Studies on monoamine oxidase. (Report XXV). Effects of alcohols on beef liver mitochondrial monoamine oxidase.

The effects of alcohols on MAO activity in beef and rat liver mitochondria were studied. MAO activity was determined manometrically using tyramine (1× 10-2 M) as substrate and expressed as O2 uptake in 60 min. At concentrations of 0.1 to 10%, methanol and ethanol increased MAO activity in both beef and rat liver mitochondria. Activation of MAO by ethanol was reversible. Ethanol increased MAO activity of beef liver mitochondria using butylamine, amylamine, benzylamine, beta-phenylethylamine, tryptamine or serotonin as substrate. The pH optima of MAO in beef liver mitochondria were at pH 7.0 and pH 7.5 in the absence and pre sence of ethanol. On addition of 1% ethanol, the Km value (2×10-2 M) did not change but the Vmax vlaue increased two-fold. The pS-activity curve of MAO was sigmoidal and on addition of ethanol, MAO was inhibited by a high concentration of substrate. Ethanol decreased the inhibition of MAO by pheniprazine or pargy line. These results suggest that addition of ethanol does not affect the dissociation constant of the enzyme-substrate complex, but accelerates reaction steps occurring after the enzyme-substrate complex has been formed. There have been four reports on the effects of alcohols on monoamine oxidase (MAO) [EC' 1. 4. 3. 4. monoanmine: oxygen oxidoreductase (deaminating)]. In 1950, Heim (1) reported that MAO in guinea-pig liver was activated by 0.18 M alcohols using tyramine as substrate. In 1960, Cotzias and Greenough (2) reported that with tyramine as substrate 1.8 x 10-s M ethanol activated MAO in a homogenate of rat liver but inhib ited MAO in the solubilized state, while with /-noradrenaline as substrate the opposite results were observed. On the other hand, in 1962, Maynard and Schenker (3) reported that MAO in mouse liver was inhibited by 100-400 mg/100 ml of ethanol, using serotonin or tyramine as substrate, and in 1964, Towne (4) also reported the inhibition of MAO in rat liver by 0.022--0.087 M ethanol, using serotonin as substrate. Thus, the exact effects of alcohols on MAO have not yet been established. In our laboratory, the effects of many organic solvents on liver mitochondrial MAO were studied in detail. It was found that at concentrations of more than 10'%ethanol inactivated mitochondria( MAO while at concentrations of less than I it activated MAO. This paper reports kinetic studies on beef liver mitochondrial MAO and also the related effects of alcohols. The effects of various alcohols on MAO of rat and beef liver mitochondria were also compared to determine whether or not these enzymes are the same. Materials Beef and rat liver were used as enzyme sources. Fresh livers were stored at ---20°C until use. The tissue was cut into small pieces and homogenized with 3 volumes of chil led 0.25 M sucrose containing 0.01 M Tris-HCI buffer, pH 8.0, in a Waring blender or a Potter-Elvehjem glass homogenizer at low temperature (0 to 4°C). The homogenate was centrifuged at 600 x g for 15 min in a refrigerated centrifuge. Then the supernatant was rapidly removed and kept in a beaker in crushed ice, while the precipitate was resuspended in sucrose solution and recentrifuged in the same way. The precipitate was discarded and the supernatants were combined and centrifuged at 8500x g for 20 min. The supernatant was discarded and the precipitate was washed with the same buffer by centrifugation and then suspended in 3 volumes of 0.1 M Tris-HC1 buf fer, pH 8.0. This suspension was then stored at -20°C and used as the preparation of mitochondrial MAO. Measures vent of MAO activity MAO activity was determined by measuring oxygen consumption rnanometrically, as described by Kamijo et al. (5). Reaction mixture, containing enzyme solution, 0.1 M Tris-HCI buyer, pH 8.0 and distilled water in the main well and substrate solution in the side arm in a total volume of 3.0 ml, was equilibrated with oxygen gas for 10 min with filter paper soaked in 3 M KOH in the inner well to absorb carbon dioxide. The reaction was started by tipping the substrate solution from the side arm into the main well of the Warburg vessel. Oxygen consumption was followed for 60 min, starting 10 min after addition of substrate. En zymatic oxidation of substrate usually prodeeced linearly during the period of measure ment. Corrections were made for non-enzymatic oxidation of the substrate and oxygen uptake by the enzyme solution with and without alcohols in the absence of added sub strate. All determinations were done in duplicate. MAO activity was expressed as the Qo2, defined as the oxygen consumption (,u]) per hr per mg dry weight of enzyme preparation. Unless otherwise stated, 0.1 M Tris-HCI buffer, pH 8.0, was used. RESULTS EfFects of ' various alcohols on beef liver mitochondrial MAO The effects of methanol, ethanol, propanol and butanol on beef liver mitochondrial MAO were studied using I 10-2 M tyrarnine as substrate. Results are shown in Table 1. Methanol at concentrations of 0.1 1 and l0 increased MAO activity by about 20%, 50 and 30°0, respectively. However, 20;(, methanol inactivated the enzyme al most completely. MAO activity also increased in the presence of low concentrations of ethanol. Ad dition of final concentrations of 0.1 % and 1 % ethanol increased the activity by about 35% and 60 °c, respectively. At concentrations of over 10'/', ethanol decreased MAO activity. For example, 10% ethanol inhibited MAO about 30°,, and 30 ethanol caused almost complete inhibition. Propanol at concentrations of 0.1 to 20 , inhibited MAO activity, inhibition be ing proportional to the concentration of propanol. As seen in the table, butanol was also inhibitory at the concentrations tested, and the inhibitory effect was proportional to the concentration. TABLE 1. Effects of some aliphatic alcohols on MAO activity in beef liver mito chondria. MAO activity was determined from the 02 consumption (PI) in 60 min in the presence and absence of the alcohol indicated, taking the MAO activity of the control as 100%. Substrate : I x 10-2 M tyramine. pH : 8.0. Temperature : 38'C. Effects of various alcohols on rat liver nritochomlrial MAO The effects of methanol, ethanol, propanol and butanol on mitochondrial MAO of rat liver were also studied using I 10-2 M tyramine as substrate, and results are shown in Table 2. MAO activity increased with increase in the concentration of methanol from 0.1 to I 0.1 , and I ",, methanol causing about 20 an 60 Increase in activity, respec tively. however, 10",, methanol onk increased the activity about I0 while 20% me thanol was completely inhibitory. MAO activity also increased with increase in the concentration of ethanol from 0.1 to 10/', 0.1% and 1 ethanol increasing activity about 40% and 60°,x, respectively. However, 10'."/ ethanol only increased activity about 30%, and 20 ethanol caused about 80'/', inhibition. Propanol had no effect on MAO activity at concentrations of 0.01 % to 0.1 %, but at concentrations above 1.0",, it caused complete inhibition. TABLU-. 2. Effects of some alcohols on MAO activity in rat liver mitochondria. Butanol at a concentration of 0.01 % had no effect on MAO activity while concentra tions of 0.1 ° ; and 1 ;o caused about 25 ° ; and 50 inhibition and concentrations of over 10% caused complete inhibition. Mechanism of activation of beef liver initochondrial MAO by ethanol a) Substrate specificity The effect of ethanol on mitochondrial MAO in beef liver was studied using tyramine, benzylamine, butylamine, amylamine, serotonin, beta-phenylethylamine and tryptamine as substrates, and results are summarized in Table 3. Concentrations of 0. 1 % to 1 % ethanol increased the oxidations of all these substrates, especially amylamine, with which 90% o increase was observed. With almost all these substrates, maximal oxidation was observed in the presence of about I ethanol, although with serotonin, activity was maximal in the presence of 0.1 % ethanol. TABLE 3. Effects of ethanol on MAO activity using various substrates. Conditions were as for Table 1. b) Reversibility of the effect of ethanol on MAO Next the reversibility of the effect of ethanol was tested using 1 x 10-2 M tyramine as substrate. A sample of a suspension of mitochondrial beef liver MAO was mixed with an equal volume of 2° ethanol, and as a control, another sample of the suspension was mixed with an equal volume of 0.1 M Tris-HC1 buffer, pH 8.0. Both mixtures were dia lyzed overnight against I 10 a M Tris-HCl buffer, pH 8.0, at 10°C after which their ac tivities were compared. Results are shown TABLE 4. Effect of dialysis on beef liver mitochondrial MAO activated with 1 % ethanol. Activity was tested before and after dialysis. Conditions were as for Table 1. in Table 4. The activity of enzyme which had been treated with 1 '/0' ethanol was the same as that of the control, so it was cncluded that activation of the enzyme by 1 ° ethanol is