Properties of aminoglycoside-3'-phosphotransferases determined by kanamycin transposones Tn 601 and Tn 5

Aminoglycoside-3'-phosphotransferase I and II (APT-3'-I and APT-3'-II) has been purified to homogenity from the cells of E. coli containing the plasmids R6 and JR67, respectively. The purification procedure involved competitive affinity chromatography on neomycin-sepharose and gel-filtration on Sephadex G-100. The specific activity of APT-3'-I with the substrates--lividomycin A, neomycin B, paromycin, ribostamycin, kanamycins A and B--are 4.3, 2.8, 2.1, 1.6, 0.9 and 0.8 mol/min. mg protein, respectively. The specific activity of APT-3'-II with the substrates--ribostamycin, paromycin, kanamycins A and B, neomycin B--are 8.0, 7.2, 4.0, 4.5 and 3.6, respectively. Mg2+ is required for the activity of both enzymes. Co2+, Zn2+ and Mn2+ are active in case of APT-3'-I; however, these cations are less active than Mg2+. The pH-optimum of APT-3'-I and APT-3'-II is 7.0--7.5. High ionic strength is required for the activity of both enzymes. The molecular weights of APT-3'-I and APT-3'-II are about 36 000 and 26 000, respectively. The amino acid composition of APT-3'-I and APT-3'-II was determined. Both enzymes contain tryptophane residues whose fluorescence intensity decreased when ATP, but not amino-glycoside antibiotics, is added. The interrelationship between the molecular weights of these enzymes and the sizes of the loops of transposones Tn 601 and Tn 5, encoding APT-3'-I and APT-3'-II, is discussed.     

Theoretical and experimental studies of the radiative properties of matter at high energy densities and their application to the problems of inertial confinement fusion

The paper presents the results of theoretical and experimental studies of the radiative properties of plasmas produced by heating and compression of various materials to high energy densities. The specific features of the theoretical plasma model known as the ion model, which is used to calculate the radiative characteristics of plasmas of complex chemical composition, are discussed. The theoretical approach based on this model is applied to the plasma produced during the explosion of the X-pinch wires. The theoretical estimate of the radiation efficiency is compared with the experimental data on the total energy yield from an X-pinch made of two different wires (NiCr and Alloy 188). The radiative characteristics of (C12 H16 O8) and (C8 H12 O6) plasmas are calculated for the temperature diagnostics of plasmas produced from porous targets employed in inertial confinement fusion experiments with the use of laser radiation and heavy-ion beams.     

Intramolecular hydrogen bonds and conformation of the lincomycin molecule in organic solvents

IR spectra (1600-1800 and 3000-3650 cm-1) of lincomycin base solutions in inert (CCl4 and C2Cl4), proton acceptor (dioxane, dimethylsulfoxide and triethyl amine) and proton donor (CHCl3, CD3OD and D2O) solvents were studied. Analysis of the concentration and temperature changes in the spectra revealed that association in lincomycin in the inert solvents was due to intramolecular hydrogen linkage involving amide and hydroxyl groups. Disintegration of the associates after the solution dilution and temperature rise was accompanied by formation of intramolecular bonds stabilizing the stable conformation structure of the lincomycin molecule. The following hydrogen linkage in the conformation was realized: NH...N (band v NH...N at 3340 cm-1), OH...O involving the hydroxyl at C-7 and O atoms in the D-galactose ring (band v OH...O at 3548 cm-1), a chain of the hydrogen bonds OH...OH...OH in the lincomycin carbohydrate moiety (band v OH...O at 3593 cm-1 and v OH of the end hydroxyl group at 3625 cm-1). Bonds NH and C-O of the amide group were located in transconformation. Group C-O did not participate in the intramolecular hydrogen linkage.     

Erythrocyte reagents in the study of Erysipelothrix antigens

The activity of species-specific and type-specific antigens in various preparations isolated from the bacterial mass of standard strains of Erysipelothrix, and also in bacterial cells was studied by means of prepared erysipeloid erythrocyte antigen (species-specific and with general type- and species-specificity) and antibody (species-specific, with general type- and species-specificity as also with type-specificity only) diagnosticums. It has been demonstrated that the activity of these antigens differs in preparations from different strains, depending on the method of extraction. An efficient method of serotyping of Erysipelothrix, based on agglutination of erythrocyte antibody diagnosticums, was proposed.     

Binding of small basic peptides to membranes containing acidic lipids: theoretical models and experimental results.

We measured directly the binding of Lys3, Lys5, and Lys7 to vesicles containing acidic phospholipids. When the vesicles contain 33% acidic lipids and the aqueous solution contains 100 mM monovalent salt, the standard Gibbs free energy for the binding of these peptides is 3, 5, and 7 kcal/mol, respectively. The binding energies decrease as the mol% of acidic lipids in the membrane decreases and/or as the salt concentration increases. Several lines of evidence suggest that these hydrophilic peptides do not penetrate the polar headgroup region of the membrane and that the binding is mainly due to electrostatic interactions. To calculate the binding energies from classical electrostatics, we applied the nonlinear Poisson-Boltzmann equation to atomic models of the phospholipid bilayers and the basic peptides in aqueous solution. The electrostatic free energy of interaction, which arises from both a long-range coulombic attraction between the positively charged peptide and the negatively charged lipid bilayer, and a short-range Born or image charge repulsion, is a minimum when approximately 2.5 A (i.e., one layer of water) exists between the van der Waals surfaces of the peptide and the lipid bilayer. The calculated molar association constants, K, agree well with the measured values: K is typically about 10-fold smaller than the experimental value (i.e., a difference of about 1.5 kcal/mol in the free energy of binding). The predicted dependence of K (or the binding free energies) on the ionic strength of the solution, the mol% of acidic lipids in the membrane, and the number of basic residues in the peptide agree very well with the experimental measurements. These calculations are relevant to the membrane binding of a number of important proteins that contain clusters of basic residues.     

Phosphate group donors for neomycin inactivation by resistant microorganism strains]

The substrate specificity of aminoglycoside phosphotransferases isolated from 3 strain of E. coli and purified was studied. All pure enzymes phosphorilated neomycin, paromomycin, lividomycin, neamine, ribostamycin, kanamycins A and B. Only ATP was the donor of the phosphate groups in these reactions, while in the non-purified extracts GTP but not UTP or CTP served as the donor of the phosphate group for inactivation of neomycin. The substrate specificity indicated that the above enzymes were aminoglycoside-3(1)-phosphotransferases. Inactivation of neomycin with the use of the phosphate group of phosphoenolpiruvate as the donor in the non-purified enzymatic preparations of the neomycin-resistant strains of E. coli and Pseudomonas was not observed.     

Determination of the antiproliferative activity of human recombinant interferon (reaferon) on diploid human cells

Dependence of the antiproliferative activity of reaferon in cultures of human diploid cells on the drug dose and duration of its action on the cells was studied by counting viable cells in the Goriaev chamber. No relationship was detected. However, such dependence was clearly evident with using tumor cells. With counting of mitoses it was shown that in doses of 10(4) and 10(3) IU reaferon significantly inhibited the cell mitotic activity when the mitotic index in the control of the strain M-19 cells exceeded 25%. When the mitotic index was lower than 25% reaferon in all the doses had no inhibitory effect on the cells. On the contrary it was noted that there was even a certain tendency to stimulation of the mitotic activity. Therefore, the line of the human diploid fibroblast cells (strain M-19) may be used for assay of reaferon antiproliferative activity in case of high mitotic activity in its monolayer (the mitotic index higher than 25%). It was also demonstrated that the dose of reaferon should be not lower than 1000 IU per 1 ml of the medium.