Effect of phosphatidylglycerol on the incorporation process of cytochrome P-450 in liposomes from dimyristoylphosphatidylcholine

Using the electron paramagnetic resonance (EPR) method with spin-labeled fatty acids and gel-penetrating chromatography, the effect of phosphatidylglycerol on cytochrome P-450 incorporation into liposomes from dimyristoylphosphatidylcholine was investigated. An addition of phosphatidylglycerol caused an increase in the protein content of the proteoliposomes as well as their accelerated formation at temperatures below and above the liposome phase transition temperature (Ts). The dependence of the proteoliposome formation rate on the phosphatidylglycerol content in the liposome mixture is described by complex kinetics with a maximum in the presence of 10 mol.% of the negatively charged phospholipid. The mechanism of proteoliposome formation is discussed in terms of asymmetric distribution and phase state of the phospholipids in the original vesicles.     

Experience in the absolute calculation of pasture ixodid ticks (Parasitiformes, Ixodidae) on the eastern macroslopes of Sikhoté-Alin]

A new technique is suggested for absolute registration of adult ixodid ticks with an improved furrow plough. Due to this the number of ticks can be estimated per square unit to various biotopes. As a result of registrations conducted in 1973 the density of ticks populations from the main landscape zones of eastern Sikhote-Alin (from watershed spruce-abietic forests to coastal oak woods) was estimated for the first time.     

The bacteriorhodopsin proton pump: effect of crosslinkings of lysine residues

All six available lysine residues in bacteriorhodopsin were amidinated with dimethyl-3,3'-dithiobispropionimidate, which is a crosslinking agent. The photocycle was studied by measuring light absorption and electric signals. The data show an essential change in the photocycle: instead of single components, the rise of the signal due to the M intermediate can be decomposed into two components, and the decay into three. The life-times and the intensities of these components and in general the proton pumping activity of bacteriorhodopsin depend only negligibly upon pH. Changes upon removing the crosslinks are not significantly different from those in the crosslinked samples. The lysine residues therefore may not be considered of primary importance in proton translocation.     

Isolation, identification and properties of a new thyroxine-binding protein--apolipoprotein A-1 from human blood serum]

A protein having a molecular mass of about 25 kWa was isolated by thyroxin (T4)-Sepharose affinity chromatography from human blood serum; its properties were found to be distinct from those of known T4-binding proteins. Using immunodiffusion, radioimmunoassay, lipid analysis, differential precipitation and electrophoresis, it was shown that the isolated protein is a component of high density lipoprotein (HDL) particles and represents an apolipoprotein A-1 (apoA-1). Using cholate-Sepharose chromatography apoA-1 was separated from the lipid moiety and contaminant proteins, and apoA-1 was effectively isolated directly from the blood serum. Apo-A-1-HDL and apoA-1 obtained by affinity chromatography as well as the HDL3 fraction isolated by a standard ultracentrifugation technique, all displayed a T4-binding activity, the affinities for the hormones being of the same order of magnitude. The T4 interaction with these preparations induced difference UV-absorption signals, altered the characteristics of apoA-1 intrinsic fluorescence without affecting the circular dichroism of the protein-hormone system. The binding of spin-labelled T4 to apo-1, apoA-1-HDL or HDI3 caused substantial changes in the shape of the ESR spectrum and an increase in the apparent rotational correlation time. The mobility of the radical fragment of spin-labelled T4 depended on the composition and properties of the protein preparation. The electron spectroscopy data suggest that the T4-HDL interaction occurs via specific mechanisms and that the molecular structures of the complexes formed thereby are not characteristic of other known T4-binding proteins.     

A new class of antivirals: antisense oligonucleotides combined with a hydrophobic substituent effectively inhibit influenza virus reproduction and synthesis of virus-specific proteins in MDCK cells.

To enhance the penetration of oligonucleotide ('oligo') into cells, the oligo was combined with the hydrophobic undecyl residue. Using the 'DNA-synthesator', we synthesized oligo, complementary to the loop-forming site of the RNA, encoding polymerase 3 of the influenza virus (type A), and combined it with the undecyl residue added to the 5' terminal phosphate group. It was found that the modified oligo effectively suppresses the influenza A/PR8/34 (H1N1) virus reproduction and inhibits the synthesis of virus-specific proteins in MDCK cells. Under the same conditions, the non-modified antisense oligo and modified nonsense oligo did not affect the virus development.     

Regulation of the catalytic activity of the monooxygenase enzyme system depending of the substrate structure and phospholipid composition of the model membrane

A comparative study of pentoxy- and benzyloxyresorufin dealkylation by a monooxygenase enzyme system in dilauroylphosphatidylcholine micelles and in proteoliposomes was carried out. In proteoliposomes whose lipid matrix is formed by double phospholipid mixtures, a cooperative regulation of the oxidation reaction was found. It was shown that the cooperativity of this process depends on the substrate type as well as on the phospholipid composition of the vesicles and decreases during peroxide oxidation of lipids. The results obtained are discussed in terms of protein-protein and protein-phospholipid interactions in the oligomer ensembles consisting of several cytochrome P-450 LM2 molecules.     

Inhibition of tryptophanyl-tRNA synthetase by modifying ATP analogs

The interaction between tryptophanyl-tRNA synthetase (EC 6.1.1.2) from beef pancreas and the ATP analogs containing alkylating or phosphorylating groups in the polyphosphate moiety of ATP was studied as an approach to investigate the structure of the enzyme active center. Some of the compounds under study were shown to irreversibly inhibit the enzyme activity; the presence of ATP in the most cases protects the enzyme against inactivation. The kinetic constants Ki and k2 of interaction of the irreversible inhibitors with the enzyme were determined. It was found that the Ki values for a number of irreversible competitive inhibitors are by 1-2 orders of magnitude less than the Km value for ATP; the k2 values were found equal to 0.02-0.04 min-1. this suggests that the compounds may be used as affinity reagents, the most efficient ones being adenosine 5'-(beta-chloroethyl phosphate) and mixed AMP-mesithylene carbonic acid anhydride. The absence of a protective effect of ATP in the case of adenosine 5'-(beta-bromoethane phosphonate) and non-competitive type of reversible inhibition inhibition of the enzyme by adenosine 5'-chloromethane phosphonate indicate that the molecule of tryptophanyl-tRNA synthetase contains sites interacting with adenine nucleotides, other than the ATP binding sites of the active center.