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V M Mazhul'

Publications and source records attributed to V M Mazhul'.

At least 19 recordsLinked to original sources
PubMed2005

[Room temperature phosphorescence of amorphous aggregates and amyloid fibrils resulting from protein misfolding].

Using actin, alpha-lactalbumin and insulin as examples, it was shown that the formation of amorphous aggregates of proteins and amyloid fibrils leads to an increase in the rigidity of tryprophan and tyrosine residues micro-environment and, consequently, to the appearance of tryptophan (tyrosine) room temperature phosphorescence (RTP). RTP was used for examining a slow intramolecular mobility of native (G-, F-form) and inactivated (I) rabbit skeletal muscle actin during the process of GdnHCl induced protein unfolding. This method made it possible to confirm that an essentially unfolded intermediate precedes the formation of inactivated actin. It has been found that the kinetic intermediate generated at the early stage of protein denaturation has no tryptophan RTP, suggesting a high lability of its structure. Symbate changes of integral intensity (relative quantum yield) and the mean lifetime of RTP during the U*-->I transition suggest a gradual increase of the number of monomers incorporated in the associate (U*-->11...-->In...-->I15), which is accompanied by an increase of protein structural rigidity. The rate of inactivated actin formation (I-->I15) is shown to increase with the increase of protein concentration. It is shown that, no matter what method of inactivation was employed (1--2 M GdnHCl or 3.0-3.5 M urea, Ca2+ removal, incubation at 70 degrees C, refolding from completely unfolded state by dialysis from 8 M urea or 6 M GdnHCl), actin transition to the inactivated state is accompanied by a significant increase in both integral intensity and the mean lifetime of RTP, suggesting the rigid structure of inactivated actin. It is shown that the lifetime of inactivated actin RTP does not depend on GdnHCl concentration within the limits from 0 to 4 M. On using insulin and alpha-lactalbumin as examples, it is shown that RTP can be used in studies of fibrillogenesis and properties of amyloid fibrils.

Actins↗
PubMed1996

[The effect of cytoskeleton modification on the process of trypsin-induced cell aggregation].

Studies were performed on the influence of cytoskeleton modification (by colchicine, vinblastine, and cytochalasin B, and by neoplastic transformation) on cell aggregation induced by small trypsin concentrations (the final concentrations equal to 1-100 mg/ml) added to a suspension of chick embryo fibroblasts dispersed from a monolayer with EDTA. It was shown that alteration of microfilaments resulted in the loss of the ability of plasma membranes to structural rearrangements. It is suggested that aggregation of these cells is realized due to a gradual exposure of adhesive sites along with proteolytic splitting of protein components of plasma membranes.

Animals↗
PubMed1980

[Mechanisms of protease-induced cell aggregation. II. The effect of trypsin on the structural state of the plasma membrane of chick embryo fibroblasts].

Using freeze-etching, registration of kinetics of tryptophane phosphorescence quenching at room temperature, and measuring the cell electrophoretic mobility, a study has been made of the influence of a short-term treatment (5-10 min) with a low concentration of trypsin (2-100 microgram/ml) on the chick embryo fibroblasts removed from the monolayer with EDTA and suspended in the Henks solution. It is shown that trypsin induces a structural rearrangement of the plasma membrane which is manifested in clustering intramembrane globular particles. Trypsin-induced rearrangement of the plasma membrane is accompanied by an increase in the negative charge of the cell surface. The results obtained by the phosphorescence method show that structural rearrangements involve alongside with plasma membranes the majority of intracellular proteins.

Animals↗
PubMed1980

[Study of the mechanisms of protease-induced cell aggregation. I. Effect of trypsin on the aggregating capacity of chick embryo fibroblasts].

The administration of trypsin (1-100 mcg/ml) to the chick embryo fibroblast suspension, removed from the monolayer by means of EDTA, causes a pronounced aggregation of the cells. The aggregation completes within a few minutes and occurs in two steps. On the first step of the trypsin-induced aggregation, the amount of aggregating cells, grows rather slowly and monotonously, whereas on the second step, a sharp and non-linear acceleration of the aggregation process is observed. The data presented favour an assumption that the second stage of the fibroblast aggregation may be due to a structural rearrangement of cytoplasmic membranes that involves a splitting of a small, critical number of peptide bonds of cell surface proteins.

Animals↗
PubMed1978

[Effect of microorganism population density on protein metabolism].

The influence of population density of yeasts Candida utilis on the protein synthesis was investigated using 14C-L-leucine. After 15--30 minutes of exposure in 14C-L-leucine, the isotope incorporation into the protein was negligible at subcritical density of cell suspension (10(4)--5X10(5) cells/mm3), but then beginning from the 10(6) cells/mm3 density, a sharp activation of incorporation was observed. With increased incubation time up to 60--120 minutes for high density suspension (more than 10(6) cells/mm3), the degradation of newly-synthesized protein is the more the higher the cell concentrations and incubation time. The changes of protein synthesis intensity seem to be not associated with the influence of such traditional factors as nutrient medium exhaustion, toxicosis, change of hydrogen ion concentration and oxygen deficiency. It is suggested that structurally functional shifts in cells initiated by intercellular interactions may underlie the dependence of protein synthesis on the suspension culture density.

Aerobiosis↗
PubMedDate not supplied

[Phosphorescent analysis of lipid peroxidation products in liposomes].

It was found that lipid peroxidation products incorporated into liposomes prepared from oxidized preparations of bovine heart phosphatidylcholine and the total lipid fraction of human erythrocyte membranes are able to phosphoresce at room temperature was studied. The temperature dependences of kinetic and spectral parameters of phosphorescence were measured. It is shown that mechanism of phosphorescence quenching of lipid chromophores has a dynamic nature. It is proposed to use endogenic molecules of the lipid peroxidation products capable of phosphorescence as intrinsic phosphorescence probes for studying the slow molecular dynamics of lipids in artificial and biological membranes in a millisecond range.

Animals↗
PubMedDate not supplied

[Phosphorescent analysis of the intramolecular dynamics of the muscle glycogen phosphorylase b].

Large-scale functionally significant changes in the intramolecular dynamics of muscle glycogen phosphorylase b (EC 2.4.1.1) in solution upon ligand binding, transition from dimeric to tetrameric form, temperature denaturation and aggregation were registered at room temperature using the tryptophan phosphorescence technique. It was shown that binding of glucose-1-phosphate (substrate, 0.25-4 mM) and glucose (competitive inhibitor, 0.5-8 mM) to the active site and temperature-induced protein aggregation decrease large-scale structural fluctuations of the protein matrix at the level of domains and subunits; whereas the transition of glycogen phosphorylase b to tetrameric form (R-conformation) leads to a dramatic increase in the structural flexibility of the peripheral parts of the protein globule.

Adenosine Monophosphate↗
PubMedDate not supplied

[Phosphorescent analysis of lipid peroxidation products in isolated human erythrocyte membranes].

The room temperature phosphorescence of lipid peroxidation products in the composition of isolated human erythrocyte membranes was registered, and its kinetic parameters were determined. The excitation and emission spectra of phosphorescence of lipid peroxidation products in the composition of erythrocyte membranes at 0 degree C measured. The nature of lipid peroxidation products possessing the phosphorescencing capacity was discussed. Based on the analysis of temperature dependences of the intensity and lifetimes of phosphorescence of lipid peroxidation products in the range -2 divided by 26 degrees C, it is concluded that the deactivation of excited triplet states of lipid chromophores was realized by the dynamic type.

Erythrocyte Membrane↗
PubMedDate not supplied

[Intramolecular dynamics and functional activity of proteins].

The intramolecular dynamics of proteins in solution and in biological membranes and cells was analyzed by the method of tryptophan phosphorescence at room temperature. Changes in protein internal dynamics in solution upon varying the pH, ionic strength and temperature of solution, transition into the apoform, binding of substrates, allosteric activator, and inhibitors, upon limited proteolysis, thermoinactivation, association, and refolding were demonstrated. The functional significance of low-frequency equilibrium fluctuations of the protein structure in enzyme catalysis was shown. A new phosphorescence method for investigation of millisecond internal dynamics of membrane proteins was developed. Functionally significant shifts of the internal dynamics of membrane proteins were detected upon the action of biologically active substances, physiologically moderate factors, under oxidative stress, and in cancer and autoimmune diseases. The role of protein internal dynamics in the process of intracellular signaling is discussed.

Catalysis↗
PubMedDate not supplied

[Tryptophan phosphorescence of nascent and inactivated actin at the room temperature].

Millisecond internal dynamics of native and inactivated actin from rabbit skeletal muscle was examined using room temperature phosphorescence. Inactivated actin was prepared by incubation of G-actin at 70 degrees C, by treatment with 4 M urea or 1.5 M guanidinium hydrochloride, renaturation from fully unfolded state or by Ca2+ ion removal. It was shown that inactivation of actin, irrespective of the denaturation procedure applied, leads to a sharp decrease of millisecond fluctuations of the protein structure. Restriction of the slow intramolecular mobility in inactivated actin can result from changes of the protein conformation and/or specific association of macromolecules.

Actins↗
PubMedDate not supplied

[Effect of serine proteinases on the intracellular dynamics of membrane lipids and aggregation of human thrombocytes].

Room temperature tryptophan phosphorescence (RTTP) of human platelets in suspension was studied. The spectral and kinetic parameters of RTTP of thrombocytes were determined. By using the RTTP, the effect of serine proteases: thrombin, trypsin, and alpha-chymotrypsin at low concentrations (0-50 micrograms/ml) on the slow internal dynamics of membrane proteins was studied. It was shown that short-term incubation of platelets with proteases induces changes in the internal dynamics of membrane proteins in situ, and the magnitude of this effect correlates well with the extent of platelet aggregation. The functional role of the changes in the internal dynamics of membrane proteins and their relationship with intracellular signal transduction were discussed.

Blood Platelets↗
PubMedDate not supplied

[Kinetics of inactivation of lectin: analysis of a method of tryptophan phosphorescence at room temperature].

The internal dynamics of muscle actin during inactivation induced by guanidine hydrochloride (0.5-1.8 M) was studied by the method of room-temperature tryptophan phosphorescence (RTTP). It was shown that the essentially unfolded actin intermediate, which appears within the first minutes of incubation with guanidine hydrochloride, exhibits no RTTP, suggesting a high lability of its structure. Subsequent accumulation of associates of inactivated actin is accompanied by a significant increase in the intensity and decay time of RTTP, which is caused by the rigidity of the structure of inactivated actin. The kinetic dependencies of the intensity and lifetime of RTTP of actin during its inactivation depended on the concentration of the protein and guanidine hydrochloride.

Actins↗
PubMedDate not supplied

[On the possibility for a protein to exist in a manifold of partially folded states].

With the help of the methods of tryptophan fluorescence and room-temperature phosphorescence and using Escherichia coli alkaline phosphatase as an example, the ability of a protein to exist in a manifold of partially folded thermodynamically stable states differing in conformation, the internal dynamics, and functional activity was shown. Such intermediate (between native and unfolded) structures may form during unfolding or folding of the protein. It was shown that the degree of destruction of the native structural organization of the globule depends on both the nature and the mode of action of the destroying agent and the structure of the protein. Conformational transitions of the globule can change the kind of the internal dynamics (fast, slow), and shifts of dynamics can initiate conformation changes of the protein and precede them. A scheme of the structural and functional transformations of the protein during denaturation is presented, which takes into account the possibility of globule transitions into a manifold of functional active and inactive partially folded states. The role of partially folded forms of cell proteins in the development of pathology is discussed.

Alkaline Phosphatase↗
PubMedDate not supplied

[Intramolecular dynamics of human erythrocyte membrane proteins upon modification of spectrin by tryptophan phosphorescence].

The slow (millisecond) protein internal dynamics of isolated human erythrocyte membranes in suspension without treatment, after deleting 95% of spectrin, after spectrin thermal denaturation upon acidification of medium in the pH range 6.0-4.0, and spectrin extracted in solution from membranes has been studied by room-temperature tryptophan phosphorescence. It has been established that integral proteins and spectrin differ in structural and dynamic state. Millisecond movements of structural elements of integral proteins are more restricted compared with those of spectrin. The removal of spectrin from the membrane led to an increase in slow fluctuations of integral protein structure. This indicates that spectrin participates in the control of the structural and dynamic state of erythrocyte membrane proteins. As medium was acidified in the pH range 6.0-4.0, the protein slow internal dynamics of membranes in native state decreased, which was explained by spectrin pH aggregation. After thermal denaturation of spectrin, no pH-induced increase of membrane protein structure rigidity was observed.

Blood Proteins↗
PubMedDate not supplied

[Effect of temperature on intramolecular dynamics and conformational state of bacterial alkaline phosphatase].

The slow internal dynamics and the conformational state of Escherichia coli alkaline phosphatase by the action of temperature in the range 0-100 degrees C have been investigated by tryptophan room temperature phosphorescence and fluorescence. It has been shown that heating an alkaline phosphatase solution in the interval 0-70 degrees C leads to a substantial increase in the slow internal dynamics. A further increase in temperature to 95 degrees C causes a reversible enhancement of internal dynamics and a partial unfolding of the globule. Heating the protein solution in a narrow temperature range 97-100 degrees C induces an irreversible conformational transition, which is characterized by total unfolding of the globule, a drastic increase in internal dynamics, and the loss of enzymatic activity.

Alkaline Phosphatase↗