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An original method for the integral evaluation of tissue and subcellular distribution of isatin binding proteins has been developed. This method is based on continuous monitoring of changes of optical characteristics that accompany complex formation between a ligand (immobilized on dextran bed of IAsys biosensor cell) and its soluble receptor. Solubilisation of tissue preparations and subcellular fractions with detergent (1% Triton X-100) is the important preconditions for the applicability of this method. The immobilisation of 5-aminoisatin was achieved by peptide bond formation between amino group of this isatin analogue and carboxyl group of the dextran bed of the biosensor cell. Addition of Triton X-100 treated preparations of membrane and soluble fractions of rat brain, liver, heart, and kidneys to the biosensor cell resulted in appearance of the characteristic response, indicating complex formation with the immobilised isatin analogue. The magnitude and a shape of kinetic curve vary in these samples. Isatin binding proteins predominated in membrane fractions of brain, liver and heart preparations whereas in the kidneys the highest isatin-binding response was detected in the soluble fraction. The distribution of isatin binding sites in the particulate fraction reduced in the following order: brainstem > brain hemispheres = cerebellum > heart > kidneys > liver. In the soluble fraction there was different rank of isatin binding activity: kidneys > heart > brainstem = brain hemispheres > liver > cerebellum. Liver outer mitochondrial membranes are characterised by the higher isatin-binding than mitochondria. Treatment of mitochondria with clorgyline and deprenyl, specifically inhibiting MAO A and B, respectively, significantly reduced the magnitude of the biosensor response and changed the shape of the kinetic curve. These data are consistent with the notion that within mitochondria MAOs are the major targets of isatin.

A rate of utilization of 4-phenyl piperidine and its 12 derivatives by brain monoamine oxidase (MAO) was studied as compared with typical neurotoxin 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP). The enzyme was isolated from P2 synaptosomal fraction of brain corpus striatum of Sprague-Dawley rats. 10 drugs were oxidized in the MAO-catalyzed reaction with the rate close or similar to the MPTP oxidation while 6 of them exhibited the neurotoxic effect. Analysis of MAO inhibition, using 1 microM of chlorgyline and/or deprenyl, enabled to evaluate the contribution of MAO-A and MAO-B forms to utilization of 1 mM content of the substances studied. MAO-B was shown to oxidize preferably the drugs radicals of which were substituted at 3rd position of the piperidine ring, while MAO-A preferred the derivatives with radical substitution at 4th position. The rate of substrate oxidation was decreased distinctly after introduction of complete substituents into the 3rd and 4th positions of the nitrogenous heterocycle; at the same time, presence of cyclic fluorine-containing structures increased the rate of utilization, similar to that of MPTP oxidation. Derivatives of 4-phenyl piperidine, which contained in a number of drugs, were oxidized in the MAO-catalyzed reactions and might exhibit direct- or side-neurotoxic effects.

Incubation of aldehyde dehydrogenase-free mitochondrial preparations with biogenic amines serotonin, tyramine, 2-phenylethylamine and 5-methoxytryptamine resulted in inhibition of enzymes activity of both outer (rotenone-insensitive NADH-cytochrome c reductase) and inner (succinate dehydrogenase, succinate cytochrome c reductase) mitochondrial membranes. Solubilization of mitochondria after the incubation did not influence the amine-induced alteration of succinate dehydrogenase activity. Pretreatment of the organelles with a mixture containing chlorgyline and deprenyl completely inhibited monoamine oxidase (MAO) activity and prevented the effects of all the amines studied on mitochondrial enzymes. MAO-dependent effects of 5-methoxytryptamine were fully reproduced by 5-methoxyindolyl-3-acetaldehyde (one of probable products of 5-methoxytryptamine deamination). The effect of the aldehyde was not prevented by chlorgyline and deprenyl. After selective inhibition of MAO-A by chlorgyline the order of MAO-B-dependent effects of biogenic amines on mitochondrial enzymes studied was as follows: tyramine greater than or equal to 2-phenylethylamine much greater than serotonin. In deprenyl pretreated mitochondria the potency of MAO-A-dependent effects of these amines was: serotonin greater than tyramine much greater than much greater than 2-phenylethylamine. The data obtained suggest that the product(s) of oxidative deamination of biogenic amines (probably the aldehydes) catalyzed by both types of MAO (MAO-A and MAO-B) are able to regulate the energy functions of mitochondria.

Chlorgyline, an inhibitor of type A monoamine oxidase, administered to rats prevented oxygen-induced decrease in gamma-aminobutyric acid brain content in hyperoxia. At the same time chlorgyline had an overall protective effect, increasing two-fold the period prior to the development of oxygen convulsions. The data suggest an important role of the modification of monoamine oxidase catalytic properties in the development of oxygen-induced intoxication and, particularly, in the decrease of gamma-aminobutyric acid content in hyperoxia.

Mitochondrial monoamine oxidases from rabbit and rat heart muscles varied distinctly in the sensitivity to the effect of selective irreversible inhibitors clorgyline and deprenyl. In presence of mitochondrial fragments from rabbit heart oxidation of phenylethylamine, tyramine, benzylamine and triptamine was highly sensitive to the effect of deprenyl. Very low concentrations of clorgyline inhibited oxidation of these amines in samples containing rat heart mitochondrial fragments. Rabbit heart mitochondrial fragments appear to contain considerable amounts of monoamine oxidases of "B" type and rat heart mitochondrial fragments--the monoamine oxidases of "A" type. A highly sensitive fluorescent method was used for estimation of the monoamine oxidase activity; it was based on the extinguishing by hydrogen peroxide in presence of peroxidase of the scopolethine fluorescence.

The denaturating effects of urea on clorgyline-produced inhibition of serotonin and tyramine deamination and deprenyl-produced inhibition of beta-phenylethylamine and tyramine oxidation were studied. It was shown that after preincubation of mitochondria with 1 and 2 M urea the intensity of inhibition by clorgyline and deprenyl of oxidation of these amines was not changed. With urea concentration of 3 and 4 M the inhibitory effect of clorgyline on deamination of serotonin and tyramine was increased, while that of deprenyl on oxidation of beta-phenylethylamine and tyramine was decreased. As a result of mitochondria treatment with 3 and 4 M urea the selectivity in inhibition by clorgyline of serotonin and tyramine deamination typical for intact mitochondria was reduced in the case of 3 M urea and eliminated in the case of 4 M urea. In intact mitochondria the intensity of inhibition by clorgyline of tyramine deamination in the presence of benzyl alcohol (competitive reversible MAO inhibitor) was increased, but the additive effect was not achieved. However, after preincubation of mitochondria with 3 M urea the summation of the inhibitory effects of clorgyline and benzyl alcohol was observed. The data obtained provide further evidence for the important role of spatial configuration of the monoamine oxidase molecule; the data are discussed in terms of arrangement on the protein molecule surface of the essential groups involved in the binding and deamination of amines for the inhibitory effects of clorgyline and deprenyl.

During hyperoxia monoamine oxidase type A acquires the ability to deaminate polyamines and histamine. A preliminary injection of clorgyline (a monoamine oxidase type A inhibitor) before hyperoxic exposure leads to a significant removal of oxygen seizures and prevents changes in the cerebral spermidine and histamine content observed in the unprotected animals. The data confirm the important role of modification of catalytic properties in monoamine oxidase in the mechanism of oxygen intoxication.

Oxidative deamination of tyramine, serotonin and benzyl amine in bovine kidney mitochondria was inhibited by low concentrations of deprenil (but not clorgylline). These data and the patterns of the plots of residual enzymatic activity against concentration of the inhibitors as well as the "mixed substrates" experiments suggest occurrence in bovine kidney mitochondria of monoamine oxidase of the B type only, which has to be considered in studies on the nature of monoamine oxidases using bovine kidney mitochondria as a rich source of monoamine oxidase activity.

The inhibition by chlorgyline and deprenyl of deamination of tyramine, i. e. substrate of two forms of monoamine oxidase (MAO) A and B, by fragments of rat liver mitochondrial membrane and the effects of competitive reversible inhibitors of the MAO activity, e. g. 4-ethylpyridine, benzyl alcohol, O-benzyl-hydroxylamine and 2-oxyquinoline, on this process were studied. It was shown that all the inhibitors used sharply increase the inhibiting effect of chlorgyline on tyramine deamination, the degree of the stimulating effect being the same irrespective of whether the inhibitors are added to the samples before or after a 30-min preincubation of chlorgyline with the enzyme at 23 degrees, i. e. after the onset of irreversible inhibition. The stimulating effect is due to the independent action of two inhibitors on the two different sites of the MAO active center: chlorgyline--on the isoalloxazine ring of FAD, that of 4-ethylpyridine, benzyl alcohol, O-benzylhydroxylamine, 2-oxyquinoline, respectively, on the hydrophobic region involved in tyramine binding. In similar experiments with deprenyl all the competitive inhibitors used, when added to the samples after a 30-min incubation of the inhibitor with the enzyme at 23 degrees, remove the inhibiting effect of deprenyl on tyramine deamination. The decrease of the inhibiting effect of deprenyl is indicative of an existence of competitive interactions between deprenyl and the above-mentioned compounds and of the reversible inhibition by deprenyl of tyramine deamination under the given experimental conditions. The data obtained revealed the differences in the type and mechanism of action of chlorgyline and deprenyl on tyramine deamination and showed that these inhibitors act on different sites of the MAO active center, responsible for tyramine oxidation. Chlorgyline blocks primarily the "flavin moiety" of the MAO molecule, essential for the catalytic act, while the effect of deprenyl is directed to the hydrophobic part of the enzyme active center essential for the enzyme binding to tyramine. In this case the irreversible inhibiting effect is achieved at a slower rate and the reversibility of tyramine oxidation by deprenyl is maintained for a longer period of time than the chlorgyline inhibition of deamination of this amine.

A simple kinetic method is developed for estimation of the monoamine oxidase (MAO) activity in solubilized mitochondria and purified preparations of MAO from rat liver tissue. The method is based on the property of alcohol dehydrogenase (from horse liver tissue) to catalyze the reduction of fatty-aromatic aldehydes (products of the MAO reaction) to appropriate alcohols in presence of NADH2 excess. The rate of the coupled reaction was evaluated spectrophotometrically by a decrease of the optic density at 340 nm. The optimal conditions were ascertained for coupling of these two reactions. Application of several amines was shown to be possible for estimation of the MAO activity by the kinetic method developed.

Statistically significant inhibition of oxidative deamination of several biogenic amines was found in mitochondrial fraction of heart muscle from rabbits and rats with experimental atherosclerosis and hypercholesterolemia, caused by various methods. Sensitivity of oxidative deamination of tryptamine to inhibitory effect of clorgyline (selective inhibitor of mitochondrial monoamine oxidases) was unaltered in rats with experimental hypercholesterolemia. The rate of oxidative deamination of beta-phenyl ethylamine, tryptamine, benzylamine as well as histamine and putrescine was inhibited after treatment of the mitochondrial fragments from healthy rabbit and rat heart muscles with preparations of oxidized linoleic and linolenic acids, concentration of which was increased in tissues under conditions of atherosclerosis.

Under conditions of hyperoxia mitochondrial monoamine oxidase (MAO) of the A type from rat brain proved to be able to deaminate gamma-aminobutyric acid (GABA); the transformation in the enzymatic properties appears to be responsible for a decrease in content of the intermediator in brain. Preadministration of chlorgiline (inhibitor of MAO of the A type) into animals before hyperoxygenation prevented completely the GABA content decrease, not affecting the glutamate decarboxylase activity, which was decreased in hyperoxia. At the same time, chlorgiline exhibited the total protective effect increasing 2-fold the period before oxygen convulsions.

A comparative study of the inhibitory effect of clorgyline and deprenyl on serotonin, beta-phenylethylamine and tyramine deamination by intact mitochondria as well as by solubilized with methylethylketone and partially purified monoamine oxidase (MAO) from rat liver was carried out. The effect of 4-ethylpyridine on this process was investigated. After solubilization of MAO by methylethylketone 7% of mitochondrial activity passes into solution and the rate of deamination of serotonin, tyramine and beta-phenylethylamine by soluble MAO preparation is selectively decreased. The corresponding residual activities are equal to 29, 63, 86 and 92%. The inhibitory effect of clorgyline on serotonin deamination by soluble MAO preparations is considerably lower than that by mitochondrial suspensions at the concentrations of the inhibitor from 1 x 10(-4) to 1 x 10(-7) M, while the inhibitory action of clorgyline on tyramine deamination after MAO solubilization by methylethylketone is increased at 10(-4) and 10(-5) M, but decreased insignificantly at 10(-6) and 10(-7) M. When solubilized MAO preparations are used, 4-ethylpyridine introduced into the sample before or after preincubation of the enzyme with clorgyline (30 min, 23 degrees) eliminates the inhibitory action of the latter on serotonin and tyramine deamination, thus suggesting the reversibility of the inhibitory effect of clorgyline. In similar experiments with mitochondrial suspensions the inhibition by clorgyline of deamination of these amines is irreversible. Similar experiments on mitochondrial suspensions showed that clorgyline irreversibly inhibits the deamination of these amines. The rate of inhibition by deprenyl of beta-phenylethylamine oxidation due to MAO solubilization by methylethylketone is not changed. 4-Ethylpyridine added to the samples before or after preincubation of deprenyl with the enzyme (30 min, 23 degrees) abolishes the inhibiting effect of the former both in soluble MOA and in mitochondrial suspensions. This suggests that under the given experimental conditions the inhibiting effect of deprenyl is reversible. Possible nature of MAO forms A and B is discussed.

The phenomenology of inhibition of FAD-containing type A monoamine oxidase by clorgyline solutions containing negligibly small amounts of clorgyline that are insufficient for stoichiometric covalent blocking of a perceptible amount of the coenzyme was studied. The nature of this phenomenon consists in the fact that at monoamine oxidase concentrations of about 10(-8) M, more than 50% of the enzyme activity in inhibited by clorgyline (less than or equal to 10(-10) M), although is accordance with a well-defined mechanism after monoamine oxidase-catalyzed tautomerization clorgyline presumably interacts with FAD at a 1:1 stoichiometric ratio. This effect termed as secondary inhibition seems to be induced not by clorgyline proper, not by changes in the solvent induced by this compound. In other words, clorgyline may initiate the synthesis of a new hypothetical inhibitor (IIC) in aqueous media which causes a reversible inhibition of the same specific inhibitory site of the enzyme. This site is responsible for the initial binding of acetylene inhibitors and catalyzes the formation of their allenic derivatives.

Chlorgyline, a selective monoamine oxidase (MAO) of the A type inhibitor, contrary to deprenil--MAO of the B type inhibitor, prevented the appearance in the rat liver mitochondrial membrane fragments incubated under aerobic conditions of qualitatively new properties to deaminate histamine or AMP. Apparently the MAO of A type but not of B type underwent a qualitative alteration (transformation) of catalytic properties under conditions favourable for partial oxidation of SH-groups.

The inhibition of the deamination of serotonin (the main substrate of monoamine oxidase (MAO) type A) by chlorgiline and deprenyl and of beta-phenylethylamine (the main substrate of the B type MAO) by fragments of rat liver mitochondrial membrane as well as the influence of 4-ethylpyridine on this process were studied. It was shown that the MAO activity of the mitochondrial membrane fragments was highly sensitive to chlorgiline, when serotonin was used as substrate, whereas a high sensitivity toward deprenyl was observed with beta-phenylethylamine as substrate. 4-Ethylpyridine (5.10(-3) M), a competitive and reversible inhibitor of the MAO activity, inhibited deamination of serotonin and beta-phenylethylamine by 34 and 30%, respectively. In experiments with chlorgiline (the specific inhibitor of MAO type A) 4-ethylpyridine (5.10(-3) M) introduced into the samples after preincubation of mitochondria with increasing concentrations of chlorgiline (30 min, 23 degrees C) decreased the inhibition by chlorgiline of the deamination of beta-phenylethylamine, but sharply increased the inhibitory effect of chlorgiline on the oxidation of serotonin. In analogous experiments with deprenyl (the specific inhibitor of MAO type B) 4-ethylpyridine (5.10(-3) M) decreased the inhibitory effect of deprenyl not only on the deamination of serotonin (substrate of MAO A), but also on the oxidation of beta-phenylethylamine (the main substrate of MAO type B). The decrease in the inhibitory effect of deprenyl on the deamination of beta-phenylethylamine after the addition of 4-ethylpyridine may be intensified upon preincubation of deprenyl with mitochondria in the presence of 4-ethylpyridine. The data obtained demonstrate the difference in the type and mechanism of inhibition of the deamination of serotonin by chlorgiline as well as in the type and mechanism of oxidation of beta-phenylethylamine by deprenyl. The possible mechanism of selective blocking of MAO activity by chlorgiline and deprenyl was discussed in terms of our previous data on the existence in the active center of mitochondrial MAO of specific sites for substrate binding, differing in their structure-functional characteristics.

Administration of monoamine oxidase type A inhibitor clorgyline to rats before hyperoxia prevented oxygen-induced increase in diene conjugate and Shiff's base brain and plasma levels in hyperoxia. This was due to antioxidative effect of clorgyline which resulted in stabilization of blood cellular membranes. Clorgyline had a normalizing effect on extraerythrocyte hemoglobin level, total peroxidase activity and glucose-6-phosphate dehydrogenase activity in the serum.