Regulation of Alternative Pathway Activity in Plant Mitochondria : Deviations from Q-Pool Behavior during Oxidation of NADH and Quinols

External NADH and succinate were oxidized at similar rates by soybean (Glycine max) cotyledon and leaf mitochondria when the cytochrome chain was operating, but the rate of NADH oxidation via the alternative oxidase was only half that of succinate. However, measurements of the redox poise of the endogenous quinone pool and reduction of added quinones revealed that external NADH reduced them to the same, or greater, extent than did succinate. A kinetic analysis of the relationship between alternative oxidase activity and the redox state of ubiquinone indicated that the degree of ubiquinone reduction during external NADH oxidation was sufficient to fully engage the alternative oxidase. Measurements of NADH oxidation in the presence of succinate showed that the two substrates competed for cytochrome chain activity but not for alternative oxidase activity. Both reduced Q-1 and duroquinone were readily oxidized by the cytochrome oxidase pathway but only slowly by the alternative oxidase pathway in soybean mitochondria. In mitochondria isolated from the thermogenic spadix of Philodendron selloum, on the other hand, quinol oxidation via the alternative oxidase was relatively rapid; in these mitochondria, external NADH was also oxidized readily by the alternative oxidase. Antibodies raised against alternative oxidase proteins from Sauromatum guttatum cross-reacted with proteins of similar molecular size from soybean mitochondria, indicating similarities between the two alternative oxidases. However, it appears that the organization of the respiratory chain in soybean is different, and we suggest that some segregation of electron transport chain components may exist in mitochondria from nonthermogenic plant tissues.     

The NADH oxidase system (external) of muscle mitochondria and its role in the oxidation of cytoplasmic NADH.

An exo-NADH oxidase system [NADH oxidase system (external)], effecting intact-mitochondrial oxidation of added NADH, was studied in pigeon heart mitochondria. Breast muscle mitochondria showed an equal specific activity of the system. The exo-NADH oxidase activity (200 micron mol of NADH/min per g of protein) equalled two-thirds of the State-3 respiratory activity with malate + pyruvate or one-seventh of the total NADH oxidase activity of heart mitochondria. The activity was not caused by use of proteinase in the preparation procedure and all measured parameters were very reproducible from preparation to preparation. The activity is therefore most likely not due to preparation artefacts. The exo-NADH oxidase system is present in all mitochondria in the preparation and is not confined to a subpopulation. The system reduced all cytochrome anaerobically and direct interaction with all cytochrome oxidase was demonstrated by interdependent cyanide inhibition. The exo-NADH oxidase system seems to be located at the outer surface of the mitochondrial inner membrane because, for instance, only this system was rapidly inhibited by rotenone, and ferricyanide could act as acceptor in the rotenone-inhibited system (reductase activity = 20 times oxidase activity). In the presence of antimycin, added NADH reduced only a part of the b-cytochromes. Freezing and thawing the mitochondria, one of the methods used for making them permeable to NADH, destroyed this functional compartmentation. The characteristics of the exo-NADH oxidase system and the malate-aspartate shuttle are compared and the evidence for the shuttle's function in heart in vivo is re-evaluated. It is proposed that oxidation of cytoplasmic NADH in red muscles primarily is effected by the exo-NADH oxidase system.     

A possible mechanism for the increased oxidation of choline after chronic ethanol ingestion

An attempt has been made to determine the location of the site at which the metabolism of ethanol interacts with that of choline to produce an increase in the oxidation of choline. The first enzyme in the oxidation pathway for choline, choline dehydrogenase, was assayed using a newly developed spectro-photometric assay and freshly isolated intact rat liver mitochondria. No changes were observed in either the ‘apparent’ V or the ‘apparent’ K_m values of choline dehydrogenase for choline after ethanol ingestion. However, when the choline oxidase system was assayed, a 28% decrease in ‘apparent’ K_m for choline and a 53% increase in ‘apparent’ V was observed. The effects of ATP on choline oxidase were studied further, and a 29.4% decrease was observed in mitochondrial ATP levels from freshly isolated mitochondria from the ethanoltreated rats. In vitro aging of mitochondria further decreased the level of ATP, and the rate of decrease was considerably faster during the first hour in the mitochondria from the ethanol-treated animals. The decreases in ATP from both control and experimental mitochondria were accompanied by increases in choline oxidase activity. The initial decrease in ATP was correlated with an increase in mitochondrial ATPase activity which may be related to an increase in mitochondrial Mg²⁺. Because chronic ethanol ingestion has resulted in decreased oxidation rates of succinate and β-hydroxybutyrate while at the same time increasing the oxidation rates of choline, the studies reported here suggest that the effect of chronic ethanol ingestion is primarily on a step that is unique to choline and which probably exists prior to the electron transport chain.     

Respiration of Mitochondria Isolated from Leaves and Protoplasts of Avena sativa

Mitochondria isolated from mesophyll protoplasts differed from mitochondria isolated directly from leaves of Avena sativa in that protoplast mitochondria (a) had a lower overall respiratory capacity, (b) were less able to use low concentrations of exogenous NADH, (c) did not respond rapidly or strongly to added NAD, (d) appeared to accumulate more oxaloacetate, and (e) oxidized both succinate and tetramethyl-p-phenylene-diamine (an electron donor for cytochrome oxidase) more slowly than did leaf mitochondria. It is concluded that cytochrome oxidase activity was inhibited, the external NADH dehydrogenase had a reduced affinity for NADH, succinate oxidation was inhibited, NAD and oxaloacetate porters were probably inhibited, and accessibility to respiratory paths may have been reduced in protoplast mitochondria. The results also suggest that there was a reduced affinity of a succinate porter for this substrate in oat mitochondria. In addition, all oat mitochondria required salicylhydroxamic acid (SHAM) as well as cyanide to block malate and succinate oxidation. Malate oxidation that did not appear to saturate the cytochrome pathway was sensitive to SHAM in the absence of cyanide, suggesting that the oat mitochondria studied had concomitant alternative and subsaturating cytochrome oxidase pathway activity.     

ULTRASTRUCTURAL DEMONSTRATION OF CYTOCHROME OXIDASE ACTIVITY BY THE NADI REACTION WITH OSMIOPHILIC REAGENTS

A new method for the subcellular and cytochemical demonstration of cytochrome oxidase has been developed with the introduction of N-benzyl-p-phenylenediamine (BPDA) and the discovery that indoanilines are osmiophilic. These indoanilines produced upon oxidation of BPDA in the presence of naphthols are highly colored compounds that yield electron-opaque coordination polymers of osmium (osmium black) that are amorphous, insoluble in water, and in organic solvents. The best methods for preparing rat tissue were in decreasing order: fixation in formaldehyde solution, fresh tissue slices, and frozen sections of fresh or fixed tissue. Ultrathin sections were counterstained by bridging with the thiocarbohydrazide-osmium tetroxide (T-O) procedure for enhancing underlying membranous structures. Cytochrome oxidase activity was noted primarily in mitochondria and occasionally in sarcotubules of heart, in mitochondria and occasionally in infoldings of the plasma membrane of renal tubular cells, and in mitochondria and, to a great extent, in endoplasmic reticulum of hepatic cells. Cytochrome oxidase activity produced deposits in droplet form, whereas dehydrogenase activity resulted in uniform staining of mitochondrial cristae, as recently demonstrated with an osmiophilic tetrazolium salt. Even more recently we have succeeded in demonstrating cytochrome oxidase activity in nondroplet staining on mitochondrial cristae with an osmiophilic benzidine-type reagent that apparently polymerizes upon oxidation (to be published later).     

A simple method for the large-scale preparation of mitochondria from microorganisms.

A simple mechanical procedure that has been developed for the large-scale preparation of intact mitochondria from yeast, is also applicable to the extraction of organelles from other organisms having cell walls. A procedure for the isolation of large quantities of pure mitochondrial DNA from these mitochondria is described. In Schizosaccharomyces pombe, further purification of the mitochondria by urografin isopycnic centrifugation leads to 50% recovery of whole cell respiration activity in a vesicular fraction of respiratory chain enzymes, with NADH oxidase activity usually greater than 10 μmol of electrons/min/mg of protein. The method has the advantage of rapidity and low cost and it is extremely healthy for the operator.     

Nature of the effect of adenosine 5''-monophosphate on the cyanide-insensitive respiration in mitochondria of Moniliella tomentosa.

The alternative oxidase of Moniliella tomentosa mitochondria is stimulated by 5'-AMP. This effect may be masked, depending on the isolation procedure of the mitochondria. The preparation of submitochondrial particles results in the expression of the 5'-AMP effect. Two more methods are now described to reveal the 5'-AMP effect whenever it would be masked: (1) switching on the myokinase activity of the mitochondria to deplete them of endogenous 5'-AMP; (2) using detergents (sodium dodecyl sulphate, sodium deoxycholate) in a controlled detergent:protein ratio, or chloroform. The alternative oxidase of detergent-solubilized mitochondria was somewhat less selective towards nucleotides than were intact mitochondria. The effect of nucleotides on quinol oxidation by mitochondrial preparations and on quinol autoxidation was also studied. Mitochondrial oxidation of succinate by the alternative oxidase and autoxidation of quinols behaved similarly in the presence of certain nucleotides. Both reactions were stimulated. Both reactions were also inhibited by salicylhydroxamic acid. These effects on quinol oxidation disappeared when bovine serum albumin or mitochondrial proteins were present. From the results obtained it is not possible to exclude quinol autoxidation as a final step of the alternative oxidase.     

CYTOCHEMICAL STUDIES OF MITOCHONDRIA : II. ENZYMES ASSOCIATED WITH A MITOCHONDRIAL MEMBRANE FRACTION

1. Mitochondria isolated from rat liver were disrupted with 0.3 per cent deoxycholate and a number of subfractions were isolated from this preparation by differential centrifugation. 2. The protein N, RNA and phospholipide content, as well as the succinoxidase, cytochrome c oxidase, adenylate kinase, and DPNH-cytochrome c reductase of these fractions were determined. 3. Two of these subfractions, found to consist of mitochondrial membranes (2), contained ~ 12 per cent of the protein N and ~ 35 per cent of the phospholipide of the whole mitochondria and accounted for ~ 70 per cent of the succinoxidase and cytochrome c oxidase activity of the original mitochondrial preparation. There was no discernible adenylate kinase, DPNH-cytochrome c reductase, or phosphorylating activities in these fractions, nor could they oxidize other substrates of the Krebs's cycle. 4. The most active fraction (60 minutes at 105,000 g pellet) had a higher phospholipide/protein value than the whole mitochondria and showed a seven-to elevenfold concentration of succinoxidase and cytochrome c oxidase activities. 5. Evidence has been given to indicate that the various components of the succinoxidase complex are present in this membrane fraction in the same relative proportions as in the whole mitochondria. 6. The implications of these findings are discussed.     

Particulate cytochromes of mung bean seedlings

Efforts have been made to solubilize cytochrome components from particulate fractions of etiolated mung bean seedlings. Low temperature spectrophotometry reveals that the cytochrome composition of mitochondria isolated from whole seedlings is the same as that reported by Bonner for mung bean hypocotyls. On the basis of the identity in position of the α-bands in low temperature difference spectra for mitochondria, for a partially purified haemoprotein from mitochondria, and for purified cytochrome b-555, it is suggested that cytochrome b-555 is an intrinsic component of mung bean mitochondria. Difference spectra show that both the mitochondrial and microsomal fractions contain at least 2 b-type cytochromes. Cytochrome b-555 is almost certainly present in the microsomes, since the low temperature difference spectrum for the cytochrome is identical with the spectrum for this particulate fraction.

By freezing and thawing mung bean mitochondria in 4% cholate and centrifuging, cytochrome oxidase activity can be concentrated in the supernatant fraction, although it is not completely solubilized. The oxidase is inhibited by high concentrations of cytochrome c. A particle-bound cytochrome c can be obtained from mitochondria by digestion with snake venom. However, the autoxidizability of the preparation indicates that the cytochrome has been solubilized in a modified form. A CO-binding pigment can be obtained from mung bean microsomes by digestion with snake venom.

     

Cyanide-insensitive Respiration in Plant Mitochondria

Pathways of electron transport have been studied in mitochondria isolated from hypocotyls of etiolated mung bean seedlings and skunk cabbage spadices that show cyanide-resistant respiratory activity. The residual flux through cytochrome c oxidase is shown to be small in comparison with the flux through an unidentified alternative oxidase that is known to have a high affinity for oxygen. This alternative oxidase is not a cytochrome. Skunk cabbage and mung bean mitochondria contain cytochromes a and a₃ that have absorption peaks differing slightly from those of animal preparations. A slow oxidation-reduction of cytochrome a₃-CN has been demonstrated. Cytochromes b undergo oxidation and reduction in the presence of cyanide but play no essential role in the cyanide-resistant pathway. Antimycin inhibits to an extent similar to that of cyanide; the respiratory chain bifurcates on the substrate side of the antimycin-sensitive site. Evidence is presented for the selective inhibition by thiocyanate, α, α′-dipyridyl, and 8-hydroxyquinoline of the alternative oxidase pathway, which may therefore contain a non-heme iron protein.     

Modulation of the Access of Exogenous NAD(P)H to the Alternative Pathway in Potato Tuber Callus Mitochondria with Triton X-100

Alternative oxidase activity in potato tuber (Solanum tuberosum L. cv Bintje) callus mitochondria with exogenous NAD(P)H as substrate is inhibited by low concentrations of the detergent Triton X-100. Alternative oxidase activity with succinate or malate as substrate is not affected by these low concentrations of Triton X-100. Cytochrome pathway activity was not influenced under these conditions, neither with endogenous nor with exogenous substrate. Washing of Triton X-100-treated mitochondria did partially restore both uninhibited and CN-resistant NADH oxidation, indicating that under these conditions Triton X-100 does not permanently remove major components from the mitochondrial membrane. Apparently, it is possible to manipulate mitochondria in such a way that the access of exogenous NADH to the alternative pathway is blocked while access to the cytochrome pathway is uninhibited. It is suggested that membrane conditions have a regulatory function (possibly via influencing the diffusion path) in the oxidation of exogenous NADH via the alternative pathway.     

UDP-glucuronosyltransferases 1A6 and 1A10 catalyze reduced menadione glucuronidation

Menadione (2-methyl-1,4-naphthoquine), also known as vitamin K3, has been widely used as a model compound in the field of oxidative stress-related research. The metabolism of menadione has been studied, and it is known that menadione undergoes a two-electron reduction by NAD(P)H:Quinone oxidoreductase 1 (NQO1) after which the reduced form of menadione (2-methyl-1,4-naphthalenediol, menadiol) is glucuronidated and excreted in urine. To investigate which human UDP-glucuronosyltransferase (UGT) isoforms participate in the glucuronidation of menadiol reduced by NQO1 from menadione, we first constructed heterologously expressed NQO1 in Sf9 cells and tested the menadiol glucuronidating activity of 16 human recombinant UGT isoforms. Of the 16 UGT isoforms, UGTs 1A6, 1A7, 1A8, 1A9, and 1A10 catalyzed menadiol glucuronidation, and, of these, UGTs 1A6 and 1A10 catalyzed menadiol glucuronidation at much higher rates than the other UGTs. Menadiol was regioselectively glucuronidated in the manner of 4-position > 1-position by UGTs 1A7, 1A8, 1A9, and 1A10. In contrast to these UGTs, only UGT1A6 exhibited 1-menadiol-preferential glucuronidating activity. The results suggest possible detoxification pathways for quinones via NQO1 reduction followed by UGT glucuronidation.     

Effect of methyl methacrylate on mitochondrial function and structure

• 1.1. Treatment of isolated rat liver mitochondria with methyl methacrylate (MM) produced membrane disruption as evidenced by the release of citrate synthase, and changes in the ultrastructure of mitochondria.
• 2.2. At concentration 0.1%, MM uncoupled oxidative phosphorylation as evidenced by stimulation of state 4 respiration supported either by pyruvate plus malate or succinate (+rotenone) and ATP-ase activity in intact mitochondria.
• 3.3. At concentration 1% MM stimulated ATP-ase activity in intact mitochondria and succinate (+rotenone) oxidation at state 4 and was without effect on this substrate oxidation at state 3.
• 4.4. MM inhibited pyruvate plus malate oxidation either at state 3 or in the presence of uncoupling agents.
• 5.5. MM inhibited the NADH oxidase of electron transport particles at a concentration which failed to inhibit either succinic oxidase or the NADH-ferricyanide reductase activity.
• 6.6. The data presented suggest that in the isolated mitochondria MM inhibits NADH oxidation in the vicinity of the rotenone sensitive site of complex I.
• 7.7. The general conclusion is that MM may block an electron transport and to uncouple oxidative phosphorylation in rat liver mitochondria. The overall in vitro effect would be to prevent ATP synthesis which could result in cell death under in vivo conditions.

     

Purification of plant mitochondria by isopycnic centrifugation in density gradients of Percoll

Mitochondria from potato tubers have been separated from contaminating organelles and membrane vesicles on self-generated Percoll gradients and in a relatively short time. The Percoll-purified mitochondria devoid of carotenoids and galactolipids showed no contamination with intact plastids, microbodies, or vacuolar enzymes. Percoll-purified mitochondria exhibited intact membranes and a dense matrix. The intactness of purified mitochondrial preparations was ascertained by the measurement of KCN-sensitive ascorbate cyt c-dependent O₂ uptake. When compared with washed mitochondria, Percoll-purified mitochondria showed improved rates of substrate oxidation, respiratory control, and ADP:O ratios. The recovery of the cyt oxidase was 70–90% and on a cyt oxidase basis the rate of succinate oxidation by unpurified mitochondria was equal to that recorded for Percoll-purified mitochondria. The great flexibility of purification procedure involving silica sols was extended from mitochondria to the isolation of intact peroxisomes.     

Properties of rat liver mitochondria with intermembrane Cytochrome c.

When rat liver mitochondria were suspended in 0.15 m KCl, the cytochrome c appeared to be solubilized from the binding site on the outside of the inner membrane and trapped in the intermembrane space. When the outer membrane of these mitochondria was disrupted with digitonin at a digitonin concentration of 0.15 mg/mg of protein, the solubilized cytochrome c could be released from mitochondria along with adenylate kinase. When mitochondria were suspended in 0.15 m KCl instead of 0.33 m sucrose, the $\text{ADP}\text{O}$ ratio observed with succinate, β-hydroxybutyrate, malate + pyruvate or glutamate as substrates was little affected. A number of cycles of State 4-State 3-State 4 with ADP was observed. The respiratory control ratios, however, were decreased, particularly when glutamate was used as the substrate. Cytochrome c oxidase activity was also decreased to 55% when assayed using ascorbate + N,N,N′,N′-tetramethyl-p-phenylene-diamine (TMPD) as substrates. Suspension of mitochondria in 0.15 m KCl resulted in an enhancement of the very low NADH oxidation by intact mitochondria and a twofold enhancement of sulfite oxidation. Trapped cytochrome c in outer membrane vesicles prepared from untreated and trypsin-treated intact mitochondria was found to be readily reduced by NADH and suggests that some cytochrome b₅ is located on the inner surface of the outer membrane. The enhanced NADH oxidase could therefore reflect the ability of cytochrome c to mediate intermembrane electron transport. The enhanced sulfite oxidase activity was sensitive to cyanide inhibition and coupled to oxidative phosphorylation ($\text{ADP}\text{O}\text{< 1}$) unlike the activity of mitochondria in sucrose medium. These results suggest that free cytochrome c in the intermembrane space can mediate electron transfer between the sulfite oxidase and the inner membrane.     

Oxidation of Reduced Nicotinamide Adenine Dinucleotide Phosphate by Potato Mitochondria: INHIBITION BY SULFHYDRYL REAGENTS

Potato tuber mitochondria oxidized exogenous NADH and exogenous NADPH at similar rates; the electron transfer inhibitor rotenone did not inhibit the oxidation of either substrate. Submitochondrial particles, prepared from potato tuber mitochondria, exhibited a greater capacity to oxidize NADH than NADPH; rotenone inhibited the oxidation of NADH by 29% and the oxidation of NADPH by 16%. The oxidation of both NADH and NADPH by potato mitochondria exhibited pH optima of 6.8, and although substantial NADH oxidase activity was observed at pH 8.0, little NADPH oxidase activity was detected at that pH. The oxidation of NADPH by the mitochondria was more sensitive to inhibition by EDTA than was the oxidation of NADH.     

Solubilization of the alternative oxidase of cuckoo-pint (Arum maculatum) mitochondria. Stimulation by high concentrations of ions and effects of specific inhibitors.

Selective solubilization of cyanide- and antimycin-insensitive duroquinol oxidase activity from cuckoo-pint (Arum maculatum) mitochondria was achieved using taurocholate. Inhibitor-sensitivities and water-forming DQH2 (tetramethyl-p-hydroquinone, reduced form): O2 stoichiometry were the same for the alternative oxidase of intact Arum mitochondria. Cyanide-insensitive oxidation of DQH2 by intact and solubilized mitochondria was stimulated by up to four-fold by high concentrations of anions high in the Hofmeister series, such as phosphate, sulphate or citrate. Optimal (0.7 M) sodium citrate increased Vmax. for DQH2 oxidation by the solubilized preparation from 450 to 2400 nmol of O2 X min-1 X mg of protein-1 and decreased the apparent Km for DQH2 from 0.53 to 0.38 mM. Inhibition of solubilized DQH2 oxidase activity by CLAM (m-chlorobenzhydroxamic acid) and SHAM (salicylhydroxamic acid) was mixed competitive/non-competitive, with apparent inhibition constants for CLAM of 25 microM (Ki) and 81 microM (KI) and for SHAM of 53 microM (Ki) and 490 microM (KI). Propyl gallate and UHDBT were non-competitive inhibitors with respect to DQH2 (apparent Ki = 0.3 microM and 12 nM respectively). Low concentrations of C18 fatty acids selectively inhibited cyanide-insensitive oxidation by intact and solubilized mitochondria, and inhibition was reversed by 1% (w/v) bovine serum albumin. Inhibition was competitive with DQH2, suggesting that fatty acids interfere reversably with the binding of DQH2 to the oxidase. These results tend to support the view that quinol oxidation by the alternative pathway of Arum maculatum mitochondria is catalysed by a quinol oxidase protein, rather than by a non-enzymic mechanism involving fatty acid peroxidative reaction. [Rustin, Dupont & Lance (1983) Trends Biochem. Sci. 8, 155-157; (1983) Arch. Biochem. Biophys. 225, 630-639].     

Inhibition of NADH oxidation by pyridine derivatives

The neurotoxicity of 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine, an impurity in an illicit drug, is expressed after its oxidation to 1-methyl-4-phenylpyridinium by monoamine oxidase. The pyridinium is concentrated by carrier-mediated transport into the mitochondria where it inhibits NADH dehydrogenase and, hence, ATP synthesis. Some structurally related compounds have been tested for their effect on the oxidation of NAD+-linked substrates in intact mitochondria, and for the inhibition of the accumulation of the pyridinium into mitochondria and of NADH dehydrogenase activity in a membrane preparation. Some pyridine derivatives are more inhibitory to NADH dehydrogenase than is 1-methyl-4-phenylpyridinium but these are not concentrated into mitochondria by the uptake system. 4-Phenylpyridine, one of the most effective inhibitors, both occurs naturally and is an environmental pollutant.     

Respiratory Chain of Plant Mitochondria: XVIII. Point of Interaction of the Alternate Oxidase with the Respiratory Chain

Oxidation of the respiratory chain carriers of anaerobic, CO-saturated skunk cabbage (Symplocarpus foetidus) mitochondria, by means of an O₂ pulse, proceeds primarily through the cyanide-insensitive alternate oxidase, since the oxidation of cytochromes a and a₃ takes place with a half-time of 3 seconds, corresponding to the rate of dissociation of CO from reduced cytochrome a₃. Ubiquinone and part of the flavoprotein are oxidized within 1 second under these conditions, and this rapid rate of oxidation is strongly inhibited by m-chlorobenzhydroxamic acid (mCLAM), a specific inhibitor of the alternate oxidase of plant mitochondria. The rate of ubiquinone oxidation under these conditions in white potato (Solanum tuberosum) mitochondria, which have no alternate oxidase, is the same as that in skunk cabbage mitochondria treated with mCLAM. Ubiquinone, thus identified as the carrier common to both the cytochrome and alternate oxidase pathways, is linked to the alternate oxidase by a flavoprotein of midpoint potential 50 millivolts more negative with which it is in equilibrium. This arrangement provides a switch for diverting electron transport primarily through the cytochrome pathway under state 3 conditions and primarily through the alternate oxidase pathway under state 4 conditions.     

Monoamine oxidase activity measurements using radioactive substrates

The use of Amberlite CG-50, Dowex 50 and solvent extraction for separation of the oxidation products of the biogenic amines are compared, and measurements of monoamine oxidase activity using ¹⁴C-labeled biogenic amines are described. K_m data for tyramine, dopamine, tryptamine, and serotonin for monoamine oxidase activity of rabbit brain mitochondria are reported. Rates of product formation from [¹⁴C]tyramine are compared with polarographic measurements of oxygen utilization using purified MAO and intact mitochondria from rabbit liver and brain. Difficulties in comparative measurements of monoamine oxidase activity and some reasons for wide variations in published data are discussed.