Irina D. Grozdova

Cold inactivation of glyceraldehyde-phosphate dehydrogenase from rat skeletal muscle

Inactivation of apo-glyceraldehyde-phosphate dehydrogenase (D-glyceraldehyde-3-phosphate: NAD+ oxidoreductase(phosphorylating) (EC 1.2.1.12) from rat skeletal muscle at 4 degrees C in 0.15 M NaC1, 5 mM EDTA, 4 mM 2-mercaptoethanol pH 7.2 is a first-order reaction. The rate constant of inactivation depends on protein concentration. With one molecule of NAD bound per tetrameric enzyme, a 50 per cent loss in activity is observed and the rate constant of inactivation becomes independent of the protein concentration over a 30-fold range. Two moles of NAD bound per mole of enzyme fully protect it against inactivation. NADH affords a cooperative effect on enzyme structure similar to that of NAD. Inactivation of 7.8 S apoenzyme is reflected in its dissociation into 4.8-S dimers. In the case of enzyme-NAD1 complex, no direct relationship between the extent of inactivation and dissociation is observed, suggesting that these two processes do not occur simultaneously; we may say that dissociation is slower than inactivation. A mechanism in which the rate-limiting step for inactivation is a conformational change in the tetramer occurring prior to dissociation and affecting only the structure of the non-liganded dimer, is consistent with the experimental observations. Inorganic phosphate protects apoenzyme against inactivation. Its effect is shown to be due to the anion binding at specific sites on the protein with a dissociation constant of 2.6 plus or minus 0.4 mM. The NaC1-induced cold inactivation of glyceraldehyde-phosphate dehydrogenase is fully reversible at 25 degrees C in the presence of 20 mM dithiothreitol and 50 mM inorganic phosphate. The rate of reactivation is independent of protein concentration. Inactivated enzyme retains the ability to bind specific antibodies produced in rabbits, but diminishes its precipitating capability.

Cytotoxicity of nonionic amphiphilic copolymers

The purpose of this study is to ascertain the relationship between the structure of an amphiphilic nonionic polymer and its toxicity for cells (cytotoxicity) growing in a culture. To this end, 16 polymers of different architectures and chemical structures are tested, namely, linear triblock copolymers of poly(ethylene oxide)-block-poly(propylene oxide)-block-poly(ethylene oxide) (Pluronics); diblock copolymers of propylene oxide, ethylene oxide, and hyperbranched polyglycerol; alternating and diblock copolymers of ethylene oxide and dimethylsiloxane; and two surfactants containing linear (Brij-35) or branched (Triton X-100) aliphatic chains. Polymer-cell interaction is assayed in a culture medium in the absence of serum. Effective concentrations of the polymers causing 50% cell death, EC50, vary within three orders of magnitude. Toxic concentrations of the alternating copolymer, Triton X-100, and Brij-35 are lower than their CMC values. In contrast, all block copolymers, regardless of their chemical structures, become toxic at concentrations above the CMC; that is, they acquire cytotoxicity only in the micellar form. The EC50 values of the copolymers depend on their hydrophilic-liphophilic balance (HLB) through the following empirical formula: EC50 × 106 = 8.71 × HLB2.1. This relationship makes it possible to predict the cytotoxic concentration region of a block copolymer of a known structure.

Effect of the structure of ethylene oxide-propylene oxide block copolymers on their interaction with biological membranes

Partition coefficients of ethylene oxide-propylene oxide block copolymers between the lipid phase and water have been estimated via equilibrium dialysis. It has been shown that for the triblock copolymer (Pluronic L61), the partition coefficient is 45 ± 9, while for the diblock copolymer (REP), this parameter is as high as 78 ± 17. The effect of the copolymers on the permeation of the charged organic ion carboxyfluorescein across the lecithin bilayer membrane changes in the same direction. Even though the triblock copolymer binding is weaker, it shows a stronger effect on the rate of transbilayer migration of lipids and on the permeation of the uncharged substance (doxorubicin). The incorporation of cholesterol into the membrane decreases its sensitivity to the action of copolymers; however, the character of changes induced by both copolymers remains invariable. The experimental data of this study indicate that the triblock structure of amphiphilic macromolecules is responsible for their higher ability to disturb lipid bilayer membranes.

The Effect of Modification with Fluorescent Groups on the Physicochemical Characteristics of Poly(alkylene oxides)

Covalent modification of poly(alkylene oxides), block copolymers of ethylene oxide and propylene oxide with M ∼ 2000, with fluororescein isothiocyanate or 7-nitrobenzo-2-oxadiazole taken in a ratio of 0.6–1.0 mol of label/mole of polymer leads to changes in the physical state of polymers and their solubility in water decreases, as well as the partition coefficient in the hexane-water system and the critical concentration of aggregation and dimensions of the formed particles.

Comparative immunochemical studies on glyceraldehydephosphate dehydrogenase isolated from rabbit and rat skeletal muscle

Antibodies were prepared in chickens against glyceraldehyde-phosphate dehydrogenase (d-glyceraldehyde-3-phosphate:NAD+ oxidoreductase (phosphorylating) EC 1.2.1.12) isolated from rabbit and rat skeletal muscle; antibodies to the rat muscle enzyme were produced in rabbits. The data obtained in gel-diffusion and enzyme-inhibition experiments carried out with different antisera demonstrate the presence of two types of antigenic determinants on the dehydrogenase molecule-interspecific, cross-reactive, and species-specific determinants.

Peroxyoxalate Chemiluminescent Reaction as a Tool for Elimination of Tumour Cells Under Oxidative Stress

The overproduction of hydrogen peroxide is an inherent feature of some tumour cells and inflamed tissues. We took advantage of this peculiarity to eliminate cells using chemiluminescent peroxyoxalate reaction. We designed dispersions containing polyoxalate and tetramethylhematoporhyrin (TMHP) in dimethylphthalate droplets stabilized with Pluronic L64. The porphyrin plays the dual role. On the one hand, it serves as an activator of the peroxyoxalate reaction of polyoxalate with intracellular hydrogen peroxide and experiences excitation as a result of the reaction. The light emitted in the reaction in the model system without cells was used to optimize the dispersion’s composition. On the other hand, TMHP acts as a photosensitizer (PS) causing cell damage. The formation of singlet oxygen led to cell elimination if the dispersions were used in combination with inducers of oxidative stress: hydrogen peroxide, paraquat, antitumour drug doxorubicin, or a nutritional additive menadione. The PS-induced cytotoxicity correlated with the level of intracellular ROS. The developed approach targeted to endogenous ROS is orthogonal to the classical chemotherapy and can be applied to increase its efficiency.

Molecular Targets of the Hydrophobic Block of Pluronics in Cells: a Photo Affinity Labelling Approach

PurposePluronics are known as inhibitors of multidrug resistance thus making tumor cells sensitive to therapeutic doses of drugs. The purpose of our study consists in revealing molecular targets of the hydrophobic poly(propylene oxide) block of pluronics in living cells and the dependence of the polymers chemosensitizing efficiency upon targeting.MethodsA photo sensitive tracer was attached to the hydrophobic poly(propylene oxide) block of 3H-labeled tert-Bu-EO-PO copolymer. The conjugate was used for treatment cells in culture. We searched for its complexes with cellular lipids or proteins using RP TLC and SDS-electrophoresis, respectively. The chemosensitizing efficiency of pluronics was evaluated by their least concentrations sufficient for MDR reversion (CMDR).ResultsThe poly(propylene oxide) block inserts in the lipid core of plasma membrane. No preferential binding of the conjugate with any cellular protein, particularly P-gp, has been detected. FITC-labeled pluronic L61 bound to alcohol insoluble cellular targets did not participate in MDR reversion. CMDR values of 13 block copolymers have been determined. These values inversely correlated with the polymers affinity toward lipids and the ability to accelerate flip-flop.ConclusionInsertion of the hydrophobic poly(propylene oxide) block of amphiphiles in the lipid core of plasma membrane and acceleration of flip-flop of lipids underlie the mechanism of MDR reversion.

Cationic nanogels as Trojan carriers for disruption of endosomes

The comparison study of interaction of linear poly(2-dimethyl amino)ethyl methacrylate and its cationic nanogels of various cross-linking with both DNA and sodium poly(styrene sulfonate) has been performed. Although all amino groups of the nanogels proved to be susceptible for protonation, their accessibility for ion pairing with the polyanions was controlled and impaired with the cross-linking. The investigation of nanogels complexes with cells in culture that was accomplished by using of calcein pH-sensitive probe revealed a successive increase in the cytoplasmic fluorescence upon the growth in the cross-linking due to calceine leakage from acidic compartments to cytosol. This regularity implies that amino groups which are buried presumably inside the nanogel are protected against the ion-pairing with polyanions of plasma membrane and hence are able to manifest buffer properties while captured into acidic endosomes, i.e. possess lyso/endosomolytic capacity. These findings suggest that network architecture makes an important contribution to proton sponge properties of weak polycations.