The influence of the lipid bilayer phase state on the p-aminohippurate (PAH) transport and the activity of the alkaline phosphatase in brush-border membrane vesicles from normal and mutant rats.

The kinetic parameters of p-aminohippurate transport and activity of the alkaline phosphatase were studied using brush-border membrane vesicles isolated from the kidney cortex of normal and mutant (strain of Campbell) rats. p-Aminohippurate (PAH) transport of both normal and mutant animals was carried out by the mechanism of facilitated diffusion. The apparent Michaelis constant at 36 degrees C was equal to 7 mM, the maximal rate of PAH transport was 15 nmol/min per mg protein and the constant of inhibition by probenecid was 0.5 mM for normal rats and, respectively, 29 mM, 62 nmol/min per mg protein and 1.4 mM for mutant rats. The Arrhenius plot for the PAH transport and activity of the alkaline phosphatase showed the breakpoints at 28-30 degrees C for normal rats and at 36-38 degrees C for the Campbell strain rats. The thermotropic phase transitions detected by the EPR method with 5-doxylstearate as a probe were recorded at 21-30 degrees C and 30-35 degrees C for normal and mutant rats, respectively. Therefore, characteristic features of the PAH carrier and alkaline phosphatase activity in normal and Campbell strain rats are determined by the difference in the phase state of their membrane lipid bilayers. We suppose that mutation in the Campbell strain gives rise to a membrane pleiotropic effect which enables us to understand the mechanism of genetic control of the lipid structure and membrane fluidity.

[Physicochemical mechanisms of organic acid transport in the apical membrane vesicles of the cells of proximal kidney tubules in rats. II. Kinetic parameters of transport and phase state of the lipid bilayer in mutant Campbell strain rats]. / Fiziko-khimicheskie mekhanizmy transporta organicheskikh kislot v vezikulakh apikal'noi membrany kletok proksimal'nykh kanal'tsev pochki krys. II. Kineticheskie parametry transporta i fazovoe sostoianie lipidnogo bisloia u mutantnykh krys linii Kempbell.

The kinetics of 14C-para-aminohippuric acid (PAH) transport in the vesicles and the influence of the temperature on the initial rate of this transport were studied using a purified fraction of the apical membrane isolated from the kidney cortex of the Campbell strain rats with an autosomic recessive gene. The transport was brought about owing to the facilitate diffusion mechanism. At 36 degrees C the apparent Michaelis constant was equal to 29 mM, the maximum rate--62 nmol/min on 1 mg of protein, the inhibition constant for the PAH-transport by probenecid--1.5 mM. The temperature dependence of the initial rate of PAH-transport in vesicles and that of the rate of substrate splitting by alkaline phosphatase show the break point on the Arrhenius plot at 36 degrees C-38 degrees C. The analysis of electron magnetic resonance reveals the thermotropic transition at temperatures near 30 degrees-35 degrees C. Therefore the affinity of the carrier to its substrates in vesicles of the Campbell strain rats is strongly reduced and the lipid layer is more viscous than in the normal rats. We decide therefore that the mutation taking place in the Campbell strain leads to pleotropic membrane reconstructions in different organs (eye, kidney). The discovery of such a mutation is of considerable biological interest and promotes bases for development of the membrane biochemical genetics.

[Distribution of the tempo-dichlorotriazine spin label in the immunoglobulin molecule]. / Raspredelenie spinovoi metki tempo-dikhlorotriazina po molekule immunoglobulina.

Spin-label 14C-TEMPO-dichlorotriazine distribution in Fab- and Fc-fragments of monoclonal myeloma immunoglobulin IgG (lambda) 1 Bel, in their heavy and light chains, and tryptic fragments of chains was investigated. Radio-active spin label in proteolytic Fab- and Fc-fragments, in heavy and light chains, and in separate peptides was detected. 3 cm and 2 mm band ESR spectra of spin-labeled IgG in water solutions was studied. It was shown that the models of highly anisotropic motion, isotropic motion and slow isotropic motion of spin label around globule or the description of experimental ESR spectra of TEMPO-dichlorotriazine spin-labeled immunoglobulins do not work.

[Analysis of existing models of spin label movement during spin labeling of biological macromolecules]. / Analiz sushchestvuiushchikh modelei dvizheniia spinovoi metki pri spin-mechenii biologicheskikh makromolekul.

The spin-labeled bovine serum albumin and IgG were studied in search of an experimental approach for comparison of different models of rotational mobility of spin label. These models are: the model of isotropic motion of spin label together with the macromolecule (IM); the model of highly anisotropic motion of spin label (HAM); and the model of slow isotropic motion of label around the binding site (SIML). The experimental spectra were measured on a common X-band ESR spectrometer and on the unique 140 GHZ (lambda = 2 mm) ESR spectrometer under the same conditions. Theoretical spectra were computer-calculated according to Freed's theory. We have found, that the results of temperature-viscosity experiments in X-band are contradictory to the model of IM both for the BSA and IgG species. The models of HAM and SIML for the BSA give identical X-band spectra. The bovine serum albumin spectra in the 2 mm region strongly contradict to the assumptions of the HAM model. Also, the SIML model fails to describe the experimental spectra in terms of isotropic motion of the spin label around the binding site. X-band spectra of IgG can not be explained by the SIML model, while the same spectra in the 2 mm region can not be explained by the HAM model.

[Spectral division of conformational states of spin-labeled tRNA Phe from Escherichia coli]. / Spektral'noe razdelenie konformatsionnykh sostoianii na spin-mechenoi tRNK Phe iz Escherichia coli.

A spectral division method of two conformational states of spin-labeled macromolecules is presented. The method is suitable in conditions of highly anisotropic motion of spin label and is based on titration of experimental spectra of spin-labeled macromolecule by theoretical ones. Theoretical spectra simulation uses the Freed theory and spin-Hamiltonian parameters, derived from independent experiments. Nomogrammes and formula for calculation of order parameter Sz and correlation time tau c in temperature-viscosity experiment are available. The method was applied to spectral division of two conformational states of spin-labeled tRNAPhe from E. coli and spectral parameters Sz and tau c were obtained for both states. ESR spectra of these conformational states at t degree = 20 degrees differ strongly from one another by order parameter Sz. The first conformer, that is characterised by a greater order parameter has no globular conformational transition (in terms of changes of the hydrodynamic macromolecule radius) between 2 degrees and 20 degrees, but local conformational changes take place in this temperature region.

[Study on the conformational state of Escherichia coli tRNA-Phe in solution by ESR-spectometry without modulation]. / Izuchenie konformatsionnoi situatsii v vodnykh rastvorakh spin-mechenoi tRNA-Phe iz Escherichia coli bezomduliatsionnym metodom EPR.

ESR-spectrometry without modulation of the magnetic field was used for registering the EST spectral line shape (with shape distortion about 0.1 percent) of spin-labeled Escherichia coli tRNAPhe. The analysis of line shape of two different spin-labels in position 8 (S4U) revealed that tRNAPhe in solution always exists as a mixture of at least two conformers, the equilibria between conformers being dependent on pH, concentration of magnesium and the biological state of tRNA (deacylated, aminoacyl- or peptidyl-tRNA). There are no large structural rearrangements upon aminoacylation or peptidylation of tRNA, the observed small changes of spectral line shape are due to the changes in conformational equilibria.

Study on conformational states of Escherichia coli tRNAPhe in solution by a modulation-free ESR-spectrometer.

A modulation free Electron Spin Resonance spectrometer was used for the registration of spectral absorption lines of a spin-labeled Escherichia coli phenylalanine tRNA in solution with low (less than 0.1%) line shape distortion. The analysis of line shape of two different spin-labels introduced into position 8 revealed that phenylalanine tRNA in solution exists as a mixture of two conformers, the equilibria between conformers being dependent on pH, concentration of magnesium and functional state of tRNA (deacylated, aminoacylated or peptidylated). There are no overall structural rearrangements upon aminoacylation or peptidylation of tRNA. The observed small changes of spectral line shape can be assigned to shifts in conformational equilibria.