Mitsuhiro Nozaki

D-amino-acid oxidase is confined to the lower brain stem and cerebellum in rat brain: regional differentiation of astrocytes.

Based on enzymatic activity, the localization and the identification of D-amino-acid oxidase-containing cells in rat whole brain was systematically studied in serial fixed sections. The oxidase activity was absent or scarce in the forebrain, was confined to the brain stem (midbrain, pons and medulla oblongata) and cerebellum, and its localization was extended to the spinal cord. In the brain stem the oxidase was mainly localized in the tegmentum, particularly in the reticular formation. The intense oxidase reactions were present in the red nucleus, oculomotor nucleus, trochlear nucleus, ventral nucleus of the lateral lemniscus, dorsal and ventral cochlear nuclei, vestibular nuclei, nuclei of posterior funiculus, nucleus of the spinal tract of the trigeminal nerve, lateral reticular nucleus, inferior olivary nucleus, and hypoglossal nucleus. In the cerebellum the activity in the cortex was much more intense than that in the medulla. In all the fields described above, the oxidase-containing cells were exclusively astrocytes including Bergmann glial cells, and neither neuronal components, endothelial cells, oligodendrocytes nor ependymal cells showed oxidase activity. These results indicated that the astrocytes regionally differentiated into two distinct types, one of which expressed oxidase in the midbrain, rhombencephalon and spinal cord, and the other which did not in the forebrain. The localization of the oxidase was inversely correlated with the distribution of free D-serine in mammalian brains (Nagata, Y., Horiike, K. and Maeda, T., Brain Res., 634 (1994) 291-295). Based on the characteristic localization of the oxidase-containing astrocytes, we discussed the physiological role of the oxidase.

An archetypical extradiol-cleaving catecholic dioxygenase: the crystal structure of catechol 2,3-dioxygenase (metapyrocatechase) from Pseudomonas putida mt-2

BACKGROUND Catechol dioxygenases catalyze the ring cleavage of catechol and its derivatives in either an intradiol or extradiol manner. These enzymes have a key role in the degradation of aromatic molecules in the environment by soil bacteria. Catechol 2, 3-dioxygenase catalyzes the incorporation of dioxygen into catechol and the extradiol ring cleavage to form 2-hydroxymuconate semialdehyde. Catechol 2,3-dioxygenase (metapyrocatechase, MPC) from Pseudomonas putida mt-2 was the first extradiol dioxygenase to be obtained in a pure form and has been studied extensively. The lack of an MPC structure has hampered the understanding of the general mechanism of extradiol dioxygenases. RESULTS The three-dimensional structure of MPC has been determined at 2.8 A resolution by the multiple isomorphous replacement method. The enzyme is a homotetramer with each subunit folded into two similar domains. The structure of the MPC subunit resembles that of 2,3-dihydroxybiphenyl 1,2-dioxygenase, although there is low amino acid sequence identity between these enzymes. The active-site structure reveals a distorted tetrahedral Fe(II) site with three endogenous ligands (His153, His214 and Glu265), and an additional molecule that is most probably acetone. CONCLUSIONS The present structure of MPC, combined with those of two 2,3-dihydroxybiphenyl 1,2-dioxygenases, reveals a conserved core region of the active site comprising three Fe(II) ligands (His153, His214 and Glu265), one tyrosine (Tyr255) and two histidine (His199 and His246) residues. The results suggest that extradiol dioxygenases employ a common mechanism to recognize the catechol ring moiety of various substrates and to activate dioxygen. One of the conserved histidine residues (His199) seems to have important roles in the catalytic cycle.

Localization of D-amino acid oxidase in Bergmann glial cells and astrocytes of rat cerebellum.

The localization of D-amino acid oxidase in rat cerebellum was systematically studied in serial fixed sections at the levels of both light and electron microscopy using a coupled peroxidation method based on the intensifying effect of nickel ions. Deposits were only seen in astrocytes and Bergmann glial cells, and not in neuronal components, endothelial cells or ependymal cells. In the molecular layer, heavy deposits were present in the profiles of Bergmann glial processes around the complexes of synapses where the parallel fiber varicosities form synapses with the thorns emerging from the spiny branchlets of Purkinje cell dendrites. In the Purkinje cell layer, the oxidase-containing processes of Bergmann glial cells enveloped basket cell axons, their terminals, the terminals of the recurrent collaterals of Purkinje cell axons and Purkinje cell bodies. In the granular layer, the cerebellar glomeruli were enveloped by the heavily stained processes of astrocytes. Based on this characteristic localization of the oxidase, we discussed the physiological role of the oxidase in connection with the function of glial cells.

Purification and properties of catechol 1,2-dioxygenase (pyrocatechase) from Pseudomonas putida mt-2 in comparison with that from Pseudomonas arvilla C-1☆

Catechol 1,2-dioxygenase (pyrocatechase) has been purified to homogeneity from Pseudomonas putida mt-2. Most properties of this enzyme, such as the absorption spectrum, iron content, pH stability, pH optimum, substrate specificity, Km values, and amino acid composition, were similar to those of catechol 1,2-dioxygenase obtained from Pseudomonas arvilla C-1 [Y. Kojima et al. (1967) J. Biol. Chem. 242, 3270-3278]. These two catechol 1,2-dioxygenases were also found, from the results of Ouchterlony double diffusion, to share several antigenic determinants. The molecular weight of the putida enzyme was estimated to be 66,000 and 64,000 by sedimentation equilibrium analysis and Sephadex G-200 gel filtration, respectively. The enzyme gave a single band on sodium dodecyl sulfate-polyacrylamide gel electrophoresis, corresponding to Mr 32,000. The NH2-terminal sequence, which started with threonine, was determined up to 30 residues by Edman degradation. During the degradation, a single amino acid was released at each step. The NH2-terminal sequence up to 20 residues was identical to that of the beta subunit of the arvilla enzyme, with one exception at step 16, at which arginine was observed instead of glutamine. The COOH-terminal residue was deduced to be arginine on carboxypeptidase A and B digestions and on hydrazinolysis. These results indicate that the putida enzyme consists of two identical subunits, in contrast to the arvilla enzyme which consists of two nonidentical subunits, alpha and beta [C. Nakai et al. (1979) Arch. Biochem. Biophys. 195, 12-22], although these two enzymes have very similar properties.

Crystallization and properties of aspartate aminotransferase from escherichia coli B

Asparate aminotransferase (EC 2.6.1 .l) has been extensively studied as a representative of the pyridoxal 5’-phosphate (pyridoxal-P&dependent enzymes. The enzyme catalyzes the reversible transfer of the amino group between L-aspartic acid and cY-ketoglutaric acid, and plays an important role in nitrogen metabolism. Animal and plant tissues contain both mitochondrial and cytosolic isoenzymes [l] . The primary structures of both isoenzymes from pig heart muscle have been elucidated [2,3] . Preliminary crystallographic data have been reported for the large and single crystals of cytosolic [4,5] and mitochondrial isoenzymes [6]. Mammalian aspartate aminotransferases are immunochemically distinct from the bacterial enzymes [7] . Structural studies of the bacterial enzymes have not been done. The enzyme has been purified to homogeneity from Pseudomonas striata and crystallized [8] . However, its low content in cells and low yield in purification prompted a search for other bacterial sources to produce it more abundantly. The aspartate aminotransferases have been highly purified from Escherichia coli K-12 [9], its mutant [lo] and Crooks strain [ 1 l] , but none of them have been obtained in a crystalline form. We describe here high yield purification, crystallization and some properties of aspartate aminotransferase from Escherichia cob B as a first approach to the comparative studies on the enzyme.

[17] Glutamate-aspartate transaminase from microorganisms

Publisher Summary Various methods have been developed for the assay of aspartate aminotransferase. Most of these depend on the determination of oxalacetate or L-glutamate. Out of these, the Karmen method is used most widely, particularly in the field of clinical analysis, in which oxalacetate is determined with malate dehydrogenase and NADH by following a decrease in absorbance at 340 nm. A new method introduced recently, determines 2-oxoglutarate produced in the reverse reaction is with 2-hydroxyglutarate dehydrogenase. A method for the determination of aspartate aminotransferase activity after electrophoretic separation of the isoenzymes has also been developed. The purification procedure consists of several steps: preparation of cell-free extract, ammonium sulfate fractionation, first Diethylaminoethyl (DEAE)-cellulose column chromatography, heat treatment, Sephacryl S-200 chromatography, second DEAE-cellulose chromatography, and hydroxyapatite chromatography.

Crystallization and properties of aromatic amine dehydrogenase from Pseudomonas sp

An amine dehydrogenase was purified to homogeneity from an extract of a bacterium of the genus Pseudomonas grown in a medium containing beta-phenylethylamine as a sole carbon source and obtained in a crystalline form with about 100-fold purification. The purified enzyme catalyzed the oxidative deamination of various aromatic amines as well as some aliphatic amines to a lesser extent. An artificial electron acceptor such as phenazine methosulfate was required for the catalysis. The molecular weight determined by sedimentation equilibrium was 103,000 and the molecule seemed to be composed of two pairs of two nonidentical subunits (Mr 46,000 and 8000). The enzyme had a dull yellow-green color with an absorption maximum at 445 nm and this chromophore appeared to be involved in the catalytic action of the enzyme.

Cysteine sulfinate transamination activity of aspartate aminotransferases.

Abstract Aspartate aminotransferases from pig heart cytosol and mitochondria, Escherichia coli B and Pseudomonas striata accepted L-cysteine sulfinate as a good substrate. The mitochondrial isoenzyme and the Escherichia enzyme showed higher activity toward L-cysteine sulfinate than toward the natural substrates, L-glutamate and L-aspartate. The cytosolic isoenzyme catalyzed the L-cysteine sulfinate transamination at 50% the rate of L-glutamate transamination. The Pseudomonas enzyme had the same reactivity toward the three substrates. Antisera against the two isoenzymes and the Escherichia enzyme inactivated almost completely cysteine sulfinate transamination activity in the crude extracts of pig heart muscle and Escherichia coli B, respectively. These results indicate that cysteine sulfinate transamination is catalyzed by aspartate aminotransferase in these cells.

A sensitive method for the detection of aspartate: 2-oxoglutarate aminotransferase activity on polyacrylamide gels

Abstract A sensitive technique for the qualitative and semiquantitative determination of the activity of aspartate aminotransferase on polyacrylamide gels after electrophoresis is described. It relies on the ability of aspartate aminotransferase to produce SO3−− in the transamination between l -cysteine sulfinate and 2-oxoglutarate. The method is based on the reduction of nitroblue tetrazolium by SO3−− using phenazine methosulfate as a coupling agent. The method has been characterized using human and pig sera, crude homogenates and crystalline preparation from pig heart muscle, and bacterial crude extracts.

Diurnal variation of dopamine content in the rat pineal gland.

Abstract Levels of norepinephrine and dopamine in the rat pineal gland were determined by a radioenzymatic assay with modifications to separate the reaction products. Catecholamines were converted to 3-O-methylated derivatives in the presence of catechol-O-methyltransferase (EC 2.1.1.1) and S-adenosyl-L-[methyl- 3 H]-methionine. Following solvent extraction of the labelled normetanephrine and 3-methoxytyramine, the amines were separated by high-performance liquid chromatography. Contents of both catecholamines in the pineal gland varied with a 24-hr rhythm. The content of norepinephrine was maximal at about 6 A.M. (lights on from 8 A.M. to 8 P.M.) and declined gradually thereafter. In contrast to the level of norepinephrine, the dopamine level was highest at about 0 A.M. and fell rapidly to reach a trough after the lights were turned on. These observations suggest that the diurnal variation of norepinephrine is generated by changes in the contents of dopamine in sympathetic nerve terminals innervating the pineal.