[Catalytic properties of acetylcholinesterase, modified by N,N-dimethyl-2-phenylaziridinium. An alkane sulfonation reaction].

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.

[Mechanism of the reaction of N,N-dimethyl-2-phenylaziridinium with acetylcholinesterase in its active site].

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.

[Solubilization by various detergents, of the muscarinic cholinoreceptor and its complex with quinuclidinyl benzilate].

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).

[Synthesis of tritium-labeled N,N-dimethyl-2-phenylaziridinium and its reaction with acetylcholinesterase].

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.

[Kinetics of the inactivation of muscarinic cholinoreceptors by solubilized digitonin].

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).

[Kinetics of the N,N-dimethyl-2-phenylaziridinium reaction with a muscarinic cholinoreceptor and acetylcholinesterases].

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.

[Effect of pH on the cholinesterase hydrolysis of different substrates].

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.

[Inhibition of acetylcholinesterase hydrolysis of cationic substrates by the reaction product].

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).

[Acetylcholinesterase of cobra venom. Determination of concentration of active sites].

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.