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CGP 4718 A (4-[5-chloro-benzofuranyl-2-]-1-methylpiperidine HCl) was found to inhibit MAO A preferentially in vitro in a competitive manner. Assessment of its in vivo effects by an ex vivo approach showed it to be a relatively weak, reversible inhibitor of MAO A. There were also effects on MAO B but they were inferior by a factor of about 10. The onset of the inhibitory effects in rat liver and brain was rapid, being maximal in about 1 h following administration of CGP 4718 A p.o. The inhibition was of relatively short duration with the effects being undetectable 24 h after treatment. CGP 4718 A also inhibited the reuptake of serotonin (5-HT) in synaptosomes in vitro and ex vivo. Evidence for 5-HT uptake inhibition was also found by using the H 75/12 depletor model. Its in vitro and in vivo potency as a 5-HT uptake inhibitor was approximately the same as that of imipramine. The effects on MAO A and on 5-HT uptake occurred over a similar dose range (above 10 mg/kg p.o.) and also had a similar time course. No evidence for inhibitory effects on noradrenaline uptake was found in vivo.

The effects of ligands on the irreversible inhibition of the NAD+-dependent isocitrate dehydrogenase from ox brain were studied. Isocitrate in the presence of Mg2+ ions was found to protect against denaturation at 40 degrees C and this protection was enhanced by ADP which also protected on its own. None of the substrates or the activator ADP afforded protection against inhibition by diethylpyrocarbonate. Inactivation by this compound and by elevated temperatures did not obey first-order kinetics with respect to time. Inhibition by iodoacetate appeared to obey first-order kinetics both with respect to time and to inhibitor concentration. Protection against inhibition was afforded by isocitrate, NAD+ and ADP. The dependence of the extent of protection on the concentration of the magnesium-isocitrate complex was sigmoid at both pH 6.5 and pH 7.5 indicating the apparent homotropic cooperativity seen in initial-rate kinetic experiments to be a reflection of cooperative binding of this substrate. ADP reduced the affinity of the enzyme for this substrate without affecting the degree of this cooperativity. The dependence of the extent of protection by ADP upon its concentration obeyed Michaelis-Menten kinetics. The significance of these observations in terms of the kinetic and allosteric mechanism followed by this enzyme is discussed in the light of previous kinetic studies.

Initial rate studies have been used to investigate the kinetic mechanism followed by the purified high-Km (AR1) form of rat brain aldehyde reductase at pH 7.0. The effects of varying the aldehyde and NADPH concentrations, together with the inhibition given by the products of the reaction, are consistent with the reduction of succinic semialdehyde and p-nitrobenzaldehyde following an ordered reaction mechanism involving the formation of an intermediate ternary complex and in which NADPH is the first substrate to bind to the enzyme. Both these aldehyde substrates inhibit the enzyme at higher concentrations. This inhibition, which is uncompetitive with respect to NADPH, suggests that many previous studies on the specificity of this enzyme, that have been based on the activity determined at a single arbitrary concentration of each substrate, may have given erroneous results.

A sensitive coupled assay for histidine decarboxylase has been developed. This method involved conversion of [3H]histidine into [3H]histamine by the enzyme sample, with methylation of this product in situ, catalysed by the enzyme histamine N-methyltransferase, to yield [3H]N-tele-methylhistamine. The radioactive product was separated from the substrate by (i) extraction into chloroform, (ii) ion-exchange chromatography and (iii) liquid cation-exchange extraction. The "no tissue" assay blank comprised 0.0007% of the substrate radioactivity. Sample material with a histidine decarboxylase activity of as little as 0.14 fmol/min/ml (measured at 1 microM histidine) gave double the blank value. More than 50 assays could be performed in one day. This assay was used to determine the in vivo changes in mouse brain histidine decarboxylase activity following irreversible inhibition with (+) alpha-fluoromethylhistidine (alpha-FMH). From the time course of recovery of enzyme activity the half-life of histidine decarboxylase in vivo was calculated to be 53 h.

The concentrations of monoamine oxidase-A and -B have been determined in mitochondria, mitochondrial outer membranes and microsomes from Sprague-Dawley and Wistar rats by determining the binding of tritium-labelled pargyline. Although the amounts of each form present depended on the source and the preparation method, this was paralleled by the specific activity such that the molecular turnover number was found to remain constant. The catalytic constants, kcat/Km, which represents the apparent second-order rate constant for the combination of enzyme and substrate, were about 0.13 and 2.1 sec-1 X microM-1 for 5-hydroxytryptamine and 2-phenethylamine, respectively, regardless of the source. Estimations of the amounts of the two forms by determining the concentrations of the inhibitors clorgyline, (-)-deprenyl, J-508 or pargyline necessary to give complete inhibition were shown to give overestimates of the true values because of the non-specific binding of these inhibitors to sites other than the monoamine oxidase active site.

CGP 11305 A (4-(5-methoxy-7-bromo-benzofuranyl)2-piperidine-HCl) inhibited serotonin (5-HT) deamination in brains and livers of pretreated rats. The ED50S were 1 and 0.7 mg/kg p.o., respectively. Phenylethylamine (PEA) deamination was only marginally affected up to doses of 100 mg/kg p.o. The duration of action of the compound was less than 48 h, and its effects did not cumulate after repeated oral treatment. Kinetic studies in mitochondrial preparations from both tissues of the rat showed that with both 5-HT and PEA as substrates the inhibition was of the competitive type when the enzyme preparation and the inhibitor were not preincubated prior to assay. These properties suggested a reversible interaction of the compound with the enzyme. However, in 'ex vivo' studies, the inhibitory activity of CGP 11305 A was not lost by dilution or dialysis of homogenates from pretreated animals and the inhibitor could not be displaced by 5-HT. Similar results were obtained when CGP 11305 A was preincubated with mitochondria or homogenates from rat liver in vitro, indicating an irreversible interaction. The apparent contradiction between the short duration of the MAO inhibitory effect of CGP 11305 A in vivo and the seemingly irreversible interaction with the enzyme under ex vivo and in vitro conditions has not yet been resolved, although a number of possible mechanisms have been considered. The short duration of action and the lack of cumulative effects of this powerful and selective monoamine oxidase-A inhibitor in vivo might, however, result in it being a valuable antidepressant.

The kinetic properties of membrane-bound glutaminase from pig brain have been compared with those of the soluble enzyme that had been preincubated with either Tris/HCl buffer or Tris/phosphate/borate buffer. The two preparations of the soluble enzyme were similar in the dependence of their activities on pH and in their inhibition by borate and some glutamine analogues. They differed markedly in their responses to phosphate and their inhibition by glutamate. The membrane-bound enzyme differed from the other preparations in showing a sigmoid dependence of initial velocity on glutamine concentration at higher pH values and being activated by borate ions. The apparently cooperative behaviour that has been previously reported for soluble preparations of glutaminase may have resulted from failure to measure the true initial rate of the reaction. The Km values for glutamine and the inhibitor and activator constants for glutamate and phosphate respectively were such as to indicate that changes in the concentrations of these compounds might affect the glutaminase activity in vivo. None of the enzyme preparations was inhibited significantly by ammonium ions at concentrations up to 50 mM. Considerable differences were found between the three preparations in their activation by compounds containing sulphate or sulphonate groups.

In the presence of glutaric acid, N2,N2'-adipodihydrazido-bis(N6-carbonylmethyl-NAD+)(bis-NAD+ ) forms cross-links between molecules of glutamate dehydrogenase, resulting in precipitation. The dependence of this process on bis-NAD+ and enzyme concentration has been investigated. This procedure has been shown to be effective in the purification of glutamate dehydrogenase from rat and ox liver, and a procedure is presented in which this affinity precipitation procedure is used instead of the affinity chromatography used in an earlier method (McCarthy, A.D., Walker, J.M. and Tipton, K.F. (1980) Biochem. J. 191, 605-611). The ox liver enzyme prepared in this way had not suffered the limited proteolysis that occurs during the preparation of the enzyme by other commonly used procedures. After the purified enzyme had been denatured by treatment with urea, guanidine hydrochloride, or low pH, no recovery of activity could be demonstrated following dilution or, in the last case, dialysis.

By use of the selective irreversible inhibitors clorgyline and selegiline [(-)-deprenyl], it is possible to determine the Km and Vmax values of the two forms of monoamine oxidase towards a variety of substrates. The validity of this procedure is demonstrated for the deamination of dopamine by rat liver monoamine oxidase -A and -B, and the specificities of the two forms are analysed in terms of their kinetic parameters towards different substrates.

The activity of beef heart mitochondrial monoamine oxidase towards 5-hydroxytryptamine (5-HT) is inhibited by the selective inhibitors clorgyline, PCO [5-phenyl-3-(N-cyclopropyl)-ethylamine-1,2,4-oxidiazole] and Deprenyl with a biphasic dependence on the inhibitor concentration. The activities towards tyramine, dopamine and tryptamine were also inhibited in a biphasic manner, but the apparent proportions of the two enzyme species active on dopamine and tryptamine depended on the inhibitor used. Phenethylamine oxidation was inhibited in a monophasic manner suggesting that only a single enzyme species was responsible for the oxidation of this substrate. The biphasic response of 5-HT oxidation to inhibition by clorgyline persisted when functionally competent mitochondria were used and was unaffected by the soluble amine oxidase inhibitors semicarbazine and aminoguanidine. These results indicate that the behaviour of the beef heart enzyme towards selective inhibitors is considerably different from that of any preparations previously studied and suggest that the classification of monoamine oxidase activites into A and B types may be only of limited usefulness.

Initial-rate studies of the low-Km aldehyde reductase-catalysed reduction of pyridine-3-aldehyde by NADPH gave families of parallel double-reciprocal plots, consistent with a double-displacement mechanism being obeyed. Studies on the variation of the initial velocity with the concentration of a mixture of the two substrates were also consistent with a double-displacement mechanism. In contrast, the initial-rate data indicated that a sequential mechanism was followed when NADH was used as the coenzyme. Product-inhibition studies, however, indicated that a compulsory-order mechanism was followed in which NADPH bound before pyridine-3-aldehyde with a ternary complex being formed and the release of pyrid-3-ylcarbinol before NADP+. The apparently parallel double-reciprocal plots obtained in the initial-rate studies with NADPH and pyridine-3-aldehyde were thus attributed to the apparent dissociation constant for the binary complex between the enzyme and coenzyme being finite but very low.

The kinetic behaviour of phosphofructokinase (ATP:D-fructose-6-phosphate 1-phosphotransferase, EC 2.7.1.11) has been studied in situ, by using rat erythrocytes and reticulocytes treated with dimethyl suberimidate and 3,3'-dithiobispropionimidate as cross-linking reagents and with digitonin as the delipidating agent. Comparison of the ATP and fructose-6-P saturation curves of phosphofructokinase in dimethyl suberimidate-permeabilized cells with those obtained in haemolysates showed the enzyme to have reduced allosteric properties under in situ conditions, although it still responded to cyclic AMP (300 microM) added as allosteric effector. Non-sigmoidal fructose-6-P saturation curves were also observed using 3,3'-dithiobispropionimidate-permeabilized erythrocytes, either in the absence or in the presence of cyclic AMP. A hyperbolic behaviour was shown after cross-linking reversal of 3,3'-dithiobispropionimidate-permeabilized erythrocytes by treatment with dithiothreitol. Specific activity values of phosphofructokinase were always lower in permeabilized cells than in haemolysates. A significant inhibition of phosphofructokinase specific activity, without any effect on its allosteric behaviour, is exerted by reaction of dimethyl suberimidate or 3,3'-dithiobispropionimidate with erythrocyte lysates in the presence of an inhibitory concentration of ATP. These results suggest that penetration of the cross-linking reagent and its subsequent reaction with intracellular phosphofructokinase will have a direct effect upon the results obtained using this in situ approach.U

Several reports have suggested that monoamine oxidase activity towards beta-phenethylamine is inhibited by high concentrations of that substrate. This inhibition is not found if initial velocities are measured, but there is a slower time-dependent inhibition at higher beta-phenethylamine concentrations. Such time-dependent inhibition is not found with tyramine as substrate or upon incubation of the enzyme with the reversible inhibitor amphetamine. The inhibition is not due to the accumulation of phenacetaldehyde, phenylethanol or phenacetic acid, or to a reaction of any of these three products either with each other or with beta-phenethylamine. Although the inhibition is time-dependent, the inactivated enzyme slowly regains activity upon removal of the beta-phenethylamine. A model is proposed to explain the observed inhibition.

A comparison of monoamine oxidase activities in the hypothalamus and striatum between malignant hyperthermia-susceptible (Pietrain) and malignant hyperthermia-resistant (Landrace/Large White) pigs showed no significant difference between the two breeds. The concentrations of noradrenaline, dopamine and their non-O-methylated metabolites did not reflect the low activities of monoamine oxidase type A differentially. The malignant hyperthermia-susceptible pigs had significantly greater concentrations of 3,4-dihydroxyphenyl glycol in the striatum, and of 3,4-dihydroxymandelic acid in the hypothalamus. Consequently, in the brain, low monoamine oxidase type A activity does not appear to be involved in susceptibility to malignant hyperthermia. In addition, monoamine oxidase activities in the heart, liver, kidney and intestinal mucosa and catechol-O-methyl transferase activities in the kidney were the same in the susceptible and resistant pigs.

Km and Vmax values of monoamine oxidase (MAO) A and B towards 5-hydroxytryptamine were determined for rat brain homogenates after the in vitro inhibition of one of the two forms by the selective inhibitors clorgyline and l-deprenyl. Km values of 178 and 1170 microM, and Vmax values of 0.73 and 0.09 nmol . mg protein-1 . min-1 towards 5-hydroxytryptamine were found for MAO-A and -B, respectively. The Ki for 5-hydroxytryptamine as a competitive inhibitor of beta-phenethylamine oxidation by MAO-B was found to be 1400 microM. The significance of these findings is discussed.

The method of Storer & Cornish-Bowden [(1974) Biochem. J. 141, 205-209] for determining the lag time in coupled enzyme assays was adapted to enable the kinetic parameters of the second (coupling) enzyme for the intermediate to be calculated. The validity and accuracy of this method of progress-curve analysis was established by comparing the Km value of glucose 6-phosphate dehydrogenase for glucose 6-phosphate generated in situ by the action of glucose phosphate isomerase on fructose 6-phosphate with that determined from initial-rate measurements. The method was applied to the determination of the Km value of ox liver cytoplasmic aldehyde dehydrogenase for N tau-methylimidazol-3-ylacetaldehyde that was generated in situ by the action of plasma amine oxidase on N tau-methylhistamine.

A through study of initial-rate data has been made on carbamoyl phosphate synthetase from bovine liver. On the basis of the results the order of substrate binding to the enzyme is ATPMg followed by HCO(3) (-), ATPMg and NH(4) (+). A model for the enzymic mechanism is proposed, and the rate equations describing it are presented. Details of the derivation of the initial-rate equation for the kinetic mechanism proposed have been deposited as Supplementary Publication SUP 50032 (6 pages) at the British Library, Lending Division (formerly the National Lending Library for Science and Technology), Boston Spa, Yorks. LS23 7QB, U.K., from whom copies may be obtained on the terms indicated in Biochem. J. (1973), 131, 5.

The activation of glutamate dehydrogenase (L-glutamate: NAD(P)+ oxidoreductase (deaminating), EC 1.4.1.3) by L-leucine has been studied. Apparently homogeneous preparations from ox liver and brain were found to respond similarly. Commercially obtained preparations of the enzyme, which had suffered limited proteolysis during the purification procedure, were shown to behave similarly to preparations which had not suffered such proteolysis when the effects of L-leucine on the oxidative deamination reaction were studied using either NAD+ or NADP+ as the coenzyme. There was also no significant difference in the responses when the reductive reaction was determined with NADPH or with 40 microM NADH. At higher concentrations of NADH (160 microM) the unproteolysed preparations were activated by L-leucine to a considerably greater extent than those which had suffered limited proteolysis. These results accord with the greater sensitivity of the former preparations to inhibition by high concentrations of NADH and the relief of such inhibition by L-leucine. This amino acid was also found to relieve the inhibition of the enzyme by GTP, resulting in an apparent increase in the activation observed in the presence of this nucleotide. In contrast, under the conditions used in this work, the apparent degree of activation by L-leucine was found to be decreased in the presence of the activators ATP or ADP. The presence of high concentrations of NADH (200 microM) potentiated the high substrate inhibition by 2-oxoglutarate, and L-leucine significantly reduced this effect. The effects of L-leucine on the activity of glutamate dehydrogenase thus appear to be composed of a direct effect on the activity of the enzyme together with a relief of high substrate inhibition. The effects of GTP and 2-oxoglutarate in potentiating inhibition by NADH can account for their effects in enhancing the apparent activation by L-leucine. The marked differences in the responses of proteolysed and unproteolysed preparations of the enzyme result from the effects of proteolysis in decreasing the sensitivity to high concentrations of NADH.

1-Methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) has been shown to produce a condition resembling idiopathic Parkinson's disease in primates, with evidence of selective destruction of the nigrostriatal dopaminergic system. It is, however, rather less toxic and selective in its actions on other experimental animals. The evidence that its toxicity involves its conversion, through the action of monoamine oxidase, to the 1-methyl-4-phenylpyridinium ion (MPP(+)), which is then taken up by dopaminergic nerve terminals, where it acts as an inhibitor of energy metabolism, is reviewed. Differences between common laboratory animals and primates which may account for the differences in sensitivity and selectivity of the actions of MPTP are considered as are other factors which may be involved in the neurotoxicity of this compound. The relevance of the use of MPTP to provide an animal model of Parkinson's disease is discussed.

The monoamine oxidase activities were found to be similar in the crypt and villous cells of the different regions of rat small intestine. In all cases the activity of the A-form of the enzyme constituted more than 70% of the total. A similar proportion of that form of the enzyme was found in homogenates of biopsy samples of human intestine. Studies with everted intestines showed that at concentrations above 10 microM over 70% of tyramine was deaminated during transport and the use of selective inhibitors confirmed the A-form of monoamine oxidase to play the dominant role in that process.

The distribution of the two principal isoenzymes of aldehyde reductase (EC 1.1.1.2) has been studied in ox brain. The more active of these, which has been termed the high-Km enzyme, has been shown to be located in the cytosol and the less abundant low-Km form has a similar localization. p-Nitrobenzaldehyde, which has been used as a substrate in previous studies, caused the reduction of NADH in the presence of the mitochondrial fraction, but mixed substrate experiments with 1,3-dinitrobenzene and the effects of pH on the activity indicate that this is due to the presence of a nitro reductase activity which has been recently described (Köchli, Wermuth & von Wartburg (1980) Biochim. Biophys. Acta 616, 133-142] rather than to the low-Km aldehyde reductase activity. Fractionation of the mitochondria indicated this activity to be largely confined to the mitochondrial inner membrane.

1. The kinetics of the reaction catalysed by fructose bisphosphatase have been studied at pH 7.2 and at pH 9.5. The activity of the enzyme was shown to respond sigmoidally to increasing concentrations of free Mg2+ or Mn2+ ions at pH 7.2, whereas the dependence was hyperbolic at pH 9.5. At both pH values the enzyme responded hyperbolically to increasing concentrations of fructose 1,6-bisphosphate, although inhibition was observed at higher concentrations of this substrate. This high substrate inhibition was shown to be partial in nature and the enzyme was found to be more sensitive at pH 7.2 than at pH 9.5. 2. The properties of the enzyme, are consistent with the enzyme obeying either a random-order equilibrium mechanism or a compulsory-order steady-state mechanism in which fructose bisphosphate binds to the enzyme before the cation. 3. Reaction of the enzyme with a four-fold molar excess of p-chloromercuribenzoate caused activation of the enzyme when its activity was assayed in the presence of MN2+ ions but inhibition when Mg2+ ions were used. Higher concentrations of p-chloromercuribenzoate caused inhibition. This activation at low p-chloromercuribenzoate concentrations, and the reaction of 5,5'-dithio-bis(2-nitrobenzoate) with the four thiol groups in the enzyme that reacted rapidly with this reagent, were prevented or slowed by the presence of inhibitory, but not non-inhibitory, concentrations of fructose bisphosphate. After reaction with a four-fold molar excess of p-chloromercuribenzoate the enzyme was no longer sensitive to high substrate inhibition by fructose bisphosphate.

1. A preparation of mitochondrial outer membranes from rat liver can be shown to contain two kinetically distinct monoamine oxidase activities. These activities are distinguishable by their different sensitivities to the irreversible inhibitor clorgyline, and by the effect of the reversible inhibitors benzyl cyanide and 4-cyanophenol. 2. The substrate specificities of the preparation and the two enzyme species have been elucidated.

The inhibition of rat liver monoamine oxidase by a number of N-propargyl and alpha-methyl amine derivatives has been examined. The results indicate that alpha-methyl-substituted primary and secondary amine derivatives tend to show selectivity as reversible inhibitors towards the A-form of the enzyme. The structural features that result in selectivity in irreversible inhibitors are less easy to define and substitution of an N-propargyl group into a compound that is a selective reversible inhibitor of monoamine oxidase will not necessarily result in retention of that selectivity. Replacement of the acetylenic group in a B-selective irreversible inhibitor by an ethylenic group resulted in a compound that was a reversible inhibitor showing slight selectivity for the A-form of the enzyme.

A reaction in which the hydron [International Union of Pure and Applied Chemistry (1986) Chem. Int. 8 (4), 21] is a reactant will have a more acidic pH optimum than the reverse reaction in which it is a product. Simple mechanistic models, made by postulating various ways in which the hydron is supplied for the forward reaction, lead to the prediction that the pK that characterizes the fall in activity on the alkaline side for the forward reaction will be equal to that characterizing the fall on the acid side for the reverse reaction. It is not possible to identify what is responsible for this pK without additional information; as is well known, different possible dispositions of hydrons in transition states cannot usually be distinguished solely by kinetics.

1.A procedure for the purification of ox liver fructose 1,6-kiphosphatase is described. A number of criteria indicate that the enzyme was not subjected to any significant degree of proteolytic attack during the purification. 2. The molecular weight, amino acid composition and subunit molecular weight are reported. 3. The activation by EDTA was shown to be due to the chelation heavy metals rather than by a more complex interaction with the enzyme as had previously been suggested.