The compound 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) causes selective destruction of nigrostriatal dopaminergic neurons in primates, giving rise to a condition resembling Parkinson's disease. The toxicity of MPTP is believed to be due to its metabolite 1-methyl-4-phenylpyridinium (MPP+). MPP+ is an inhibitor of mitochondrial respiration at the NADH-ubiquinone oxidoreductase site and this, together with its selective transport into dopaminergic nerve terminals, accounts for its neurotoxicity. In this paper an electrode selective for MPP+ was developed and used to measure the rate of uptake and the steady-state accumulation of MPP+ in rat liver mitochondria. The initial rates of MPP+ uptake were not saturable, confirming previous work that the transport of MPP+ is not carrier-mediated. The membrane potential of mitochondria respiring on succinate was decreased by MPP+ and the steady-state accumulation ratio of MPP+ did not come to equilibrium with the mitochondrial transmembrane potential gradient (delta psi). The effect of the cation exchanger tetraphenylboron (5 microM) was to increase the initial rate of MPP+ uptake by about 20-fold and the steady-state accumulation by about 2-fold. This suggests that there may be a mechanism of efflux of MPP+ from mitochondria which allows MPP+ to cycle across the membrane and thus decrease delta psi. These data indicate that MPP+ interacts with mitochondria independently of its inhibition of NADH-ubiquinone oxidoreductase, and these alternative interactions may be of relevance for its mechanism of neurotoxicity.

The steady-state kinetics of purified cytoplasmic aldehyde dehydrogenase (EC 1.2.1.3) from human erythrocytes have been studied at 37 degrees C. Previous studies of the enzyme from several mammalian sources, which used a lower assay temperature, have been difficult to interpret because of the substrate activation by acetaldehyde which led to complex kinetic behaviour. At 37 degrees C the initial-rate data do not depart significantly from Michaelis-Menten kinetics. Studies of the variation of initial rates as a function of the concentrations of both substrates and studies of the inhibition by NADH were consistent with a sequential mechanism being followed. High-substrate inhibition by acetaldehyde was competitive with respect to NAD+. The enzyme was not inhibited by the product acetate and thus the results of these studies, although consistent with an ordered mechanism in which NAD+ was the first substrate to bind, were inconclusive. That such a mechanism was followed was confirmed by determination of the initial-rate behaviour in the presence of acetaldehyde and glycolaldehyde as alternative substrates. When the reciprocal of the initial rate of NADH formation was plotted against the acetaldehyde concentration at a series of fixed ratios between that substrate and glycolaldehyde, a linear ‘mixed inhibition’ pattern was obtained, confirming the mechanism to be ordered with NAD+ being the leading substrate and with kinetically significant ternary complex-formation.

Initial velocity and product inhibition studies have been performed on soluble catechol-O-methyltransferase which has been partially purified from pig liver. The results are consistent with an ordered reaction mechanism, in which S-adenosyl-L-methionine (AdoMet) is the leading substrate. The enzyme is irreversibly inhibited by maleimide derivatives in a biphasic manner, which suggests a differential reaction with two thiol groups. N-(3,4-Dihydroxyphenyl)maleimide, which has a reactive moiety (maleimide ring) and an affinity moiety (catechol ring), acts as an affinity labelling compound on the more reactive SH group; AdoMet and Mg2+ protect against this modification. Total protection of this SH group results in a pseudo-first-order inhibition of the enzyme, with the apparent rate constant being proportional to the inhibitor concentration. All the other maleimide derivatives studied inhibited the enzyme by reacting with one of the two SH groups in a non-specific manner. The reaction of the other, more reactive, SH group was either specific (active-site-directed) or non-specific, depending on the substituent present in the affinity moiety and also on the length of an intermediate chain of methylene groups present between this moiety and the reactive maleimide ring. In the presence of both AdoMet and Mg2+, 3,5-dinitrocatechol, a reversible inhibitor of the enzyme which is competitive with respect to the catechol substrate, protects the enzyme from inactivation by any of the maleimide derivatives. The adducts of these maleimide derivatives formed with dithiothreitol inhibit the enzyme reversibly, showing inhibition patterns that are consistent with the mechanism deduced from the initial velocity and product inhibition studies.

Glutamate dehydrogenase in disrupted mitochondrial preparations is activated by L-leucine to a much greater extent than is the purified enzyme. A factor, or factors, responsible for modulating the sensitivity of L-leucine is lost during the purification of the enzyme. Although both cardiolipin and phosphatidylserine are inhibitors of the enzyme, only the inhibition by the former phospholipid is reversed by L-leucine. The inhibition of glutamate dehydrogenase by its binding to cardiolipin in the disrupted mitochondrial preparations and its relief by L-leucine could account for the greater sensitivity of such preparations to activation by that amino acid.

Impure preparations of rat intestinal peroxidase were shown to aggregate at low ionic strengths and to disaggregate at higher values. This aggregation was accompanied by a decrease in specific activity, which could lead to hysteretic behaviour of reaction progress curves. Advantage was taken of this reversible aggregation to obtain a relatively pure extract, which was subsequently purified to apparent homogeneity by affinity chromatography on concanavalin A-Sepharose followed by hydrophobic chromatography. The purified enzyme did not show the ionic-strength-dependent aggregation behaviour, behaving as a monomer of Mr 50,000. The purified enzyme was shown to catalyse the peroxidatic conversion of the commonly used antioxidant 2-t-butyl-4-methoxyphenol (butylated hydroxyanisole, BHA) to form 3,3′-di-t-butyl-2,2′-dihydroxy-5,5′-dimethoxybiphenyl, with a Km value of 176 microM and a maximum velocity of 8 mumol/min per mg. The specificity constant, kcat./Km, for this substrate was similar to that shown towards the substrate guaiacol.

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.

The involvement of Mg2+ ions in the reaction catalysed by phosphofructokinase from Trypanosoma brucei was studied. The true substrate for the enzyme was shown to be the MgATP2-complex, and free Mg2+ ions are also required for enzyme activity. At concentrations of MgATP2- of 2.92 mM and greater, and a fructose 6-phosphate concentration of 1 mM and in the presence of EDTA as a Mg2+ buffer, the Km value for Mg2+ was determined to be 294 +/- 18 microM. Neither MgATP nor free ATP is an inhibitor of the enzyme, although apparent inhibition by the latter can be observed as a consequence of the decrease in free Mg2+ by chelation.

The steady-state kinetics of the reaction catalysed by the bloodstream form of Trypanosoma brucei were studied at pH 6.7. In the presence of 50 mM-potassium phosphate buffer, the apparent co-operativity with respect to fructose 6-phosphate and the non-linear relationship between initial velocity and enzyme concentration, which were found when the enzyme was assayed in 50 mM-imidazole buffer [Cronin & Tipton (1985) Biochem. J. 227, 113-124], are not evident. Studies on the variations of the initial rate with changing concentrations of MgATP and fructose 6-phosphate, the product inhibition by fructose 1,6-bisphosphate and the effects of the alternative substrate ITP were consistent with an ordered reaction pathway, in which MgATP binds to the enzyme before fructose 6-phosphate, and fructose 1,6-bisphosphate is the first product to dissociate from the ternary complex.

The compound 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP), which produces symptoms resembling Parkinson's disease in humans, acts both as a substrate and an enzyme-activated irreversible inhibitor of the B-form of monoamine oxidase from rat liver. Analysis of the inhibitory process showed the compound to be considerably more efficient as a substrate than as an irreversible inhibitor, with about 17000 mol of product being formed per mol of enzyme inactivated. The half-time of the inhibitory process was about 22 min. With the A-form of the enzyme, the compound had a lower Km value and a considerably lower maximum velocity than the corresponding values obtained with the B-form. Under the conditions used in the present work the inhibition of the A-form of the enzyme was largely reversible.

Histamine N-methyltransferase (EC 2.1.1.8) was purified 1100-fold from ox brain. The native enzyme has an Mr of 34800 +/- 2400 as measured by gel filtration on Sephadex G-100. The enzyme is highly specific for histamine. It does not methylate noradrenaline, adrenaline, DL-3,4-dihydroxymandelic acid, 3,4-dihydroxyphenylacetic acid, 3-hydroxytyramine or imidazole-4-acetic acid. Unlike the enzyme from rat and mouse brain, ox brain histamine N-methyltransferase did not exhibit substrate inhibition by histamine. Initial rate and product inhibition studies were consistent with an ordered steady-state mechanism with S-adenosylmethionine being the first substrate to bind to the enzyme and N-methylhistamine being the first product to dissociate.

Kinetic constants were determined for commercially available samples of ox liver glutamate dehydrogenase, which had previously been shown to have suffered limited proteolysis during preparation, with a range of substrates and effectors. These were compared with the values obtained with enzyme preparations purified in such a way as to prevent this proteolysis from occurring [McCarthy, Walker & Tipton (1980) Biochem. J. 191, 605-611]. The Km values and maximum velocities determined with different substrates revealed little difference between the two preparations although the proteolysed enzyme had lower Km values for NH4+ and glutamate when the activities were determined with NADPH and NADP+ respectively. This preparation was more sensitive to inhibition by Cl- ions but less sensitive to inhibition by high concentrations of the substrate NADH. The two preparations also differed in their sensitivities to allosteric effectors, with the proteolysed enzyme being the less sensitive to inhibition by GTP. At high concentrations of NADH, this preparation was also more sensitive to activation by ADP and ATP.

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.

Phosphofructokinase (EC 2.7.1.11) from Trypanosoma (Trypanozoon) brucei brucei was purified to homogeneity by using a three-step procedure that may be performed within 1 day. Proteolysis, which removes a fragment of Mr approx. 2000, may occur during the purification, but this can be prevented by including antipain, an inhibitor of cysteine proteinases, in the buffers during the purification. The subunits of the enzyme appear to be identical in size, with an Mr of 49 000. The Mr of the native enzyme was estimated to be approx. 220 000, suggesting a tetrameric structure. Kinetic studies showed the activity to depend hyperbolically on the concentration of ATP but sigmoidally on the concentration of fructose 6-phosphate. Although cyclic AMP, AMP and ADP stimulated the enzyme activity at low concentrations of fructose 6-phosphate, the last two nucleotides were inhibitory at high concentrations of this substrate. Phosphoenolpyruvate behaved as an allosteric inhibitor of the phosphofructokinase. Citrate, fructose 1,6-bisphosphate, fructose 2,6-bisphosphate and Pi did not influence significantly the activity of the enzyme.

The effects of ATP and GTP on the activities of ox liver and brain glutamate dehydrogenase were determined in the absence and presence of added Mg2+ ions. Although GTP was an inhibitor of the enzyme reaction assayed in the direction of NAD+ reduction, the magnesium complex of this nucleotide had no effect on the activity. Similarly the magnesium complex of ATP was without effect on the activity of the enzyme although the free nucleotide was an activator. These results suggest that it is important to take account of magnesium complex formation when considering the regulatory actions of these nucleotides.

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.

3-(4-[(3-Chlorophenyl)methoxy]phenyl)-5-[(methylamino)methyl]-2- oxazolidinone methanesulphonate (compound MD 780236) is a selective inhibitor of the B-form of monoamine oxidase. Inhibition involves an initial non-covalent interaction between enzyme and inhibitor followed by a time-dependent process resulting in irreversible inhibition. The initial, reversible, phase of inhibition was found to be competitive with respect to phenethylamine and 5-hydroxytryptamine, and a comparison of the Ki values indicated the affinity of the inhibitor for the B-form of the enzyme to be some 7-fold greater than its affinity for the A-form. This selectivity was considerably enhanced by preincubation of the enzyme and inhibitor. Time courses showed that complete inhibition was not achieved under conditions where the inhibitor concentration was over 100-fold greater than that of the enzyme. Assay of the activity of monoamine oxidase by determining the release of hydrogen peroxide fluorometrically showed compound MD 780236 to be a substrate for, as well as an inhibitor of, monoamine oxidase, and kinetic analysis revealed that the rate of product formation was some 530-fold greater than that of the process leading to irreversible inhibition of the B-form of the enzyme.

The concentration-dependent aggregation behaviour of purified ox liver and brain glutamate dehydrogenase preparations was compared with that of commercially-obtained preparations of the liver enzyme, which have recently been shown to have suffered proteolytic cleavage. Although there were no significant differences in these effects, the presence of 3 mM-GTP and 3 mM-NADH had markedly different effects on the two types of preparation. In this situation, at higher protein concentrations the commercially obtained preparations existed in a higher degree of aggregation than those which had not suffered proteolysis. Studies of the effects of GTP and NADH concentrations on the sedimentation coefficients at a fixed enzyme concentration suggested these effects to be largely due to differences in the affinities of the two preparations for nucleotides.

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.

A procedure is described that yields an apparently homogeneous preparation of the high-Km aldehyde reductase from rat brain. This procedure is also applicable to the purification of this enzyme from rat liver and ox brain. In the latter case, however, the purified preparation could be resolved into two protein bands, both of which had enzyme activity, by polyacrylamide-gel electrophoresis. Since a sample of the ox brain enzyme from an earlier step in the purification procedure only showed the presence of a single band of activity after electrophoresis, this apparent multiplicity probably results from modification of the enzyme, possibly by oxidation, during the final step of the purification. A number of properties of the rat brain enzyme were determined and these were compared with those of the enzyme from rat liver. The two preparations were similar in their stabilities, behaviour during purification, kinetic properties, electrophoretic mobilities and amino acid compositions. Antibodies to the rat liver enzyme cross-reacted with that from brain and the inhibition of both these preparations by the antiserum was similar, further supporting the view that the enzymes from these two sources were closely similar if not identical.

1. A rapid procedure, involving ion-exchange chromatography on DEAE-cellulose and affinity chromatography on GTP-Sepharose, was used to purify glutamate dehydrogenase from ox brain and liver. 2. Preparations purified in this way differed from those of the ox liver enzyme that were obtained from commercial suppliers in their mobilities on polyacrylamide-gel electrophoresis in the presence of sodium dodecyl sulphate. This difference appears to result from the occurrence of limited proteolysis during the preparation of the latter enzyme samples. 3. N-Terminal sequence analysis showed the presence of four amino acid residues in the enzyme prepared in this study that were not present in those obtained from the commercial sources and which have not been reported in previous studies on the sequence of the ox liver enzyme. 4. A preliminary examination of the enzyme prepared in this way indicated that the Michaelis constants for the substrates are similar to those obtained from the commercial preparation, but that the response to allosteric effectors was modified.

1. In an enzyme that has two independent binding sites for a ligand, any inhibitor that binds solely to the free enzyme will give rise to positive co-operativity. 2. A model is considered for the allosteric control of enzymes by effectors in which their effects are mediated by ligand-induced perturbations of the ionization constants of a group or groups involved in the binding of substrate to the active site. 3. The model described offers a plausible explanation for the observation that the sigmoidal initial-rate curves reported for some regulatory enzymes are not expressed at all pH values where the enzyme is catalytically active.

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.

1. The kinetics of benzylamine oxidation by a soluble preparation of rat liver mitochondrial monoamine oxidase were investigated and were shown to conform to adouble-displacement (or Ping Pong) mechanism. 2. The pathway differs in detail from that followed by other amine oxidases, including the membrane-bound enzyme in rat liver mitochondrial outer membranes. 3. It is suggested taht the conformation of the protein in the soluble state differs from that in the membrane-bound state. 4. The full rate equations for this mechanism have been deposited as Supplementary Publication SUP 50039 (5pages) at the British Library (lending Division) (formely the National Lending Library for Science and Technology), Boston Spa, Yorks, LS237BQ, U.K.. from whom copies can be obtained on the terms indicated in Biochem. J (1975) 145,5.

The metabolism of a number of biogenic amines has been simulated by using data obtained from studies of the individual enzymes from pig brain. It is shown that β-hydroxylated amines such as noradrenaline and octopamine are metabolized primarily to the alcoholic metabolite whereas amines lacking this group [e.g. dopamine (3,4-dihydroxyphenethylamine) and 5-hydroxytryptamine] are metabolized at low concentrations to give the corresponding acid. Increase in the amine concentration results in an increase in the proportion of the alcoholic metabolite formed and this may in part account for the effects of the drug reserpine on amine metabolism. The effects of disulfiram (Antabuse) and ethanol (acting through its metabolite acetaldehyde) on amine metabolism may be understood in terms of this simulated model. It is shown that drugs that affect this system also cause alterations in the steady-state concentrations of the intermediate aldehydes and the possible implications of this are discussed.

1. The time-courses of thermal denaturation of human oxyhaemoglobins A, A 2 , C and S at 45°C were studied by following the increase in protein fluorescence. Haemoglobins S and C were less stable than haemoglobin A, whereas haemoglobin A 2 was considerably more stable. 2. The time-courses of denaturation did not follow first-order kinetics and could be fitted most simply to a co-operative scheme in which the partial denaturation of the α chain preceded that of the β chain. 3. The denaturation of these haemoglobins was studied as a function of temperature by using optical rotatory dispersion. Haemoglobin A 2 was again more stable than the others. The addition of small quantities of haemoglobin A 2 had a disproportionate effect on the stability of haemoglobin C. 4. The thermodynamic parameters of the denaturation process were calculated.

1. NADPH-linked aldehyde reductase from pig, ox and rat brain exhibits non-linear reciprocal plots when partially purified enzyme preparations are studied. 2. In pig brain this non-linearity is due to the presence of two distinct aldehyde reductases, which can be separated by DEAE-cellulose chromatography. 3. These two enzymes can be distinguished by several criteria, including pH optima, Michaelis constants for substrates and their inhibitor sensitivity. 4. The probable role of these enzymes in the metabolism of the aldehydes derived from the biogenic amines is discussed.

1. In the presence of the substrate benzylamine, phenethylhydrazine has been shown to be a competitive inhibitor of monoamine oxidase from rat liver and pig brain. 2. Phenethylhydrazine is also a substrate for monoamine oxidase. Reciprocal plots for hydrazine oxidation give families of intersecting lines in contrast with the parallel lines previously reported for tyramine oxidation. 3. Two possible modifications of the mechanism obeyed by tyramine oxidation are suggested, but the product inhibition results are insufficient to distinguish between these two mechanisms.

1. Ox heart phosphofructokinase catalyses isotope-exchange reactions at pH6.7 between ADP and ATP, and between fructose 6-phosphate and fructose 1,6-diphosphate, the latter reaction being absolutely dependent on the presence of the magnesium complex of ADP. 2. The reaction kinetics are hyperbolic with respect to substrate concentration for both exchange reactions (within the experimental error). 3. The influence of pH, AMP and citrate suggests that the fructose 6-phosphate–fructose 1,6-diphosphate exchange is subject to effector control, and is abolished by dissociation of the enzyme. 4. These results are discussed in relation to the reaction mechanism of the enzyme.

The NADP-dependent isocitrate dehydrogenase from pig liver soluble fraction was purified over 500-fold with an overall yield of 25%. The purified enzyme, which is homogeneous by all the usual criteria, has a molecular weight of about 75000 and is composed of two identical subunits. This has been demonstrated by ultracentrifugation, fluorescence titration and peptide `fingerprinting'. The maximal turnover number, extinction coefficients at 280nm and 260nm and amino acid analysis are described.

A continuous-flow apparatus is described for automatically plotting substrate saturation curves, and is suitable for use with a variety of enzymes. A linear concentration gradient of the variable substrate is combined with a fixed proportion of the other substrates and the enzyme, and after passing through a reaction coil the product concentrations are measured spectrophotometrically. Use of a 4cm. flow cell and modified spectrophotometer permits accurate measurement of NADH concentration in the region of 0·1μ m . Precise control over reaction times and substrate concentration is achieved by using power-driven syringes with an integral mixer. Specimen results are given for yeast alcohol dehydrogenase.

1. The optical rotatory dispersion of carnitine acetyltransferase is altered in the presence of l -carnitine or acetyl- l -carnitine. These changes, which include an increase in the reduced mean residue rotation at 233nm. ([ M ′] 233 ), suggest that substrate binding causes the enzyme to unfold. 2. CoA and acetyl-CoA have no immediate effect on [ M ′] 233 and CoA has no effect on the change in this parameter induced by l -carnitine. 3. The change in [ M ′] 233 was used as a measure of the degree of saturation of the enzyme with carnitine substrates. Dissociation constants for the enzyme complexes with l -carnitine, d -carnitine and acetyl- l -carnitine were determined in this way. 4. Prolonged incubation of carnitine acetyltransferase in the presence of CoA leads to a small increase in the value of [ M ′] 233 accompanied by irreversible inhibition of the enzyme. 5. Optical-rotatory-dispersion studies of two specifically inhibited enzyme forms are reported.

1. The specimen of polygalacturonic acid used in these studies was shown to contain very little neutral sugar, methyl ester groups or ash, and only residues of galacturonic acid. Its electrophoretic homogeneity was examined in pyridine–acetic acid buffer at pH6·5 and in borate buffer at pH9·2. The distribution of effective particle weights was shown to be fairly narrow. 2. The pH-titration curve of the polymer gave a p K value of 3·7. 3. The interaction of the polymer with Ruthenium Red was studied and titration curves were obtained for the spectral shifts associated with the formation of a complex. 4. Optical-rotatory-dispersion studies showed that the Drude constant, λ c , was dependent on pH. 5. Polygalacturonic acid was shown to display non-Newtonian properties in solution and to have an anomalously high relative specific viscosity at low concentrations. 6. Studies were made of the pH-dependence of the sedimentation coefficient of the polymer. 7. These results are discussed in terms of the structure of the molecule and their relevance to the properties of pectic substances.

Activities of the enzymes monoamine oxidase (EC 1.4.3.4), α-glycerophosphate dehydrogenase (EC 1.1.99.5) and cytochrome oxidase (EC 1.9.3.1) were determined in homogenates and in the mitochondrial fraction prepared from individual regions of pig brain. The variation in the activity of α-glycerophosphate dehydrogenase paralleled that of cytochrome oxidase, but this was not the case with monoamine oxidase. The differences in the activities of the enzymes among homogenates of the various regions of the brain persisted in mitochondria prepared from these homogenates. The purification of these three enzymes paralleled each other when mitochondria were prepared, suggesting that the three enzymes are bound to the same particles.