[The study of effects of a novel acetylcholinesterase inhibitor on electrical activity of the heart].

We have investigated effect of a representative of the novel class of selective acetylcholinesterase inhibitors A 1,3-bis[5(diethyl-o-nitrobenzyl ammonio) penthyl]-6-methyluracildibromide (compound 547) on duration and rhythm of sequence of right atrial action potential (AP) as well as on kinetics of acetylcholinesterase catalyzed reaction in homogenates of skeletal muscle (m. extensor digitorum longus) and cardiac muscle in the rat. We have shown that contrary to classical acetylcholinesterase inhibitors armin and proserin none of studied concentrations (1, 10 and 100 nM) of compound 547 exerted significant effect on AP configuration and rate of sinus rhythm. Compound 547 belongs to noncompetitive type with K1(heart)=3.6 x 10(-4) M and K1(EDL)=1.3 x 10(-8) M. Proserin exerts comparable inhibitory action on reaction in the heart and skeletal muscle, its K1(heart)=0.73 x 10(-5) M and K1(EDL) = 0.4 x 10(-5) M. Thus low sensitivity of myocardium to compound 547 in electrophysiological experiments is not related to lesser availability of synaptic acetylcholinesterase in the heart compared with acetylcholinesterase in skeletal muscles but reaction catalyzed by cardiac acetylcholinesterase is actually to a substantial degree less prone to inhibition by compound 547.

[Selective labeling of barstar in a T7 polymerase system by 15N-isotopes].

It has been shown that the use of a special growth medium enriched with amino acids and an inhibitor of aminotransferases alpha-aminooxyacetic acid makes possible the selectivity of labeling of barstar with 15N-leucine and 15N-tryptophan. The system of selective labeling, which was previously optimized with respect to the time of introducing the label relative to the time of introducing the inductor IPTG and the inhibitor of cell polymerase rifampicin, was substantially refined by the use of the transamination inhibitor. The inhibition of aminotransferases enables one to completely eliminate the redistribution of the isotope, which is a necessary step in NMR studies even if the strongly metabolizable 15N-leucine is used. The suppression of the redistribution of the isotope by alpha-aminooxyacetic acid is a successful approach to preparation of any selectively labeled proteins in the T7 polymerase system.

[Kinetic study of the mechanisms of eliminating substrate inhibition of lactate dehydrogenase by anions and pH]. / Kineticheskie issledovaniia mekhanizma sniatiia substratnogo ingibirovaniia laktatdegidrogenazy anionami i pH.

The dependence of lactate dehydrogenase inhibition at high pyruvate concentrations on pH and neutral salt anions was studied. It was shown that Cl- anions compete with the substrate within the ternary inhibitory complex, ENADpyr in equilibrium ENADCl-, as a result of which the pyruvate-induced inhibition is eliminated. The KD values for Cl- (50 mM) and I- (27 mM) were calculated from the substrate velocity curves at high concentrations of pyruvate. It was supposed that pyruvate inhibition elimination by OH- proceeds via the same kinetic mechanism. The pK value (7.1 +/- 0.1) calculated from this model corresponds to pKn of essential His-195. The additivity of OH- and Cl- function was demonstrated.

[Kinetic manifestations of slow conformation rearrangements of lactate dehydrogenase. An experiment and a mathematical model]. / Issledovanie kineticheskikh proiavlenii medlennykh konformatsionnykh perestroek v laktatdegidrogenaze. Eksperiment i matematicheskaia model'.

Non-steady-state kinetics of lactate dehydrogenase (LDH) catalyzed reaction was investigated for a wide time interval (from 100 msec to 1-3 min) by using stopped-flow methods. A two-stage character of LDH reaction, slow changes like a lag-period on kinetic curves at pH 8.0, flexions on kinetic curves after pre-mixing LDH with NAD+ and pyruvate have been revealed. The graph theory for mathematical analysis of experimental data was applied, which has been developed for the non-steady-state kinetics. An enzyme model of the two-conformer LDH structure was used. The reaction scheme with a preferential inhibition of one of the conformers (pH 8.0) is suggested. The obtained values of kinetic constants prove that transitions between LDH conformers must be slow.

[Biphase reaction of enzymatic pyruvate reduction during inhibition]. / Bifaznost' reaktsii fermentativnogo vosstanovleniia piruvata v usloviiakh ingibirovaniia.

At least two stages have been revealed in the reaction of enzymatic reduction of pyruvate under inhibition. A fast process during the first stage of the reaction was finished in a period of "dead time" with routine spectroscopic measurements. A reaction rate at this stage decreased by three times. The second stage was characterized by a constant rate, which changed by less than 10%. The analysis of neutral salt influences on the inhibition complex showed, that the latter decomposed rapidly. A model suggested earlier, was used for interpreting the reversible product inhibition of the reaction catalysed by LDH.