[Catalytic properties of acetylcholinesterase, modified by N,N-dimethyl-2-phenylaziridinium. An alkane sulfonation reaction]. / Kataliticheskie svoistva atsetilkholinésterazy, modifitsirovannoi N,N-dimetil-2-fenilaziridiniem. Reaktsiia alkansul'fonilirovaniia.

By means of affinity labelling with N,N-dimethyl-2-phenylaziridinium ion (DPA) two forms of acetylcholinesterase were synthesized that contained one or two molecules of the label covalently attached to the enzyme. The reaction of native and covalently modified acetylcholinesterases with n-alkane sulfonyl chlorides CnH2n + 1SO2Cl at n = 1 -4 was used to characterize the reactivity and properties of the enzymes. It was found that labelling of acetylcholinesterase with one molecule of DPA did not affect the enzyme's reactivity. Acetylcholinesterase containing two labels (the second one presumably located at the anionic centre of the enzyme) displayed enhanced and more specific reactivity towards alkane sulfonyl chlorides. It was found that the phenomenon of acceleration caused by affinity modification is analogous to the influence of n-tetraalkylammonium ions on the same reaction. Therefore, the mechanism of regulation of the properties of the esteric centre, caused by affinity labelling of the enzyme at the anionic centre, is the same as in the case of n-tetralkylammonium ions.

[Mechanism of the reaction of N,N-dimethyl-2-phenylaziridinium with acetylcholinesterase in its active site]. / Mekhanizm reaktsii N,N-dimetil-2-fenilaziridiniia s atsetilkholinésterazoi v ee aktivnom tsentre.

Effect of temperature on the rate of the bond-breaking step of acetylcholinesterase modification with N,N-dimethylaziridinium ion was studied within 8 to 45 degrees C temperature interval. For this reaction measured by irreversible inhibition of the acetylcholinesterase-catalyzed hydrolysis of acetylthiocholine the activation parameters delta H not equal to = 94 kJ/mole and delta S not equal to (25 degrees C) = -9.4 J/mol X deg were obtained. Processing of these data together with our earlier results on spontaneous solvolysis of the aziridinium ion in various water-solvent mixtures showed that all these reactions form a common isokinetic series. That gave evidence of the SN1 mechanism of the alkylation reaction occurring at the acetylcholinesterase active centre. Kinetics of spontaneous decomposition of the covalent bond between the aziridinium reagent and protein molecule was studied. This reaction followed the first-order kinetics and lead to complete liberation of the label from the enzyme, thus suggesting that a single carboxylic or amide group in the active centre was modified by the aziridinium ion.

[Solubilization by various detergents, of the muscarinic cholinoreceptor and its complex with quinuclidinyl benzilate]. / Soliubilizatsiia nekotorymi detergentami muskarinovogo kholinoretseptora i ego kompleksa s khinuklidinilbenzilatom.

The possibilities to solubilize the rat brain cortex muscarinic acetylcholine receptor and its complex with [3H]-L-quinuclidinyl benzilate (QNB) were studied, using 14 detergents. It was shown that the native muscarinic cholinoreceptor was solubilized in addition to digitonin, also by CHAPS, with a 6% yield. Besides, the receptor-QNB complex was solubilized with the detergents Triton X-100, -102, -114, -165 (with 30% and 50% yields) and within a narrow concentration range with sodium dodecyl sulfate (50% yield). Some detergents of the Tween series, e.g., Triton X-45 and -305, as well as sodium deoxycholate and sodium oxycholate, did not solubilize the native receptor and its complex with QNB. It was found that yield of receptor solubilization did not exceed half of the total number of the receptor sites in the membranes, despite the fact that different concentrations of detergents were applied. The solubilization yield did not increase, when different mixtures of detergents were used. It was assumed that incomplete solubilization of the receptor protein reflects its heterogeneity in the membrane structure.

[Synthesis of tritium-labeled N,N-dimethyl-2-phenylaziridinium and its reaction with acetylcholinesterase]. / Sintez mechenogo tritiem N,N-dimetil-2-fenilaziridiniia i ego reaktsiia s atsetilkholinésterazoi.

The synthesis of tritium-labeled N,N-demethyl-2-phenylaziridinium has been described. The specific radioactivity of the product obtained was 1,06 TBq/mmole. Kinetics of incorporation of this radioactive label into acetylcholinesterase of cobra venom (Naja naja oxiana) has been studied at 1,05 mM ligand concentration (25 degrees C, pH 7,50. 0,15 M phosphate buffer). Under these conditions two molecules of the radioactive label have been found to react with the enzyme. One molecule incorporates fast with half-life of 4,8 min, not affecting the enzymatic activity. Incorporation of the second label is a slow reaction with half-life of 6 hr and leads to complete inactivation of acetylcholinesterase. Molecular mass of the modified enzyme is 63 +/- 4 kDa and coincides with that of native one.

[Kinetics of the inactivation of muscarinic cholinoreceptors by solubilized digitonin]. / Kinetika inaktivatsii soliubilizirovannogo digitoninom muskarinovogo kholinoretseptora.

The kinetics of spontaneous inactivation of the digitonin-solubilized rat brain muscarinic cholinoreceptor was investigated at 4 degrees, 15 degrees, 25 degrees, 35 degrees and 45 degrees C. The inactivation process was followed by the loss of specific L-[3H] quinuclidinyl benzilate binding capacity after incubation of the receptor at an appropriate temperature. Since the inactivation process of the receptor inactivation obeys the first order kinetics, it was possible to determine the values of inactivation rate constants (kappa in). It was shown that the inactivation rate does not depend on the detergent excess in a reaction mixture and is characterized by the apparent activation energy, Ea = 158 +/- 7 . KJ/mole and entropy, delta S not equal to = 249.8 J/K . mole. These values are in good agreement with those obtained for the water-soluble proteins, but differ essentially from the analogous values for the spontaneous activation of the membrane-bound receptor.

[Kinetics of the N,N-dimethyl-2-phenylaziridinium reaction with a muscarinic cholinoreceptor and acetylcholinesterases]. / Kinetika reaktsii N,N-dimetil-2-fenilaziridiniia s muskarinovym kholinoretseptorom i atsetilkholinésterazami.

Kinetics of the reaction of N,N-dimethyl-2-phenylaziridinium ions with soluble acetylcholinesterases from cobra venom and electric eel as well as with the membrane-bound acetylcholinesterase and the muscarinic acetylcholine receptor from the cerebral cortex of rat brain was investigated at pH 7,5 and 25 degrees C in 0,15 M phosphate buffer. The inhibition reaction involves the non-covalent binding step followed by the irreversible alkylation step. The spontaneous hydrolysis of the aziridinium compound and the reversible inhibition of the enzymes with the hydrolytic product were taken into account in data treatment. The aziridinium ions were found to bind with similar effectiveness in the active centers of acetylcholinesterases and the muscarinic acetylcholine receptor, however, for the latter the alkylation step is more than 10-fold faster. This difference can be explained by different solvation effects exerted by the active centers of these proteins. The rate constants of the protein alkylation reaction are compared with the kinetic data for decomposition reaction of the aziridinium ion in several solvent-water mixtures, studied separately in the case of five solvents. The basicity of the solvents was found to be the most important factor accelerating the solvolysis.

[Effect of pH on the cholinesterase hydrolysis of different substrates]. / Vliianie pH na gidroliz kholinesterazoi razlichnykh substratov.

The effect of pH on the kinetic constants of cholinesterase-catalyzed hydrolysis of isoamylacetate and acetylthiocholine was investigated. For the first substrate the rate-limiting step is the enzyme acetylation reaction, while the overall hydrolysis rate of the second substrate is limited by the deacetylation reaction. It was concluded that a basic group is involved in the deacetylation reaction and in the non-covalent binding step of both ionic and non-ionic substrates. An acidic group participates in the both reaction steps. It was suggested that protonation of the basic group leads to a conformational transition of the free enzyme and can also participate in the catalytic mechanism. The second basic group characterized by Ka3 can be the functional group of the enzyme anionic site. Taken together, the influence of pH on cholinesterase catalysis can be generalized by the overall reaction scheme.

[Inhibition of acetylcholinesterase hydrolysis of cationic substrates by the reaction product]. / Ingibirovanie atsetilkholinesterazy produktami gidroliza kationnykh substratov.

The kinetics of acetylcholinesterase-catalyzed hydrolysis of the two cationic substrates (I and II in Russian text) was analyzed by means of the integrated Michaelis equation (3). The constants kII, kcat Km and the enzyme-product complex dissociation constant Ki were determined. (Table 1). It was shown that acetylcholine (II) binds to to the enzyme active center more effectively than the alcohol product of its hydrolysis. In case of the pipecholine derivative (I) reversed situation occurs. The different dependence of the ester substrate and appropriate alcohol binding effectiveness upon the reagent structure indicates the dissimilar location of the molecules in the active center of acetylcholinesterase. Some structural implications of the enzyme active center were discussed.

[Acetylcholinesterase of cobra venom. Determination of concentration of active sites]. / Atsetilkholinesteraza iada kobry. Opredelenie kontsentratsii aktivnykh tsentrov

O-Nitrophenyl dimethylcarbamate and organophosphorus inhibitors O-n-propyl-p-nitrophenyl methylphosphonate, O-n-butyl-p-nitrophenyl methylphosphonate, O,O-diethyl-p-nitrophenyl phosphate, O-n-butyl-S-(beta-ethylmercaptoethyl) methylthiophosphonate, methysulphate of O,O-diethyl-S-(beta-phenyldimethylammoniumethly) thiophosphate were used in the titration of acetylcholinesterase active site concentratration in Naja naja oxiana venom. No side reactions with the acetylcholinesterase molecule as well as with other components of the venom were observed. In titration the effective concentrations of organophosphorus inhibitors with asymmetric phosphorus were 50% of their analytical concentrations, since cobra venom cholinesterase showed practically absolute stereoselectivity against the compounds.