The biosynthesis of tyramine glucuronide by liver microsomal fractions.

Labelled tyramine glucuronide was synthesized in vitro from UDP-[14C]glucuronic acid, [14C]tyramine or [3H]tyramine. The glucuronidation was carried out at pH9.2 in the presence of a monoamine oxidase inhibitor, trans-2-phenylcyclopropylamine. The Km values for tyramine were 69 and 125 micrometer and those for UDP-glucuronic acid were 260 and 290 micrometer respectively for guinea-pig and rat liver microsomal preparatons. The specific activities of microsomal glucuronyltransferase measured in fresh hepatic preparations of guinea pig, mouse and rat were respectively 601, 251 and 235 pmol of [14C]tyramine glucuronide/min per mg of protein. Increase in activity ranged from 2- to 6-fold in preparations which were frozen and thawed once and 5.4- to 10-fold when the freezing and thawing was repeated. Rabbit liver has very low activity, and monkey liver and intestine were completely devoid of this conjugating capacity.     

THE DISTRIBUTION AND PROPERTIES OF THE NONSPECIFIC N-METHYLTRANSFERASE IN BRAIN

Abstract— The distribution and properties of a nonspecific N -methyltransferase in the rat brain are described. The enzyme N -methylates tryptamine and N -methyltryptamine as well as β-phenylethylamine, phenylethanolamine, tyramine and octopamine. The enzyme exhibits a pH optimum of 7·9 with phosphate buffer and has a K_m for tryptamine of 28 μM. There are potent inhibitors to the enzyme that can be removed by dialysis. Enzymatic activity is present in the brains of a number of species including man, rat, mouse, guinea-pig and frog. Its activity is unevenly distributed in the brain with the highest activity in the cerebral cortex and striaturn of the rat and in the subcortical white matter in man. Studies of its subcellular distribution indicate that most of the N -methylation activity is released into the soluble fraction. Enzymatic activity is also present in a number of peripheral tissues of the rat.     

Some parameters affecting the activity of monoamine oxidase in purified bovine brain mitochondria.

Abstract— Some parameters affecting the activity of monoamine oxidase (MAO) in purified beef brain mitochondria were investigated, and diversities in enzyme properties were found as a function of substrate. The deamination of the biogenic amines: serotonin, dopamine, tyramine, tryptamine, phenylethylamine and two non-physiological amines, kynuramine and m-iodobenzylamine, was studied. Anions in high concentrations inhibited enzyme activity with kynuramine being the substrate most affected. Among the biogenic amines, the activity with the indolalkylamines showed greater sensitivity to mono-valent anions such as chloride than to polyvalent ions such as phosphate whereas the opposite was true with the phenylalkylamines. However, pyrophosphate ion had little or no effect on MAO activity, regardless of substrate. The inhibition of kynuramine and serotonin deamination was non-competitive but mixed competitive inhibition was found with tyramine and phenylethylamine. The activity of MAO was markedly affected by pH, and it had been previously reported that the substrates showed different pH optima in their oxidation. The effect of pH on activity has been attributed in part to changes in the ionization of the substrate and the hypothesis that the true substrate is the non-protonated amine. This was reflected in kinetic studies showing high substrate inhibition with increased pH. It was calculated that phenylethylamine would have the highest percentage of un-ionized amine at pH 8.2 and 9.1. At these pHs, there was more pronounced inhibition with high substrate concentrations of phenylethylamine than with the other substrates. In contrast, there was little inhibition with high substrate concentrations of tyramine which was the most ionizable of the substrates tested. When K_m values obtained at pH 7.4, 8.2 and 9.1 were corrected for ionization of the substrate, the corrected K_m was lowest at pH 7.4 for all substrates. Less than 50% of MAO activity was lost when beef brain mitochondria was heated at 50°C for 20 min. However, there was only a slight variation with substrate in the thermal inactivation experiments. It is concluded that the mitochondrial membrane environment surrounding the enzyme imposes certain restrictions on the enzymatic activity with respect to the different substrates which, in turn, are also affected by such parameters as pH and ions. The results are discussed in terms of the relationship of these factors to the question of enzyme multiplicity.     

Sulfate Conjugation of Monoamines in Human Brain: Purification and Some Properties of an Arylamine Sulfotransferase from Cerebral Cortex

An arylamine sulfotransferase (PST-M) from human brain cortex that is involved in the formation of O-sulfate esters of monoamines has been purified 272-fold by ammonium sulfate fractionation, gel filtration, DEAE-cellulose ion-exchange chromatography, chromatofocussing, and hydroxyapatite chromatography. A molecular weight of 62,000, pK of pH 5.8, and an optimum pH for the reaction at 7.8-8.0 with respect to tyramines have been determined. This enzyme possesses an extremely high affinity for dopamine and m-tyramine based on the low Km values and is moderately active toward noradrenaline and p-tyramine. Serotonin is a poor substrate. In contrast, another sulfotransferase, PST-P, which has been separated from PST-M and partially purified, exhibited a very high affinity for phenol and nitrophenols but was inactive toward the amine sulfate acceptors. In the human brain the specific activity toward dopamine as well as the ratio of activity toward dopamine/phenol was considerably higher than those for rat, hog, and bovine brains.     

Purification and properties of rabbit brain and liver 4-aminobutyrate aminotransferases isolated by monoclonal-antibody immunoadsorbent chromatography.

The use of a monoclonal-antibody immunoaffinity column for the rapid isolation of 4-aminobutyrate aminotransferases (EC 2.6.1.19) from rabbit brain and liver is described. Homogeneous enzyme protein is eluted from the immunoadsorbent with 100mM-citrate buffer, pH5, and remains stable at 4 degrees C for several days. One such column (bed volume 8 ml) has been used 40 times in a 9-month period to isolate 10-15 units of enzyme activity (specific activity approx. 3.5-7.5 units/mg) per extraction. Kinetic and spectral analysis of the enzymes from the two tissues revealed a close similarity. Sodium dodecyl sulphate/polyacrylamide-gel electrophoresis showed the isolated enzyme to have a monomeric Mr of 52 000, and this was confirmed by h.p.l.c. gel exclusion at pH 5.0. The results of Sephadex G-100 chromatography at different pH values are taken to indicate that the enzyme behaves as a dimer at pH 7.0 and above, but as a monomer at pH 5.0. 4-Aminobutyrate aminotransferase isolated from the brain by the procedure of Fowler & John [(1981) Biochem. J. 197, 149-152] is more stable than the immunoaffinity-purified material, and has been shown to contain a contaminant protein of Mr 84 000 that exhibits succinic semialdehyde dehydrogenase activity.     

Enzymic inactivation of neurotensin by hypothalamic and brain extracts of the rat.

A sensitive and specific radioimmunoassay has been developed to study inactivation of neurotensin by hypothalamic and brain peptidases. Degrading activity of peptidases from both hypothalamus and brain seems to have similar activity. These peptidases are temperature- and time- dependent. Brain and hypothalamic enzymes of particulate fractions can be differentiated on the basis of the pH effects; brain peptidase(s) has (have) maximal activity at pH 7.4 and hypothalamic peptidase(s) displaying a maximal activity at pH 5.8. Kidneys and liver extracts contain enzyme(s) degrading neurotensin.     

The enzymatic system of phospholipid deacylation-reacylation in rat liver lysosomal membranes

It has been shown for the first time that lysosomal (tritosomal) membranes of rat liver contain enzymes that are responsible for the deacylation-reacylation of phospholipids; their activity optimum lies at pH 7.0. Deacylation of lysosomal membrane phospholipids is controlled by a cascade of enzymatic reactions involving Ca2(+)-dependent phospholipase A1 which exhibits the maximal activity at 2.5 mM Ca2+ and at neutral values of pH, as well as lysophospholipase. Reacylation of lyso-derivatives of phospholipids is catalyzed by Mg2(+)-activated oleoyl-CoA:lysophosphatidylcholine acyltransferase having an activity optimum at pH 7.2.     

Disorders of the dynamics of postnatal development of monoamine oxidase activity in the brain as affected by the antenatal action of alcohol

The activity of different types of monoamine oxidase (MAO)-MAO, type A (substrate serotonin) and two types of mixed MAO forms using tyramine or dopamine as substrates, in different brain regions of the rat offspring exposed prenatally to ethanol was investigated on the 30th and 60th day postnatally. The present study has revealed differences in the development of brain MAO activity during ontogenesis. Disturbances in the activity of all MAO types investigated as well as the distortion of their postnatal development have been observed in the brain of the rat offspring exposed prenatally to ethanol. The possible teratogenic effect of ethanol on the developing fetal brain is discussed.     

A SENSITIVE ENZYMATIC-ISOTOPIC METHOD FOR THE ANALYSIS OF TYRAMINE IN BRAIN AND OTHER TISSUES

Abstract— An enzymatic-isotopic assay for the measurement of tyramine with a sensitivity of 1.0 ng has been developed. Using this assay, the endogenous content of tyramine in various tissues from adult rats has been determined. The highest tyramine content was found in rat heart atria, followed by salivary gland, kidney, and brain. Within the brain the distribution of tyramine is heterogeneous and the highest tyramine content was localized in the striatum.     

Tyrosine aminotransferase of chicken liver: purification and properties

Tyrosine aminotransferase has been purified from chicken liver to homogeneity by a 5-step procedure. The resultant enzyme preparation has a specific activity (256 units activity/mg protein) comparable to results published for the enzyme purified from rat liver and represented an overall recovery of 35-40%. In terms of structure (native and subunit molecular weights, immunological reactivity, and kinetic parameters) (apparent Michaelis constants for L-tyrosine and 2-oxoglutarate, oxoacid specificity, pH optimum) the purified enzyme from chicken liver exhibits remarkable similarities to tyrosine amino-transferase from rat liver.     

Purification and characterization of deacetylipecoside synthase from Alangium lamarckii Thw

Deacetylipecoside synthase (DIS), the enzyme catalyzing the condensation of dopamine and secologanin to form the (R)-epimer of deacetylipecoside, has been purified 570-fold from the leaves of Alangium lamarckii and partially characterized. The isolated enzyme is a single polypeptide with Mr 30,000, and has a pH optimum at 7.5 and a temperature optimum at 45 degrees C. The apparent Km values for dopamine and secologanin are 0.7 and 0.9 mM, respectively. DIS exhibits high substrate specificity toward dopamine, whereas neither tyramine nor tryptamine are utilized. The enzyme activity is not inhibited by its substrate dopamine, but is inhibited by alangimakine and dehydroalangimakine with similar I50 values of 10 microM. DIS presumably provides (R)-deacetylipecoside for the formation of tetrahydroisoquinoline monoterpene glucosides that also possess an (R)-configuration at the same chiral center.     

Purification and characterization of an NADP+-linked alcohol oxido-reductase which catalyzes the interconversion of gamma-hydroxybutyrate and succinic semialdehyde.

Abstract— An NADP⁺ -linked enzyme, capable of interconverting γ-hydroxybutyrate and succinic semialdehyde, has been isolated from hamster liver and brain. The enzyme which was isolated from liver has been purified 300-fold and exhibits a single band by polyacrylamide gel electrophoresis. The molecular weight of the enzyme is - 31,000 as estimated from gel filtration and 38,000 as estimated from sodium dodccyl sulfate gel electrophoresis. The enzyme is inhibited by amobarbital, diphenylhy-dantoin, 2-propylvalerate, and diethyldithiocarbamate, but not by pyrazole. The enzymes from brain and liver appear to be very similar with regard to their molecular weights and their kinetic constants for γ-hydroxybutyrate and succinic semialdehyde.     

The kinetics of phenethylhydrazine oxidation by monoamine oxidase

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.     

A major phosphotyrosyl-protein phosphatase from bovine heart is associated with a low-molecular-weight acid phosphatase

The phosphotyrosyl [Tyr(P)]-immunoglobulin G (IgG) phosphatase activity in the extracts of bovine heart, bovine brain, human kidney, and rabbit liver can be separated by DEAE-cellulose at neutral pH into two fractions. The unbound fraction exhibits a higher activity at acidic than neutral pH while the reverse is true for the bound fraction. Of all tissues examined, the Tyr(P)-IgG phosphatase activity in the unbound fraction measured at pH 5.0 is higher than that in the bound fraction measured at pH 7.2. The acid Tyr(P)-IgG phosphatase activity has been extensively purified from bovine heart. It copurified with an acid phosphatase activity (p-nitrophenyl phosphate (PNPP) as a substrate) throughout the purification procedure. These two activities coelute from various ion-exchange and gel filtration chromatographies and comigrate on polyacrylamide gel electrophoresis, indicating that they reside on the same protein molecule. The phosphatase has a Mr = 15,000 by gel filtration and exhibits an optimum between pH 5.0 and 6.0 when either Tyr(P)-IgG-casein or PNPP is the substrate. It is highly specific for Tyr(P)-protein with little activities toward phosphoseryl [Ser(P)]- or phosphothreonyl [Thr(P)]-protein. The enzyme activities toward Tyr(P)-casein and PNPP are strongly inhibited by microM molybdate and vanadate but insensitive to inhibition by L(+)-tartrate, NaF, or Zn2+. The molecular and catalytic properties of the acid Tyr(P)-protein phosphatase purified from bovine heart are very similar to those of the low-molecular-weight acid phosphatases of Mr = 14,000 previously identified and purified from the cytosolic fraction of human liver, placenta, and other animal tissues.     

Effects of pH changes and charge characteristics in the uptake of norepinephrine by synaptosomes of rat brain.

The pH dependence of the initial uptake of norepinephrine by rat whole brain synaptosomes was studied using short incubation times at 37 degrees C in order to examine the possible involvement of the phenolic OH group. The pH vs. uptake profile exhibits a maximum near pH 8.2 in H2O medium. When the medium was changed to 2H2O, the profile showed a shift of maximum corresponding to the pKa change of the phenolic OH group. The pH vs. uptake profile of tyramine was quite different from that of norepinephrine. These pH effects on uptake were explained as manifestations of the involvement of the phenolic OH group in the process. The amine and phenolic hydroxyl groups in norepinephrine were studied separately by employing two series of compounds structurally related to catecholamines, amphetamine-like and catechol-like, for their inhibitory effects on the uptake. The inhibitions were affected by changes in pH with changes in opposite directions found for the two series indicating the need for a positive charge in the side chain and suggesting an effect of the negative charge on the ring. These charge characteristics agreed with the pH profile observed in uptake. Consequently, the two groups with opposite charge characteristics in norepinephrine both appear to function in the uptake process.     

Oxidation of putrescine, spermidine and spermine by diamino-oxidase from mouse liver]

Optimal conditions to determine the activity of diaminooxidase in mouse liver homogenate are described. Maximal oxidation rate for putrescine was found to take place at a concentration of 20 mM and pH 9.5, and for spermidine and spermine--at 10 mM concentration and pH 9.2. The rate of tyramine oxidation was maximal at pH 7.8. Apparent KM values were 4.98-10(-3 M, 1-10(-3) M and 0.8-10(-3) M for putrescine, spermidine and spermine respectively. Hydroxylamine did not inhibit the rate of putrescin oxidation at optimal pH value.     

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.     

Purification and Properties of Bovine Brain Dopamine β-Hydroxylase

Abstract: Dopamine β-hydroxylase (DBH) was purified from bovine brain by a series of steps including extraction with 0.5% Triton X-100, ammonium sulfate fractionation, and serial chromatographies with Concanavalin A (Con A)-Sepharose, Biogel A-1.5 m, DEAE-Sephadex, and phenyl-Sepharose. The overall purification was approximately 4200-fold and the final specific activity was 147 nmol/min/mg protein. Bovine brain DBH was apparently a glycoprotein and interacted with immobilized Con A. Furthermore, the enLyme bound to phenyl-substituted agarose by hydrophobic interaction. An approximate molecular weight was estimated to be 400,000 by gel filtration; the protein eluted earlier than bovine adrenal DBH with a molecular weight estimated to be 290,000. The K_m values toward tyramine and ascorbate were 1.53 and 1.42 mM, respectively, the optimal pH was 5.0 in the presence of 20 mM tyramine as substrate. Immunological titration studies indicated that bovine brain and adrenal DBH had common antigenic sites. Our data showed a close similarity between the bovine brain and adrenal enzymes.     

THE BIOSYNTHESIS OF OCTOPAMINE-CHARACTERIZATION OF LOBSTER TYRAMINE β-HYDROXYLASE

—Tyramine β-hydroxylase catalyzes the biosynthesis of octopamine in the lobster nervous system. This enzyme has been characterized and a rapid microassay, based on the enzymic release of tritiated water from [1,2-(side chain) ³H] tyramine, has been developed. Lobster tyramine β-hydroxylase resembled mammalian dopamine β-hydroxylase. The most conspicuous differences were that the lobster enzyme was inhibited by anions, particularly fumarate, and had a higher affinity for substrates. Tyramine β-hydroxylase activity was present in both particulate and soluble fractions of homogenates of the lobster nervous system. Bound activity, extracted by repeated freezing and thawing, was partially purified. The enzyme had the following properties: (1) The optimum pH for the conversion of tyramine to octopamine was 7·4. (2) The apparent Michaelis constant for tyramine was 0·15 mm and for ascorbic acid was 0·2 mm at pH 6·6. (3) The purified enzyme was inhibited by salts; the degree of inhibition was sensitive to the anion and decreased in the order chloride ? fumarate > sulphate > acetate. (4) Tyramine β-hydroxylase was inhibited by metal chelating agents and by cupric sulphate at concentrations greater than 10^?4m ; N-ethylmaleimide had no significant effect on activity in concentrations up to 3 mm . (5) The purified enzyme also β-hydroxylated dopamine to form norepinephrine, with an apparent Michaelis constant of 0·24 mm . This activity co-purified with tyramine β-hydroxylase, suggesting that a single enzyme catalyzed both reactions.     

Monoamine Oxidase Activities in Catfish (Parasilurus Asotus) Tissues

Abstract

The substrate- and inhibitor-related characteristics of monoamine oxidase (MAO) were studied for catfish brain and liver. The kinetic constants for MAO in both tissues were determined using 5-hydroxytryptamine (5-HT), tyramine and β-phenylethylamine (PEA) as substrates. For both tissues, the V_max values were highest with 5-HT and lowest with PEA. The K_m value for the brain was highest with 5-HT, followed by tyramine and PEA; but for the liver its value was highest with PEA, followed by 5-HT and tyramine, although all values were in the same order of magnitude. The inhibition of MAO by clorgyline and deprenyl by use of 5-HT, tyramine and PEA as substrates showed that the MAO-A inhibitor clorgyline was more effective than the MAO-B inhibitor deprenyl for both catfish tissues; a single form was present since inhibition by clorgyline or deprenyl with 1000 μM PEA showed single phase sigmoid curves. It is concluded that catfish brain and liver contain a single form of MAO, relatively similar to mammalian MAO-A.