Binding of monovalent cations induces large changes in the secondary structure of Na+,K+-ATPase as probed by Raman spectroscopy

Raman spectra of active Na+,K+-ATPase from pig kidney in media containing Na+ (E1), K+ (E2) or without exogenous ions (E1 conformation) were recorded in order to calculate the changes in the enzyme's secondary structure induced by binding of monovalent cations. It is demonstrated that: (i) K+ binding to the E1 form of the enzyme leads to conversion of approximately 100 peptide groups from the beta-structure to alpha-helical conformation; (ii) the transition is reversible and fully reproducible in the E1----E2----E1 and E2----E1----E2 experimental schemes. Predictional calculations revealed polypeptide chain segments involved in the alpha----beta transformations. These segments reside mainly in the two highly conserved regions of the alpha-subunit in the cytoplasmic domain of Na+,K+-ATPase. A possible role for the beta-subunit is discussed.     

Endogenous pyrogen formation by bone marrow cells

The cells of the rabbit bone marrow produced endogenous pyrogen in response to stimulation with bacterial lipopolysaccharide. Incubation of the cells in medium No 199 containing a 15% homologous serum is optimal for the release of pyrogen. It is supposed that the cells of the bone marrow take part in the formation of endgenous pyrogen and in the mechanism of pyrexia in the organism.     

Immunochemical study of the heterogeneity of trophoblastic beta 1-glycoprotein

It has been found that in mild acid (pH 6.6) and mild-alkaline media (pH 7.7) both pregnancy proteins form complete precipitates. In more alkaline buffer solutions the form of alpha 2-glycoprotein (alpha 2-GP) precipitate is preserved, while trophoblastic beta 1-glycoprotein (TBG) shows three immunochemically identical components with different electrophoretic mobility. The form with beta-globulins mobility predominates, and minor fragments are presented by alpha- and gamma-components. All TBG forms are clearly seen at pH 8.6. In more alkaline medium (pH 10.0) the clarity of the precipitates drastically decreases. It is shown that heparin introduction into the gel of first dimension electrophoresis increases anode electrophoretic mobility of both proteins at polysaccharide concentration of at least 0.1 mg/ml. Large amounts of heparin cause the increase in TBG alpha-component precipitate area and the decrease in the form with beta-globulins mobility. At the same time alpha 2-GP precipitate area and form remain unchanged.     

Effective Size of Subpopulations in Early-Run Sockeye Salmon Oncorhynchus nerka from Azabach'e Lake (Kamchatka): The Effect of Relative Reproductive Success of Different-Year Cohorts

The effect of variation in reproductive success of cohorts of different year of birth (within generation) on the effective subpopulation (breeding group) size in early-run sockeye salmonOncorhynchus nerka from Azabach'e Lake (Kamchatka). The annual variation in census size and overlapping of year classes reduced the ratio of the effective subpopulation size to the census size by 7 to 88% in different subpopulations. The total effect of the variance of reproductive success in individual years and the variance of reproductive success of different cohorts reduced the effective size/census size ratio by 68–96%.     

Modeling of peptides and proteins in a membrane environment: II. Structural and energetic aspects of glycophorin A in a lipid bilayer

The conformational space of a hydrophobic peptide fragment of glycophorin A in a lipid membrane was studied with the Monte Carlo method using the solvation model described in the first communication of this series. The simulation was performed for various starting orientations of the peptide relative the membrane bilayer: outside, inside, partially immersed, and transbilayer. We showed that the membrane substantially stabilizes the α-helical conformation of the central hydrophobic part of the glycophorin A molecule, which for the most part is immersed in the apolar core of the bilayer. For various conformational states, energy values were calculated and the orientations of the peptide relative to the membrane were characterized. Depending on the thickness of the bilayer, either an entirely α-helical conformation in transbilayer orientation or a conformation with a kink in the central part of the helix with theN- andC-termini exposed on one side of the membrane corresponds to the minimal-energy structure. The transmembrane orientation of glycophorin A is energetically advantageous when the membrane thickness is close to the length of its hydrophobic helical portion, which is consistent with the effect ofhydrophobic match observed experimentally. The prospects for further refinement of the model are discussed. For communication I, see [1].     

Possibility of the unification of directed drug transport as illustrated by liposome transport to target antigens

A new approach to the targeted drug delivery is described. Unlike previous methods, associated with the necessity of specific immunoglobulin immobilization on the surface of drug-containing microcontainer, the present approach permits targeted transport of standardized container to a set of target antigens, using intermediate molecules-mediators possessing high and specific affinity to both vector antibody and standardized container. It was shown that simultaneous targeting of 14C-labeled liposomes to three target antigens using avidin-biotin system permits the increase in liposome binding to target monolayer by 30-50%, as compared to targeting of the same amount of liposomes to one antigen. The method developed is particularly promising in cases when relative availability of target antigens in the target organ is unknown.     

The coupling mechanism of respiratory complex I — A structural and evolutionary perspective

Complex I is a key enzyme of the respiratory chain in many organisms. This multi-protein complex with an intricate evolutionary history originated from the unification of prebuilt modules of hydrogenases and transporters. Using recently determined crystallographic structures of complex I we reanalyzed evolutionarily related complexes that couple oxidoreduction to trans-membrane ion translocation. Our analysis points to the previously unnoticed structural homology of the electron input module of formate dehydrogenlyases and subunit NuoG of complex I. We also show that all related to complex I hydrogenases likely operate via a conformation driven mechanism with structural changes generated in the conserved coupling site located at the interface of subunits NuoB/D/H. The coupling apparently originated once in evolutionary history, together with subunit NuoH joining hydrogenase and transport modules. Analysis of quinone oxidoreduction properties and the structure of complex I allows us to suggest a fully reversible coupling mechanism. Our model predicts that: 1) proton access to the ketone groups of the bound quinone is rigorously controlled by the protein, 2) the negative electric charge of the anionic ubiquinol head group is a major driving force for conformational changes. This article is part of a Special Issue entitled: 17th European Bioenergetics Conference (EBEC 2012).     

The coupling mechanism of respiratory complex I - A structural and evolutionary perspective

Complex I is a key enzyme of the respiratory chain in many organisms. This multi-protein complex with an intricate evolutionary history originated from the unification of prebuilt modules of hydrogenases and transporters. Using recently determined crystallographic structures of complex I we reanalyzed evolutionarily related complexes that couple oxidoreduction to trans-membrane ion translocation. Our analysis points to the previously unnoticed structural homology of the electron input module of formate dehydrogenlyases and subunit NuoG of complex I. We also show that all related to complex I hydrogenases likely operate via a conformation driven mechanism with structural changes generated in the conserved coupling site located at the interface of subunits NuoB/D/H. The coupling apparently originated once in evolutionary history, together with subunit NuoH joining hydrogenase and transport modules. Analysis of quinone oxidoreduction properties and the structure of complex I allows us to suggest a fully reversible coupling mechanism. Our model predicts that: 1) proton access to the ketone groups of the bound quinone is rigorously controlled by the protein, 2) the negative electric charge of the anionic ubiquinol head group is a major driving force for conformational changes. This article is part of a Special Issue entitled: 17th European Bioenergetics Conference (EBEC 2012).     

Lateral clustering of lipids in hydrated bilayers composed of dioleoylphosphatidylcholine and dipalmitoylphosphatidylcholine

Investigation of lateral heterogeneities (clusters) in cell membranes is an important step toward understanding the physical processes that lead to the formation of lipid domains and rafts. Computer modeling methods represent a powerful tool to solve the problem, since they can detect clusters containing only a few lipid molecules—the situation that still resists characterization with modern experimental techniques. Parameters of clustering depend on lipid composition of a membrane. In this work, we propose a computational method to detect and analyze parts of membrane with different packing densities. Series of one- and two-component fluid systems containing lipids with the same polar heads and different acyl chains, dioleoylphosphatidylcholine (18 : 1) and dipalmitoylphosphatidylcholine (16 : 0), were chosen as the objects under study. The developed algorithm is based on molecular dynamics simulation of hydrated lipid bilayers in all-atom mode. The method is universal and could be applied to any other membrane system with arbitrary lipid composition. Here, we demonstrated that the studied lipid bilayers reveal small lateral dynamic clusters composed of just several (most often, three) lipid molecules. This seems to be one of the most important reasons determining the “mosaic” nature of the membrane-water interface.