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V M Tishchenko

Publications and source records attributed to V M Tishchenko.

16 recordsLinked to original sources

Human immunoglobulin light chains lambda form amyloid fibrils and granular aggregates in solution.

Myeloma nephropathy is a disorder characterized by deposition of monoclonal immunoglobulin light chains in the kidneys. The chains deposited form either amyloid fibrils or granular (amorphous) aggregates. Distinct molecular mechanisms leading to the formation of different aggregate types in kidney of patients with multiple myeloma are poorly understood. Here we describe the self-association kinetics of human monoclonal immunoglobulin light chains lambda (GRY) isolated from urine of a patient with multiple myeloma. Under physiological conditions, the isolated light chain exists predominantly in a form of covalent dimer with apparent molecular mass of 50.1 kD. Spectral probe binding, analytical gel filtration, Western blot analysis, and electron microscopy indicate that GRY dimer aggregation occurs via two different pathways producing either amyloid fibrils or amorphous aggregates depending on microenvironment. Incubation of GRY (25 microM) for 4-14 days at 37 degrees C in phosphate buffered saline (PBS), pH 7.0, or in PBS containing urea (0.8 M), pH 6.5, leads to amyloid fibril formation. Under electron microscopy, the fibrils show unbranched thread-like structures, approximately 60-80 x 1000 A in size, which can bind thioflavin T and Congo Red. GRY maintained in acetate buffer, pH 3.5, forms granular aggregates. The structure of GRY oligomers formed during the early stage of amyloid fibril formation (1-4 days) has been examined by means of protein cross-linking with homobifunctional reagents. These oligomers are predominantly trimers and tetramers.

Amyloid↗

[Effect of immunoglobulin G1 Pro 290 residue on structural and biological characteristics of its SH2 domain].

Tryptic hydrolysis of only one of 11 studied Fc fragments of human myeloma immunoglobulins G1 (IgG1) provided an intact CH2 domain in a high yield (up to 40% as opposed to 2-3% for other IgG1s). The only structural difference of this domain was shown to be the substitution of Pro for Lys290. This decreased the capacity of the IgG1 Sem Fc fragment to interact with proteins of the complement system.

Amino Acid Substitution↗

Mechanisms of generation of antibody diversity as a cause for natural selection of homoiothermal animals in the process of evolution.

The temperature-dependence of domain interactions in the Fab, Fc, Fb and Fv fragments from human myeloma immunoglobulins (IgG) samples was investigated by scanning microcalorimetry, NMR and difference spectroscopy. The fragments were found to be very sensitive to temperature changes. Lowering the temperature below the physiological value (37 degrees C) considerably decreases the energy of interaction of the variable VH and VL domains, resulting at times in their dissociation. Since the association energies of VH and VL pairs can be affected by the result of somatic recombination and mutation events affecting antibody genes, immunoglobulins can fortuitously acquire the properties of cryoglobulins or cold autoantibodies and induce severe pathological states. It is postulated that this property of immunoglobulins, and by extension, of T-cell antigen receptors might have been one of the causes for the possible natural selection of homoiothermal animals. In these, the high conformational sensitivity of immunoglobulins to temperature change may be important in the mechanisms of induction of secondary functions in immune responses.

Antibody Diversity↗

[Isolation of the fifth IgG-binding domain from Staphylococcus aureus protein A].

Using affinity chromatography on IgG-Sepharose at pH 5.0, a new fragment capable of binding to IgG (domain E) was isolated from trypsin hydrolysate of protein A. Trypsinolysis of protein A was performed at low temperatures. Thus, the intact structure of protein A was found to include six domains, of which five interact with IgG.

Amino Acids↗

[Interaction between CH2 and CH3-domains of human immunoglobulin G1 in glycosylated and aglycosylated Fc-fragments].

Heat denaturation of glycosylated and aglycosylated human immunoglobulin G1 Fc fragments was investigated by differential scanning microcalorimetry. The enthalpy of the interaction between aglycosylated CH2 and CH3 domains is significantly reduced at 37 degrees C (but not at 0 degree C) as compared to the glycosylated form. The temperature dependence is consistent with the data on restricted proteolysis by trypsin.

Calorimetry, Differential Scanning↗

[Domain structure of CAF1M protein from Yersinia pestis. Structure and the role of various domains].

The cooperative structure of Caf1M from Yersinia pestis was studied using scanning microcalorimetry, fluorescence, and limited proteolysis. It was shown that, in Caf1M-Hg (a derivative in which the disulfide bond is replaced by an S-Hg-S bond), the first to melt is the N-domain. Then the C-domain melts. After renaturation in a buffer with a low NaCl concentration, only the C-domain is in the native state, and it can be obtained by limited proteolysis. After renaturation in a buffer with a high NaCl concentration, only the N-domain is in the native state, and it can be obtained by limited proteolysis. Both domains have native structure; however, only the N-domain interacts with Cafl (natural substrate for Caf1M).

Bacterial Proteins↗

[Hydrodynamic parameters of native C-reactive protein molecule in a solution].

The hydrodynamic properties of the C-reactive protein in solution (pH 6.8) were studied using quasi-elastic light scattering and size-exclusion liquid chromatography. It was shown that the solution containing the C-reactive protein represents a polydisperse system. The values of the translation diffusion coefficient and the apparent molecular weight of the C-reactive protein in solution at pH 6.8 were determined. The values of the translation diffusion coefficient, molecular weight and the hydration radius obtained suggest that the native pentameric C-reactive protein is the major form of the protein in solution at pH 6.8.

C-Reactive Protein↗

[Aggregation of C-reactive protein in solutions at acid pH].

The hydrodynamic properties of the C-reactive protein (CRP) at different pH were studied using quasi-elastic light scattering, size-exclusion liquid chromatography, and nonreducing gel electrophoresis. It was shown that a CRP solution at pH 5.0-7.2 presents a polydisperse system the major component of which is the native pentameric CRP. At pH 4.0-4.5, CRP exists in two states having different hydrodynamic properties: the native pentameric form with a molecular mass of 120 kDa and with the hydrodynamic radius of 4.03 nm and high-molecular-weight aggregates with a wide range of their molecular weight distribution. The interaction of the C-reactive protein with monoclonal antibodies to it indicates that conformation-dependent surface epitopes of the protein lose the native structure at pH 5.0-5.5. The aggregation of CRP is an irreversible process, which begins in a narrow pH range of pH 5.0-4.5 and is not accompanied by the dissociation into subunits but is determined by intermolecular interactions of its quasi-native pentamers.

Acids↗

[Effect of Ca2+ ions on hydrodynamic properties of pentamer and decamer of C-reactive protein in solution].

The molecular mass and sedimentation coefficient of native C-reactive protein in solution were determined by analytical ultracentrifugation in the presence and absence of calcium ions. Pentameric C-reactive protein was shown to be the major macroscopic form of this protein in solution. The removal of calcium ions from solution caused decompaction of the protein accompanied by changes in its hydrodynamic parameters. The sedimentation coefficient s20(0), w of pentameric C-reactive protein in solution containing 2 mM--Ca2+ (6.6S) exceeded that for C-reactive protein in solution containing 2 mM EDTA (6.4S). Analysis of average molecular masses Mw and Mz obtained from sedimentation data demonstrated that the solution of highly purified protein was not homogeneous. As shown by intermolecular crosslinking, the solution also contained the 241-kDa decamer of C-reactive protein (9.5S) as a separate macroscopic form, whose share hardly reached 10% in the presence of 2 mM Ca2+ and increased after removal of calcium ions. The decamers were shown to result from intermolecular association of the pentamers.

C-Reactive Protein↗

[Number of hydrogen bonds in the structure of collagen].

The kinetics of hydrogen exchange of collagens from different animals was studied by the radioisotopic method (tritium) and infrared spectroscopy (deuterium). It has been shown that collagens from different animals (rat, pike, cod, carp, frogs) differ in amino acid composition and thermostability but are similar in the amount of slowly exchanged hydrogens. All the studied collagens have (1.00 +/- 0.05) very slowly exchanged hydrogens per triplet and (0.6 +/- 0.1) slowly exchanged hydrogens per triplet. Identifying the quantity of slowly exchanged hydrogens with the quantity of hydrogen bonds in the macromolecule, it can be concluded that collagens differing in stability do not differ by the quantity and composition of intramolecular hydrogen bonds.

Animals↗

[Calorimetric study of heat denaturation of beta-trypsin].

Thermal denaturation of beta-trypsin was studied by scanning microcalorimetry. It is shown that the denaturational transition is well described by the one-step model of transition between two macroscopic states. The specific enthalpy of denaturation is a linear function of temperature, the extrapolation to 110 degrees leading to a value of 54.2 J/g, i. e. to the value being characteristic for small compact globular proteins.

Calorimetry, Differential Scanning↗

[Quaternary structure of butyrylcholinesterase from horse blood serum].

The molecular weight of butyrylcholinesterase from horse blood serum was determined using the method of analytical ultracentrifugation. The molecular weight of the enzyme subunit was also determined by its mobility in gel containing sodium dodecyl sulphate. The number of active sites was determined by titrating butyrylcholinesterase with a specific inhibitor, namely di-isopropyl phosphorofluoridate. A conclusion is made that butyrylcholinesterase is a tetramer with one active site per unit.

Animals↗

[Theoretical and experimental studies on the conformation of the hinge regions in human immunoglobulin G subclasses].

Using the methods of difference adiabatic scanning microcalorimetry and difference thermal perturbation spectrophotometry it was established that both the Fc subunits within the intact immunoglobulins G of different subclasses, and the Fc fragments corresponding to them significantly differ in conformational properties. These differences are associated with a different energy of interactions between the CH2 and CH3 domains and also with a different rigidity of structure of the N- and C-terminal parts of the CH2 domains. The analysis of these data allowed to suggest that the "hinge region" interacts with the CH2 domains and the difference in the structure of this region affect the conformation of the Fc subunits. Possible models of the "hinge regions" in immunoglobulins G of the first, second and fourth subclasses, and also their arrangement relative to the CH2 domains were proposed. The analysis of curves of the small-angle scattering of X-rays shows an agreement between the theoretical models and experimental data.

Calorimetry, Differential Scanning↗