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I N Gorshkova

Publications and source records attributed to I N Gorshkova.

12 recordsLinked to original sources
PubMed2000

Probing the lipid-free structure and stability of apolipoprotein A-I by mutation.

To probe the secondary structure of the C-terminus (residues 165-243) of lipid-free human apolipoprotein A-I (apoA-I) and its role in protein stability, recombinant wild-type and seven site-specific mutants have been produced in C127 cells, purified, and studied by circular dichroism and fluorescence spectroscopy. A double substitution (G185P, G186P) increases the protein stability without altering the secondary structure, suggesting that G185 and G186 are located in a loop/disordered region. A triple substitution (L222K, F225K, F229K) leads to a small increase in the alpha-helical content and stability, indicating that L222, F225, and F229 are not involved in stabilizing hydrophobic core contacts. The C-terminal truncation Delta(209-243) does not change the alpha-helical content but reduces the protein stability. Truncation of a larger segment, Delta(185-243), does not affect the secondary structure or stability. In contrast, an intermediate truncation, Delta(198-243), leads to a significant reduction in the alpha-helical content, stability, and unfolding cooperativity. The internal 11-mer deletion Delta(187-197) has no significant effect on the conformation or stability, whereas another internal 11-mer deletion, Delta(165-175), dramatically disrupts and destabilizes the protein conformation, suggesting that the presence of residues 165-175 is crucial for proper apoA-I folding. Overall, the findings suggest the presence of stable helical structure in the C-terminal region 165-243 of lipid-free apoA-I and the involvement of segment 209-243 in stabilizing interactions in the molecule. The effect of the substitution (G185P, G186P) on the exposure of tryptophans located in the N-terminal half suggests an apoA-I tertiary conformation with the C-terminus located close to the N-terminus.

Amino Acid Substitution↗
PubMed1996

Alterations in the physiochemical characteristics of low and high density lipoproteins after lipolysis with phospholipase A2. A spin-label study.

Human low and high density lipoproteins (LDL and HDL, respectively) were treated with porcine pancreatic phospholipase A2 (PLA2) in the presence of albumin resulting in hydrolysis of 40-84% of the lipoproteins phospholipids. The resulting PLA2-treated LDL and HDL and concurrent control lipoproteins incubated without PLA2 were reisolated by ultracentrifugation and labelled with 5-doxyl- and 16-doxyl-stearic acid, and with a spin-labelled analogue of maleimide. Analysis of ESR spectra showed that phospholipid hydrolysis both of LDL and HDL resulted in an increase in order, micro-viscosity and polarity of lipid regions in the surface monolayer of the particles. In the temperature range from 3 degrees C to 50-60 degrees C, Arrhenius plots of a spectral parameter of LDL and HDL labelled with 5-doxyl-stearate exhibited alterations which suggest an increase in free cholesterol content near the surface of the lipoproteins after PLA2-treatment. ESR spectra of the maleimide analogue bound covalently to the protein moiety of the lipoproteins have demonstrated that, following phospholipid hydrolysis, the conformation of the apoproteins became more condensed, with more masked domains. The possible implications of the revealed alterations for enhanced delivery of LDL and HDL cholesterol to cells after phospholipolysis of the lipoproteins are discussed.

Ascorbic Acid↗
PubMed1988

[Physico-chemical and functional characteristics of a subfraction of low-density lipoproteins isolated by ion-exchange column chromatography].

Fractionation of human blood plasma low density lipoproteins (LDL) was performed by ion-exchange chromatography, using a linear NaCl gradient. It was shown that the binding of LDL subfractions eluted with a low ionic strength buffer (i.e., containing the particles with a lower negative charge) to B, E-receptors of fibroblasts was more effective than that of subfractions eluted with a high ionic strength buffer (i.e., containing the particles with a higher negative charge). The LDL particles with a lower negative charge had lower values of flotation coefficients (according to analytical ultracentrifugation data), smaller dimensions (according to gradient gel electrophoresis data) and a lower phospholipid/protein ratio (w/w). The experimental results suggest that LDL subfractions having different electrical parameters of the particle surface also differ in other physicochemical properties and seem to play a different role in atherogenesis.

Cells, Cultured↗
PubMedDate not supplied

[Changes in high density lipoprotein subfractions during interaction with fibroblasts and hepatoma G-2 in various dyslipidemias].

Subfractional alterations of high density lipoproteins (HDL) were studied after incubation of blood serum from patients with normal lipid spectrum and with four types of dyslipidemia (hypercholesterolemia, hypertriglyceridemia, hypo- and hyper-alpha-cholesterolemia) in mixtures containing human skin fibroblasts and G-2 hepatoma cells used as typical populations of peripheric and liver cells. Incubation of normolipidemic blood sera with fibroblasts overloaded with cholesterol led to conversion of small HDL3 particles into large HDL2 subfractions arising due to the lipoprotein acception of cholesterol. At the same time, incubation of these blood sera with the hepatoma cells resulted in a decrease of the large particles ratio in total pool of HDL because of their absorption by the cells. No distinct differences were detected in formation of large particles from small subfractions when cholesterol was accepted from fibroblasts under conditions of hypercholesterolemia, hypertriglyceridemia and hyper-alpha-cholesterolemia, while formation of the largest particles HDL2b was impaired in hypo-alpha-cholesterolemia. These HDL2b particles interacted less effectively with hepatoma cells, thus suggesting the decreased cholesterol transport function of HDL in hypo-alpha-cholesterolemia. Content of HDL2b in total pool of HDL was unaltered if blood serum from patients with hyper-alpha-cholesterolemia was incubated together with the hepatoma cells. Antiatherogenic effect of hyper-alpha-cholesterolemia was caused mainly by active transfer of cholesterol from low density lipoproteins to HDL and a decrease in the LDL concentration but not by increased absorption of HDL particles by liver cells.

Adult↗
PubMedDate not supplied

[A study of thermoinduced changes in human high-density lipoproteins in hypo-alpha-lipoproteinemia and ischemic heart disease by the spin label method].

Thermo-induced structural reorganization in human high density lipoproteins subclasses HDL2 and HDL3 have been studied by means of electronic paramagnetic resonance method. A number of spin labels were used: 5- and 16-DOXYL stearates, DOXYL-labelled derivatives of pentadecane, phosphatidyl choline and sphingomyelin. In hypoalphalipoproteinemia one of the thermo-induced transitions in HDL2 (at 23-30 degrees) was not observed. In coronary heart disease the critical temperature for HDL3 was shifted from the physiological values and located at 42-48 degrees. The data obtained are discussed from the point of view of their potential role in atherogenesis.

Adult↗
PubMedDate not supplied

[Interaction between plasma lipoproteins and bilayer lipid membranes. The role of surface charge].

Interactions of planar BLM with different thickness and surface charge were analysed theoretically. Drawing together of the membranes is accompanied with the appearance of intramembrane potential jumps which may cause destruction and breakdown of the membranes. The theory is extrapolated to the interaction between spherical lipoprotein particles and planar BLM. Experimentally calculated (by means of ESR) surface charges of lipoproteins of low density (LLD) (--0,3 . 10(-2) C/m2) and lipoproteins of high density (LHD) (--2 . 10(-2) C/m2) enabled calculation of the interaction energy between the particles and BLM as well as of the values of intramembrane potential jumps. The latter cause local reconstructions of the membranes in the contact region and fusion of the particles with them. The earlier obtained experimental data were proved by the finding that LHD adsorption as compared with LLD is impeded due to the existence of a high energetic barrier. These peculiarities of the particles manifested during their interactions with BLM seem to be one of the factors responsible for atherogenic function of LLD and antiatherogenic one of LHD.

Electron Spin Resonance Spectroscopy↗