-phosphate dehydrogenase by non-covalent binding to specific antibodies and Fab-fragments coupled to Sepharose]. / Nekovalentnaia immobilizatsiia glitseral'degid-3-fosfatdegidrogenazy na antitelakh i Fab-fragentakh antitel, sviazannykh s sefarozoi.

Rabbit antibodies to rat skeletal muscle glyceraldehyde-3-phosphate dehydrogenase, as well as monovalent Fab fragments of these antibodies were coupled to CNBr-activated Sepharose 4B. Rat skeletal muscle glyceraldehyde-3-phosphate dehydrogenase was then immobilized on a matrix by non-covalent binding to specific antibodies. Immobilized enzyme retains approximately 90% catalytic activity of the soluble dehydrogenase; pH optimum of activity and the Km value observed are changed as compared to the enzyme in solution. Glyceraldehyde-3-phosphate dehydrogenase immobilized on specific antibodies is shown to undergo adenine nucleotide-induced dissociation into dimers. The immobilized dimeric form of the enzyme thus obtained is catalytically active and capable of reassociating with the dimers of apoglyceraldehyde-3-phosphate dehydrogenase added in solution to the suspension of Sepharose.

[Enzymatic splitting of antibodies bound to immobilized antigen as a means of Fab fragments preparation]. / Fermentativnoe rasshcheplenie antitel, sviazannykh s immobilizovannym antigenom, kak sposob polucheniia Fab-fragmentov

A method of Fab fragments preparation by enzymatic splitting of antibodies bound to specific antigen immobilized on an insoluble support is described. The complex of rat muscle glyceraldehyde-3-phosphate dehydrogenase (GAPD), immobilized on Sepharose 4B, with anti-rat GAPD rabbit antibodies was digested with papain. The antigen was inaccessible to proteolysis under conditions employed. After 4 hrs of incubation with papain the antibody was completely split into non-precipitating fragments. The products of proteolysis not bound to Sepharose, were eluted with 0.1 M givcine buffer pH 2.5, and shown to correspond to Fab fragments.