The mechanism of coupling of the organic anion exchange to Na(+)-dicarboxylate symport in basolateral membrane vesicles.

The rates of p-aminohippurate (PAH) and of uric acid uptake by basolateral membrane vesicles isolated from proximal tubules of rat kidney have been investigated. Accumulation of both substrates against the concentration gradient within the vesicles was shown to occur in the presence of alpha-ketoglutarate (alpha-KG) and Na+ gradient in the incubation medium. The mechanism of the coupling between Na(+)-dicarboxylate symport and organic anion transport is discussed based on the differences between the rates of PAH and of uric acid uptake. It is proposed that the limiting step of coupling between two transporters is the step of alpha-KG-organic anion exchange.

Affinity identification of organic anion transporters in brush-border membrane vesicles from rat kidney.

The inhibitory properties of bromoacetyl-p-aminohippuric acid as the affinity probe of the organic anion transport system were studied. Bromoacetylated p-aminohippurate was shown to be able to inhibit irreversibly the p-aminohippurate (PAH) uptake in brush-border membrane vesicles. The inhibition depends on both the time of treatment and the affinity probe concentration. The treatment of brush-border membrane with 1 mM bromoacetyl-p-aminohippurate for 1.5 h results in 100% irreversible inhibition of PAH transport but no changes were observed in the activity of alkaline phosphatase, gamma-glutamyltranspeptidase or maltase. The affinity labelling of the organic anion transporters was performed with bromoacetyl-p-amino[3H]hippuric acid. It was shown, by means of SDS-polyacrylamide gel electrophoresis, that the probe bound covalently to the brush-border membrane proteins with molecular masses of 28 kDa, 63 kDa, 98 kDa, and > 150 kDa. The data obtained with SITS and probenecid as the organic anion transport inhibitors indicate that brush-border membrane proteins of 28 kDa, 63 kDa, 98 kDa may correspond to the organic anion transport system.

[A quantitative study of the nonspecific component in p-aminohippurate transport in the apical membrane vesicles]. / Kolichestvennoe izuchenie nespetsificheskoi sostavliaiushchei transporta p-aminogippurata v vezikulakh apikal'noi membrany.

Separate components of the p-aminohippurate (PAH) nonspecific uptake were studied quantitatively using brush border membrane vesicles of the rat kidney cortex. It is shown that nonspecific PAH uptake is due only to membrane surface sorption, when diffusion and the Donnan ion distribution take no part in PAH uptake by vesicles. The magnitude of sorption depends on the incubation time and may reach as much as 25% of equilibrium value of the total uptake of PAH.

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.

[Physico-chemical mechanisms of organic acid transport in the apical membrane cells of the proximal kidney tubules in rats. I. Kinetic transport parameters and effect of the lipid phasic state on transport]. / Fiziko-khimicheskie mekhanizmy transporta organicheskikh kislot v vezikulakh apikal'noi membrany kletok proksimal'nykh kanal'tsev pochki krys. I. Kineticheskie parametry transporta i vliianie fazovogo sostoianiia na transport.

The kinetics of the transport of 3H-para-aminohippuric acid (PAH) and the influence of the temperature on the initial rate of transport were studied on the vesicles of a purified fraction of the apical membrane isolated from cells of kidney proximal tubules. The PAH transport is accomplished owing to the facilitate diffusion mechanism. The apparent Michaelis constant at 36 degrees C was equal to 7.0 + 1.0 mM, the maximum rate was 15 nmol/min on 1 mg of protein, the inhibition constant for the PAH transport by probenecid being 0.5 mM. At 22 degrees C the apparent Michaelis constant was drastically increased. When the temperature dependence of the initial rate of PAH transport into vesicles was replotted in the form of the Arrhenius plot, there was a turning-point of the line at 28-30 degrees C. The same turning-point is shown on the Arrhenius plot for temperature dependence of alkaline phosphatase activity (a marker enzyme for the apical membrane). The electron paramagnetic resonance spectra analysis of 5-doxylstearate-labeled apical membrane preparation reveals a thermotropic transition near 21-29 degrees C. It is concluded that the function of the carrier and the activity of alkaline phosphatase depend on the phasic state of membrane lipids; the normal function of membrane proteins is possible under the liquid-crystalline state of the lipid bilayer.

[Effect of a NaCl gradient on the transport of para-aminohippuric acid into the vesicles of the basolateral membrane of the kidney cortex]. / Vliianie gradienta NaCl na transport paraaminogippurovoi kisloty v vezikuly bazolateral'noi membrany kory pochki.

Basolateral membrane vesicles were isolated from the rat kidney cortex by a modified method of cation precipitation. Different steps of preparation were analysed using the marker enzymes: Na+,K+-ATPase (for basolateral membrane), alkaline phosphatase (for apical membrane), glucose-6-phosphatase (for membranes of endoplasmic reticulum) and succinate dehydrogenase (for mitochondria). The basolateral membrane was purified by a 8-9-fold treatment with Na+,K+-ATPase, while other membrane contaminations were as low as 2% (as compared to homogenate). The transport of 3H-p-aminohippurate (3H-PAH) by basolateral membrane vesicles was measured under different experimental conditions. The 3H-PAH uptake was found to be Na-gradient dependent. The initial rate of 3H-PAH uptake in the presence of NaCl gradient (500 pM/mg X min) was higher than without the gradient (88 pM/mg X min). It is concluded that the PAH transfer across the basolateral membrane may be energized by the Na+ chemical gradient.

Effects of microwave irradiation on some membrane-related processes in bacteria.

In a series of experiments performed on intact cells or spheroplasts of E. coli and Bac. subtilis a possibility of non-thermal effects induced by continuous microwave irradiation of a low power density (at wave length range from 0.0 to 7.8 mm) was studied. Thymidine and thymine uptake, leakage of potassium and hydrogen ions as well as the uptake of the transforming DNA by the component cells of Bac. subtilis were shown to be affected in a way typical of that due to heating of a sample. No specific dependence of the effects observed on wavelength was found.

[Mechanism of catalysis by o-diphenol oxidase]. / Mekhanizm kataliza o-difenoloksidazoi.

The reaction of terminal oxidation of the substrate (catechol) by molecular oxygen catalyzed by o-diphenoloxidase (o-diphenol: oxygen oxydoreductase; EC 1.10.3.1) is found to occur via a free radical mechanism. The copper of the active center changes its valency during the reaction. The spectra of substrate radicals and of the Cu2+ ions were registered by means of a high sensitivity ESR-spectrometer and their concentrations were determined.

On the active transport of organic acids (fluorescein) in the choroid plexus of the rabbit.

The kinetics of active transport of an organic acid (fluorescein) through the membranes of the choroid plexus from the lateral ventricules of the brain of rabbit was studied both morphologically and functionally. It was shown that fluorescein is actively translocated through the apical and basal membrane of the epithelium and is accumulated in blood capillaries at a concentration exceeding one order of magnitude that in the incubation medium. The kinetic curves displaying saturation and the demonstration of inhibition by other acids shows that a specific carrier is involved in the transfer across the membrane. The active transport of fluorescein at 20 degrees C was found to be sodium independent. Total exclusion of sodium from the incubation medium does not change the Michaelis constant (Km) and maximal velocity (V). The active transport depends on the operation of (Na+ + K+)-ATPase as energy source but obviously no specific complexes with the participation of sodium are involved.

[Mechanism of action of D-amino acid oxidase. II. Evidence for the free radical mechanism of the reaction catalysed by the monomer form of the enzyme]. / Izuchenie mekhanizma deistviia oksidazy D-aminokislot. II. Dokazatel'stvo svobodno-radikal'nogo mekhanizma reaktsii monomernoi formy fermenta.

D-Amino acid oxidase was shown to dissociate into subunits in 2 M urea retaining the catalytic activity. This makes possible the direct observation of ESR spectra of the intermediate radical state of the enzyme when interacting with the substrate. We have shown that these radicals are really observable. Using the reversibility of the reaction and an equilibrium shift the amount of radicals can be increased up to 10% of all flavin groups present. The dependence of the radicals concentration on the amount of substrate and product can be predicted. The theory is confirmed by experimental data.

[The mechanism of action of d-amino acid oxidase. I. Evidence for a free radical mechanism of the reaction catalyzed b a dimeric form of the enzyme]. / Izuchenie mekhanizma deistviia oksidazy d-aminokislot. I. Dokazatel'stvo svebodno-radikal'nogo mekhanizma reaktsii, kataliziruemoi dimernoi formoi fermenta

d-Amino acid oxidase can oxidize the substrate to a ketoacid in the absence of oxygen. The stoichiometry of this reaction is precisely 1 molecule of keto acid for 1 molecule of enzyme, containing two flavin groups. Hence, the flavin must be in the semi-reduced free radical state. But these free radicals cannot be visualized by ESR spectroscopy because of closeness and strong interaction. After the acid denaturation of the protein the coenzyme is released as a semi-reduced free radical. An alternative method of registration is the transfer of the free radical state to an added excess of free flavin molecules. By both methods it is quantitatively determined that each flavin of the enzyme is reduced to a free radical. Therefore, we believe to have evidenced unambiguously that this enzymatic reaction proceeds via a free radical transition state.