On rat liver mitochondrial monoamine oxidase activity and lipids

Following earlier observations on the retention of 5-hydroxytryptamine oxidizing activity by a purified preparation of monoamine oxidase from rat liver mitochondria, this fraction has been obtained in a water-soluble form by Triton X-100 gradient gel filtration and DEAE-Bio-Gel A chromatography. The soluble fraction appears to depend on Triton X-100 and phospholipids for its activity. The results seem to implicate membrane lipid components in the expression of rat liver mitochondrial monoamine oxidase activity.     

Kinetic properties of membrane-bound and Triton X-100-solubilized human brain monoamine oxidase

The kinetic properties of membrane-bound and Triton X-100-solubilized human brain mitochondrial type A and B monoamine oxidase were examined. These studies reveal that the K_m values for phenylethylamine and benzylamine, type B monoamine oxidase substrates, were only slightly increased by the solubilization procedure. The K_m value for 5-hydroxytryptamine, a type A monoamine oxidase substrate, was similarly increased by treatment with Triton X-100. The K_m values for oxygen with all three amine substrates were unaffected by solubilization of the oxidase. Similarly, the optimum pH for deamination of substrates for the B isoenzyme was essentially unaltered in the solubilized preparation as compared to the membrane-bound enzyme whereas that for 5-hydroxytryptamine metabolism was decreased from pH 8.5 to approximately 7.75 on solubilization. The energy of activation with all three substrates was altered on solubilization of the oxidases with Triton X-100. The energy of activation for the B monoamine oxidase substrates increased whereas that for 5-hydroxytryptamine decreased. These data support the contention that the lipid environment surrounding the two forms of monoamine oxidase controls, in part, the activity and kinetic properties of the enzymes.     

Optimal assay and subcellular location of phosphatidylglycerol synthesis in lung

Synthesis of phosphatidylglycerol from CDPdiacylglycerol and glycerol 3-phosphate by membranous subcellular fractions of rat lung and liver was optimal when assayed in the presence of bovine serum albumin and Triton X-100. Specific activities of glycerolphosphate phosphatidyltransferase in all membranous subcellular fractions of lung were several times higher than the corresponding fractions from liver. Distribution of this enzyme in subcellular fractions of lung or liver closely parallel the activity of the mitochondrial enzymes monoamine oxidase and succinate cytochrome c reductase. The phosphatidylglycerol-synthesizing activity in microsomes of both lung and liver was a minor fraction of total tissue activity and could be interpreted as due either to contamination with outer mitochondrial membrane or to a small amount of activity innate to microsomes. These results suggest that phosphatidylglycerol, which is believed to be a component of pulmonary surfactant, is synthesized by lung at a rapid rate relative to liver and that the subcellular distribution of its synthesis is similar in both tissues, with mitochondria as the major site.     

Effect of the nonionic detergent Triton X-100 on mitochondrial succinate-oxidizing enzymes

Specific activities of succinate:coenzyme Q reductase, ubiquinone:cytochrome c reductase, cytochrome oxidase, succinate:cytochrome c reductase, succinate oxidase, and ubiquinol oxidase have been measured in rat liver mitochondria in the presence of Triton X-100. The last three activities are much more sensitive to Triton X-100 than the first ones; the data suggest that the electron transport chain components cannot react with each other in the presence of the detergent. At least in the case of succinate:cytochrome c reductase, reconstitution of the detergent-treated membranes with externally added phospholipids reverses the inhibition produced by Triton X-100. These results support the idea that the respiratory chain components diffuse at random in the plane of the inner mitochondrial membrane; the main effect of the detergent would be to impair lateral diffusion by decreasing the area of lipid bilayer. When detergent-treated mitochondrial suspensions are centrifuged in order to separate the solubilized from the particulate material, only the first three enzyme activities mentioned above are found in the supernatants. After centrifugation, a latent ubiquinol:cytochrome c oxidase activity becomes apparent, whereas the same centrifugation process produces inhibition of cytochrome c oxidase in the presence of certain Triton X-100 concentrations. These effects could be due either to a selective solubilization of regulatory or catalytic subunits or to a conformational change of the enzyme-detergent complex.     

Identity of the active site flavin-peptide fragments from the human “A”-form and the bovine “B”-form of monoamine oxidase

The monoamine oxidase present in the mitochondria of human placenta was verified to be the clorygyline sensitive or A form. This property was not abolished following extensive phospholipase treatment and Triton X-100 extraction. Proteolytic digestion of the partially purified enzyme using trypsin and chymotrypsin and isolation of a peptide containing the covalently linked flavin coenzyme permitted determination of the amino acid composition and sequence of the flavin region of the catalytic site of the enzyme. The structure of the flavin peptide was found to be identical to that of the bovine liver enzyme which is clorgyline insensitive, hence, the B form. The flavin peptide segment of mitochondrial monoamine oxidase is thus conserved between the two forms and among mammalian species.     

Isolation of pure,catalytically active human liver monoamine oxidase B: Antibody complex

Monoamine oxidase B was purified from human liver mitochondria using a monoclonal antibody, MAO B-1C2, which recognizes monoamine oxidase B but not A. Triton X-100 extracts of mitochondria were incubated with purified MAO B-1C2 (IgG₁), and the catalytically active enzyme:antibody complex was isolated by affinity chromatography on Protein A-Sepharose. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis of the complex revealed the presence of four polypeptide bands (monoamine oxidase B, 57,900 dalton; antibody heavy chain, 52,200 dalton; and two light chains, 29,400 and 27,700 dalton), and indicated a 1:1 stoichiometric ratio of enzyme to antibody. This method gave 154-fold purification of the enzyme from mitochondria.     

Assay and subcellular localization of the arylsulphatases in rat brain

—The properties and subcellular localization of type I (nitrophenyl) and type II (nitrocatechol) arylsulphatases were investigated in brain tissue of the rat, and optimal assay conditions were established. Sulphate, phosphate and sulphite ions inhibited the nitrocatechol sulphatases; nitrophenyl sulphatase was inhibited only by sulphite. The presence of latent enzyme activity was demonstrated for the nitrocatechol sulphatases, beta-glucuronidase, and beta-glycerophosphatase in rat and mouse brain homogenates. These hydrolases were highly sensitive to mechanical and osmotic damage; and Triton X-100 was very effective in releasing their latent (bound) activities, a finding suggestive of a lysosomal localization. Activity of nitrophenyl sulphatase was unaffected by osmotic changes or Triton X-100, characteristics suggesting a membranous association for this enzyme. Total activity of nitrophenyl sulphatase was approximately twice as great in canine gray matter as in canine white matter; the converse obtained for beta-glucuronidase activity. Values for total enzymic activity of the nitrocatechol sulphatases in canine white and gray matter were similar. Fractionation of homogenates from rat brain by differential centrifugations and separation of crude mitochondrial fractions by sucrose density gradient centrifugations revealed the following: (1) most of the nitrocatechol sulphatase activity (93 per cent) and all of the nitrophenyl sulphatase activity were sedimentable; (2) crude mitochondrial fractions exhibited the highest relative specific activity (RSA = 1·38) for the nitrocatechol sulphatases, whereas microsomal fractions displayed the highest RSA for nitrophenyl sulphatase (1·89); (3) the lightest fraction (A + B) and the densest fraction (E) from the sucrose density gradient contained most of the activity for both the type I and type II arylsulphatases, whereas the RSA of cytochrome oxidase was greatest in the intermediate density regions (fractions C and D); (4) the highest RSA for beta-glucuronidase and beta-glycerophosphatase occurred in gradient fraction C; (5) appreciable activity of beta-glycerophosphatase was found in a nerve ending fraction (M₃). It is suggested that the hydrolases in heterogeneous tissue like brain might be associated with lysosomal particles of differing enzyme compositions and varying populations, and that the data on distribution lend credence to the concept of bimodal and possible trimodal particle affinity for the hydrolases of brain tissues.     

Guanine-deaminase activity in rat brain and liver

1. Guanine deaminase in rat brain and liver was distributed among all the subcellular fractions: nuclei, `heavy' mitochondria, `light' mitochondria, microsomes and the supernatant fluid. The greater part of the activity passed into the soluble fraction. Among the particulate components, the `light' mitochondria constituted the richest fraction. 2. The sum of the enzymic activities of the component fractions obtained on differential centrifugation was considerably greater than the activity of guanine deaminase in the whole homogenate. 3. The `heavy'-mitochondrial fraction had a powerful inhibitory effect on the guanine-deaminase activity of the supernatant fraction. 4. All the sedimented fractions, except the microsomes, gave rise to higher guanine-deaminase activity on treatment with Triton X-100. 5. The inhibitory capacity of the `heavy' mitochondria increased on treatment with Triton X-100; the detergent-treated nuclear fraction also brought about inhibition of the 5000g supernatant. 6. Guanine-deaminase inhibitor from the `heavy' mitochondria was solubilized by high-speed grinding of the particles, followed by treatment with Triton X-100. The inhibitor appeared to be protein in nature, since it was precipitated by trichloroacetic acid and by half-saturation with ammonium sulphate, and was non-diffusible. It was inactivated by heating at 50° for 5min. 7. It is possible that the guanine deaminase associated with particles differs from the soluble enzyme in its response to inhibitor.     

Separation of synaptic junctional complexes from thin layers of rabbit cerebral cortex

Synaptic junctional fractions were separated from rabbit brain by procedures based on combining the methods of Cotman and Taylor [4], Orosz et al. [16, 17] and Lisman et al. [13]. Thin layers of cerebral cortices were homogenized to obtain a crude mitochondrial-synaptosomal fraction. The sedimentation rates of mitochondria and mitochondria containing synaptosomes were increased by raising the density of mitochondria with an insoluble dense formazan deposit inside mitochondria after iodo-nitrotetrazolium treatment. The synaptic plasma membrane fraction isolated by this method contained no mitochondrial contamination. After Triton X-100 treatment the insoluble residues of the detergent were centrifuged through discontinuous sucrose gradients. A great enrichment of morphologically identifiable intact synaptic junctions was observed in some of the obtained interface layers.     

The heterogeneity of rat brain mitochondria isolated on continuous sucrose gradients

Abstract— The distribution of a series of enzymes in the post-nuclear supernatant of rat brain homogenates was investigated following continuous density-gradient centrifugation. The enzymes studied were acetyl coenzyme A synthetase, glutamic dehydrogenase, glutamine synthetase, glutaminase I, succinic dehydrogenase and monoamine oxidase. Each of these enzymes with the exception of glutamine synthetase appears predominantly in the mitochondrial region of the gradient. Although about 20 per cent of this enzyme is present in the crude mitochondrial pellet, on density gradient centrifugation no special association of glutamine synthetase with any of the mitochondrial fractions was observed. Each of the other enzymes studied was found to have a characteristic distribution in the gradient; this suggests that brain mitochondria may be heterogeneous both in buoyant density and in their enzyme content. Three principal fractions are described: (i) dense particles containing high concentrations of acetyl coenzyme A synthetase and glutamic dehydrogenase; (ii) a fraction comprising the bulk of the mitochondria with high levels of monoamine oxidase, succinic dehydrogenase and glutaminase I; and (iii) particles in the synaptic ending region of the gradient characterized by relatively high levels of monoamine oxidase and succinic dehydrogenase and containing only small amounts of the other enzymes studied. If the mitochondrial heterogeneity that is observed on centrifugation reflects the existence within brain cells of mitochondria with specialized function, a partial explanation may be available for multiple pools of tricarboxylic acid cycle intermediates which have been postulated from isotopie labelling experiments.     

Early changes in mitochondrial monoamine oxidase activity of rat liver during 2-acetylaminofluorene feeding

The activities of mitochondrial type A and B monoamine oxidase were determined in the liver of rats fed a diet containing 2-acetylaminofluorene (AAF). Three days after the initiation of AAF-feeding, there was a significant decrease of type B monoamine oxidase activity without affect on type A enzyme. The decreased activity of type B monoamine oxidase, which reached a minimum after three weeks, was sustained for as long as AAF-feeding was continued. Sex-related difference in response to AAF was seen in the rat with respect to the onset and the intensity of the decreased type B monoamine oxidase activity, male rats being more sensitive to the carcinogen than female rats. In contrast to the in vivo effect, AAF showed a potent inhibitory effect on type A monoamine oxidase, rather than on type B enzyme, when added in vitro. The pI₅₀ values were estimated to be 7.5 against type A monoamine oxidase and 4.1 against type B enzyme, respectively. The in vitro inhibition of both types of monoamine oxidase by AAF was competitive. The K_i values for AAF were calculated to be 9.51 · 10^?9 M for type A monoamine oxidase and 1.30 · 10^?5 M for type B enzyme, respectively. In accordance with the potent inhibitory effect of AAF on type A monoamine oxidase in vitro, a single administration of the carcinogen, at a dose of 50 mg/kg, resulted in a marked and temporal decrease of the enzyme activity in the mitochondria of male rat liver. Recovery of the decreased type B monoamine oxidase activity was slow, and the enzyme activity did not return to control levels, even if rats were fed the basal diet for 2 or 4 weeks after the cessation of AAF-feeding.     

Enhancement and inhibition of enzymes of heme metabolism by diethyl maleate in the rat kidney

The acylation of sn-glycerol 3-phosphate with palmityl-CoA was compared in mitochondria and microsomes isolated from rat liver. Polymyxin B, an antibiotic known to alter bacterial membrane structure, stimulated the mitochondrial glycerophosphate acyltransferase but inhibited the microsomal enzyme. When mitochondrial and microsomal fractions were incubated at 4–6 °C for up to 4 h, the mitochondrial enzyme remained virtually unchanged while the microsomal enzyme lost about one-half of its activity. Incubations at higher temperatures also revealed that the mitochondrial enzyme was comparatively more stable under the conditions employed. The mitochondrial acyltransferase showed no sensitivity to bromelain, papain, Pronase, and trypsin, all of which strongly inhibited the microsomal enzyme. The differential sensitivity to trypsin was observed in mitochondria and microsomes isolated from other rat organs. However, the liver mitochondrial glycerophosphate acyltransferase was inhibited by trypsin in the presence of either 0.05% deoxycholate or 0.1% Triton X-100. The trypsin sensitivity of the mitochondrial glycerophosphate acyltransferase in the presence of detergent was not due to the presence, in the mitochondrial fraction, of a trypsin inhibitor which became inactivated by Triton X-100 or deoxycholate. The results suggest that the catalytic site of mitochondrial glycerophosphate acyltransferase is not exposed to the cytosolic side and it is located in the inner aspect of the outer membrane.     

Modulation of brain mitochondrial function by deprenyl

The present study shows that deprenyl, a known inhibitor of monoamine oxidase B (MAO B), may generate changes in mitochondrial function. Brain submitochondrial membranes (SMP), synaptosomes and cytosolic fractions were incubated with different deprenyl concentrations and nitric oxide synthase (NOS) activity was measured. The effect of deprenyl on oxygen consumption, calcium-induced permeability transition and hydrogen peroxide (H(2)O(2)) production rates was studied in intact mitochondria. Respiratory complexes and monoamine oxidase activities were also measured in submitochondrial membranes. Incubation of brain submitochondrial membranes with deprenyl 10, 25 and 50 microM inhibited nitric oxide synthase activity in a concentration-dependent manner. The same effect was observed in cytosolic fractions and synaptosomes. Monoamine oxidase activity was inhibited at lower deprenyl concentrations (from 0.5 microM). Cytochrome oxidase (complex IV) activity was found 42% increased in the presence of 25 microM deprenyl in a condition of maximal nitric oxide synthase activity. Incubation of brain mitochondria with deprenyl 25 microM produced a 60% increase in oxygen uptake in state 3, but no significant changes were observed in state 4. Pre-incubation of brain mitochondria with deprenyl 0.5 and 1 microM inhibited calcium-induced mitochondrial permeability transition and decreased hydrogen peroxide production rates. Our results suggest that in vitro effects of deprenyl on mitochondrial function can occur through two different mechanisms, involving nitric oxide synthase inhibition and decreased hydrogen peroxide production.     

Oxygenation of mitochondrial membranes by the reticulocyte lipoxygenase. Action on monoamine oxidase activities A and B

Incubation of isolated rat liver mitochondria with the pure rabbit reticulocyte lipoxygenase caused a time-dependent inactivation of the monoamine oxidase activities A and B. Furthermore, a conversion of the monoamine oxidase into a diamine oxidase was observed. The inactivation kinetics for both monoamine oxidase activities A and B showed a biphasic behaviour; a reversible short-term inhibition during the first 5 min of incubation was followed by an irreversible inactivation of the enzyme. The kinetic studies suggest that the slow irreversible inactivation of the monoamine oxidase activities is due to secondary reactions subsequent to the initial attack of the lipoxygenase on the mitochondrial outer membrane. During the interaction of the lipoxygenase with the mitochondria, only about 1.5% of the polyenoic fatty acids present in the mitochondrial membranes were oxygenated. The predominant products formed during the interaction of the lipoxygenase with the mitochondrial membranes are (13S)-hydro(pero)xy-9Z,11E-octadecadienoic acid and (15S)-hydro(pero)xy-5,8,11,13(Z,Z,Z,E)-eicosatetraenoic acid.     

Effect of disulfiram (DS) on mitochondria from rat hippocampus: Metabolic compartmentation of DS neurotoxicity

This experiment was designed to study the acute effects of disulfiram on mitochondrial enzymes in nonsynaptic and synaptic mitochondria from rat hippocampus. Cytochromec oxidase, monoamine oxidase-B, glycerolphosphate acyltransferase and betahydroxybutyrate dehydrogenase were studied. Differences in enzyme activity were seen in controls. Cytochromec oxidase activity was higher in synaptic mitochondria whereas glycerolphosphate acyltransferase activity was higher in nonsynaptic mitochondria. Mitochondria from disulfiram treated rats, particularly synaptic mitochondria, exhibited lower specific activities of cytochromec oxidase and monoamine oxidase-B. These alterations were not limited to either the inner or outer mitochondrial membrane. Transmission electron microscopy revealed that mitochondria from disulfiram treated rats were severely altered in isolated preparations as well as in those from whole tissue. This study shows that disulfiram exerts a differential effect on mitochondrial subpopulations.     

Localization and identification of covalently bound flavoproteins in rat liver mitochondria by prelabeling of their flavin moiety

The covalently bound flavoproteins in rat liver mitochondria were prelabeled by injecting [14C]riboflavin into a rat, then liver mitochondria were obtained and further labeled with [3H]pargyline, a suicide inhibitor of monoamine oxidase. When the mitochondria were subjected to osmotic lysis, two covalently bound flavoproteins having molecular weights of 110,000 and 94,000 were found in the supernatant. These proteins were identified as sarcosine dehydrogenases. Upon treatment of the membranous fraction with 1% Triton X-100, succinate dehydrogenase with a molecular weight of 70,000 was found in the soluble fraction, while two well-separated proteins doubly-labeled with 14C and 3H were found in the insoluble fraction. Their molecular weights were 61,000 and 57,000. By isoelectric focusing, two 3H peaks were observed with pI values of 8.3 and 8.4. The former corresponded to the 61,000-dalton protein, and the latter, to the 57,000 one. From the data obtained by using selective inhibitors, deprenyl and clorgyline, the [3H]pargyline-binding proteins with molecular weights of 61,000 and 57,000 were assigned to proteins of monoamine oxidases of type A and type B, respectively.     

Changes in non-synaptosomal and synaptosomal mitochondrial membrane-linked enzymatic activities after transient cerebral ischemia

Non-synaptosomal and synaptosomal mitochondrial membrane-linked enzymatic activities, NADH-cytochrome c reductase rotenone insensitive (marker of the outer membrane) and cytochrome oxidase (marker of the inner membrane), were measured in rat brain hippocampus and striatum immediately after and 1, 4, and 7 days following the induction of complete transient ischemia (15 min) by the four vessel occlusion method. Furthermore citrate synthetase activity was measured with and without Triton X-100 in order to qualitatively evaluate the membrane permeability. Nonsynaptosomal mitochondrial membranes showed reduction of both activities only in the late reperfusion phase: NADH-CCRRi decreased in striatal mitochondria after 4–7 days and only after 7 days in the hippocampus. COX activity decreased only in striatal mitochondria 7 days after ischemia. Non-synaptosomal mitochondrial membrane permeability did not show changes. Synaptosomal mitochondria showed a decrease of NADH-CCRRi only at 7 days of reperfusion both in hippocampus and striatum, while COX activity decreased only during ischemia and returned to normal levels in the following days in the two areas considered. In summary, free mitochondria showed insensitiveness to ischemia but they risulted damaged in the late reperfusion phase, while mitochondria from the synaptic terminal showed ischemic damage, partially restored during reperfusion. The striatal mitochondria showed a major susceptibility to ischemia/repefusion damage, showing changes earlier than the hippocampal ones.     

Isolation of synaptic junctional complexes of high structural integrity from rat brain

A new method has been developed for isolating synaptic junctional complexes (SJC) of high structural integrity. The major step in the isolation involves homogenization of a synaptosomal membrane (SM) fraction in a biphasic system consisting of Freon 113 and an aqueous phase containing 0.2% Triton X-100. Well-preserved SJCs, along with membrane vesicles, were recovered in the aqueous phase after low-speed centrifugation of the homogenate. The membranes were subsequently separated from the SJCs by centrifugation on a discontinuous sucrose density gradient. The purity and identity of subcellular fractions were monitored by thin sectioning electron microscopy, using specific and nonspecific staining methods. From the electron microscope studies we conclude that SJCs and their components occupy about 65% of the area covered by structures in this fraction. The assay of enzyme activities indicates that homogenization in Triton-Freon and subsequent steps of the isolation procedure affect the activities of Na, K-ATPase, cytochrome oxidase, and acid phosphatase to different extents, but do not cause total inactivation. Electrophoresis of the SJC-enriched fraction on sodium dodecyl sulfate-polyacrylamide gels has demonstrated that a polypeptide which co-migrates with tubulin is the major component in this fraction, and that a polypeptide co-migrating with actin is also present.     

Subcellular distribution and properties of carbonyl reductase in guinea pig lung

On subcellular fractionation, carbonyl reductase (EC 1.1.1.184) activity in guinea pig lung was found in the mitochondrial, microsomal, and cytosolic fractions; the specific activity in the mitochondrial fraction was more than five times higher than those in the microsomal and cytosolic fractions. Further separation of the mitochondrial fraction on a sucrose gradient revealed that about half of the reductase activity is localized in mitochondria and one-third in a peroxidase-rich fraction. Although carbonyl reductase in both the mitochondrial and microsomal fractions was solubilized effectively by mixing with 1% Triton X-100 and 1 M KCl, the enzyme activity in the mitochondrial fraction was more highly enhanced by the solubilization than was that in the microsomal fraction. Carbonyl reductases were purified to homogeneity from the mitochondrial, microsomal, and cytosolic fractions. The three enzymes were almost identical in catalytic, structural, and immunological properties. Carbonyl reductase, synthesized in a rabbit reticulocyte lysate cell-free system, was apparently the same in molecular size as the subunit of the mature enzyme purified from cytosol. These results indicate that the same enzyme species is localized in the three different subcellular compartments of lung.     

Studies on the control of 4-aminobutyrate metabolism in ''synaptosomal'' and free rat brain mitochondria.

1. The specific activities of 4-aminobutyrate aminotransferase (EC 2.6.1.19) and succinate semialdehyde dehydrogenase (EC 1.2.1.16) were significantly higher in brain mitochondria of non-synaptic origin (fraction M) than those derived from the lysis of synaptosomes (fraction SM2). 2. The metabolisms of 4-aminobutyrate in both 'free' (non-synaptic, fraction M) and 'synaptic' (fraction SM2) rat brain mitochondria was studied under various conditions. 3. It is proposed that 4-aminobutyrate enters both types of brain mitochondria by a non-carrier-mediated process. 4. The rate of 4-aminobutyrate metabolism was in all cases higher in the 'free' (fraction M) brain mitochondria than in the synaptic (fraction SM2) mitochondria, paralleling the differences in the specific activities of the 4-aminobutyrate-shunt enzymes. 5. The intramitochondrial concentration of 2-oxoglutarate appears to be an important controlling parameter in the rate of 4-aminobutyrate metabolism, since, although 2-oxoglutarate is required, high concentrations (2.5 mM) of extramitochondrial 2-oxoglutarate inhibit the formation of aspartate via the glutamate-oxaloacetate transaminase. 6. The redox state of the intramitochondrial NAD pool is also important in the control of 4-aminobutyrate metabolism; NADH exhibits competitive inhibition of 4-aminobutyrate metabolism by both mitochondrial populations with an apparent Ki of 102 muM. 7. Increased potassium concentrations stimulate 4-aminobutyrate metabolsim in the synaptic mitochondria but not in 'free' brain mitochondria. This is discussed with respect to the putative transmitter role of 4-aminobutyrate.