[Anticonvulsive activity of antiepileptic drugs and quantum-chemical modelling of their interaction with GABA(A) receptor]. / Antikonvul'santnaia aktivnost' protivoépilepticheskikh preparatov i kvantovo-khimicheskoe modelirovanie ikh vzaimodeistviia s retseptorom GAMK(A).

The results of experimental analysis of the clinical activity of the antiepileptic drugs' (Phenobarbital, Carbamazepine, Valproic acid, Lamotrigine, Topiramate, Felbamate) widely used in clinic, that was carried out using the standard convulsion test with bicuculline in vivo were compared with characteristics of these drugs' interaction with the key aminoacids of GABA(A) receptor calculated by quantum chemical method (program HyperChem7, semi-empirical method AM1 technique). The correlation between the activity of the drugs in the experiment in vivo and energy of system's interaction of the drugs with aminoacid residue Thr201-Thr202-Gly203- Ala204-Tyr205-Pro206 was found out.

Kinetic characterization of the hydrolytic activity of the H+-pyrophosphatase of Rhodospirillum rubrum in membrane-bound and isolated states.

Substrate hydrolysis by the H+-pyrophosphatase (pyrophosphate phosphohydrolase, H+-PPase) of the photosynthetic bacterium Rhodospirillum rubrum follows a two-pathway reaction scheme in which preformed 1:1 and 1:2 . enzyme . Mg2+ complexes (EMg and EMg2) convert dimagnesium pyrophosphate (the substrate). This scheme is applicable to isolated enzyme, uncoupled chromatophores and chromatophores energized by a K+/valinomycin diffusion potential. Tris and other amine buffers exert a specific effect on the bacterial H+-PPase by increasing the Michaelis constant for substrate binding to EMg by a factor of 26-32, while having only small effect on substrate binding to EMg2. Formation of EMg requires a basic group with pKa of 7.2-7.7 and confers resistance against inactivation by mersalyl and N-ethylmaleimide to H+-PPase. The dissociation constants governing EMg and EMg2 formation, as estimated from the mersalyl-protection assays and steady-state kinetics of PPi hydrolysis, respectively, differ by an order of magnitude. Comparison with the data on soluble PPases suggests that, in spite of gross structural differences between H+-PPase and soluble PPases and the added ability of H+-PPase to act as a proton pump, the two classes of enzyme utilize the same reaction mechanism in PPi hydrolysis.

Differential sensitivity of membrane-associated pyrophosphatases to inhibition by diphosphonates and fluoride delineates two classes of enzyme.

1,1-Diphosphonate analogs of pyrophosphate, containing an amino or a hydroxyl group on the bridge carbon atom, are potent inhibitors of the H(+)-translocating pyrophosphatases of chromatophores prepared from the bacterium Rhodospirillum rubrum and vacuolar membrane vesicles prepared from the plant Vigna radiata. The inhibition constant for aminomethylenediphosphonate, which binds competitively with respect to substrate, is below 2 microM. Rat liver mitochondrial pyrophosphatase is two orders of magnitude less sensitive to this compound but extremely sensitive to imidodiphosphate. By contrast, fluoride is highly effective only against the mitochondrial pyrophosphatase. It is concluded that the mitochondrial pyrophosphatase and the H(+)-pyrophosphatases of chromatophores and vacuolar membranes belong to two different classes of enzyme.

[Superoxide dismutase in liver ischemia]. / Superoksiddismutaza pri ishemii pecheni.

The properties of Cu,Zn-superoxide dismutase (SOD) from rat liver after 2-hour total ischemia or after ischemia with subsequent 24-hour reperfusion were studied. Two hours after ischemia the specific activity of SOD decreases drastically (about 3-fold) - from 510 +/- 11 u./mg in normal tissue and 196 +/- 33 u./mg after ischemia showing a further increase after reperfusion (276 +/- 40 u./mg). Using competitive immunoenzymatic analysis, the relative contents of SOD in the cytosol were determined. After ischemia the SOD content in the cytosolic fraction decreased (approximately 3-fold) but returned to the initial level after reperfusion. Polyacrylamide gel electrophoresis revealed that in control samples active SOD is heterogeneous and produces 3-4 bands, similar to the purified SOD from rat liver. After the ischemia the intensity of minor fast band IV increased and a new band V of a still higher mobility appeared. After the reperfusion the electrophoretic patterns were similar to control. Two or three times more SOD antigen from ischemia liver cytosol was absorbed to the surface of polystyrol plate in a direct sorption enzyme immunoassay procedure as compared to that from intact liver cytosol. It is suggested that the decreases of amount and the activity as well as changes of properties of SOD could be due to its oxidative modification and degradation of the modified enzyme.

Use of biotinylated inorganic pyrophosphatase for detection of biotin bound to solid support.

A colorimetric procedure to detect biotin bound to microtiter plates with a sensitivity down to 10(-16) mol was developed using biotinylated inorganic pyrophosphatase of Escherichia coli. Reaction of pyrophosphatase with 1 mM N-biotinyl-6-aminocaproic acid N-hydroxy-sulfonosuccinimide ester yielded a stable 87% active enzyme containing 5.6 mol biotin/mol. In the measurements of human immunoglobulin G, a biotinylated pyrophosphatase.streptavidin complex provided a sensitivity superior to that of conventional enzyme immunoassay due to low nonspecific binding. The new procedure was also more sensitive compared with that using biotinylated alkaline phosphatase. Together with high thermostability of pyrophosphatase and its substrate, low background staining allowed measurement of enzymatic activity to be performed at 60 degrees C for 4 h resulting in a marked increase in assay sensitivity.

[A comparison of pyrophosphatase and peroxidase as markers in the immunoenzyme analysis of the tick-borne encephalitis virus and antibodies to it]. / Sravnenie pirofosfatazy i peroksidazy kak markerov v immunofermentnom analize virusa kleshchevogo éntsefalita i antitel k nemu.

The authors compare the sensitivities of pyrophosphatase and peroxidase conjugates with immunoglobulins in assays of tick-borne encephalitis (TBE) virus antigens and anti-TBE antibodies in human sera. Pyrophosphatase conjugates are similar to peroxidase ones in their enzymic and immunochemical characteristics and are not inferior to them in sensitivity in titration of antibodies and superior in assays with antigens. A number of advantages of pyrophosphatase conjugates, i.e. a simple preparation procedure, stable substrate and enzymic reaction product, color range convenient for visual recording, etc., make these conjugates preferable vs. the peroxidase ones and recommend them for wide use at diagnostic laboratories.

[Use of inorganic pyrophosphatase as a marker in enzyme immunoassays]. / Ispol'zovanie neorganicheskoi pirofosfatazy v kachestve markera v immunofermentnom analize.

A technique of heterogeneous enzyme immunoassay with the E. coli inorganic pyrophosphatase as marker enzyme and Malachite green dye and acidic molybdate as color reagent is developed. Color change (light-yellow/greenish blue) is extremely suitable for visual perception, in some cases making unnecessary the measuring device. Assays with pyrophosphatase are 5-10 times more sensitive than with peroxidase. Further advantages of pyrophosphatase include high thermostability, insensitivity to sodium azide, low value of Michaelis constant (5 microM), substrate stability. Examples are given of use of the pyrophosphatase for assays of human alpha-fetoprotein and immunoglobulin.

A malachite green procedure for orthophosphate determination and its use in alkaline phosphatase-based enzyme immunoassay.

An improved procedure for phosphate determination based on a highly colored complex of phosphomolybdate and malachite green is described. All necessary reagents are combined in one concentrated solution, making the assay sensitive and convenient. The procedure is based on the finding that the dye is easily soluble and stable in the presence of 6 N acid. The addition of Tween 20 is required to stabilize the dye-phosphomolybdate complex at phosphate concentrations above 10 microM. The time of color development at 25 degrees C is about 3 min. The procedure was adopted to measure alkaline phosphate activity in heterogeneous enzyme immunoassay with rho-nitrophenyl phosphate and pyrophosphate as substrates. In both cases, a 4-fold increase in sensitivity in terms of absorbance readings was obtained compared to the standard method based on rho-nitrophenol measurement. In visual analysis, the gain in sensitivity was as high as 20-fold, due to contrast color change (yellow to greenish blue).