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.     

RELATIONSHIP BETWEEN THE RATE OF AXOPLASMIC TRANSPORT AND SUBCELLULAR DISTRIBUTION OF ENZYMES INVOLVED IN THE SYNTHESIS OF NOREPINEPHRINE

The net rate of proximo-distal transport of tyrosine hydroxylase, dopamine β-hydroxylase, DOPA decarboxylase and choline acetyltransferase was determined by measuring the accumulation of these enzymes proximal to a ligature of the rat sciatic nerve. The rate of accumulation was constant for at least 12 h. For the enzymes involved in the biosynthesis of norepinephrine the rate of transport was correlated to their subcellular distribution and a close correlation between these two parameters was found. Dopamine β-hydroxylase, an enzyme mainly localized in the particulate fraction of the sciatic nerve, showed the fastest rate of transport (1·94 mm/h) whereas DOPA decarboxylase, exclusively located in the high-speed supernatant fluid, gave the slowest (0·63 mm/h) rate of transport. Tyrosine hydroxylase, predominantly located in the non-particulate fraction of the sciatic nerve was transported much slower (0·75 mm/h) than dopamine β-hydroxylase but still significantly (P < 0.005) faster than DOPA decarboxylase. The subcellular distribution of dopamine β-hydroxylase in ganglia did not differ significantly (0·45 > P > 0·40) from that in the sciatic nerve, but in nerve endings a greater proportion of dopamine β-hydroxylase was localized in particulate fractions. Tyrosine hydroxylase and DOPA decarboxylase were found exclusively in the non-particulate fractions of ganglia. In the nerve endings of the effector organs a small but consistent portion of tyrosine hydroxylase was found in particulate fractions, whereas DOPA decarboxylase was exclusively localized in the high-speed supernatant fluid.     

Spectroscopic Analysis of the Weak Light Generated in Autoxidation of Linseed Oil

Nucleoside oxidase purified from Pseudomonas maltophilia LB-86 had mol. wt. = 130.000 and was composed of one each of four non-identical subunits: subunit α, 76,000; subunit β, 33,000; subunit γ, 18,000; subunit δ, 14,000. The enzyme contains 1 mol of covalently bound FAD, 2g atoms of nonheme iron, 2 mol of labile sulfides, and 1 mol of heme per mol enzyme protein. The absorption spectrum of nucleoside oxidase had maxima 278 and 390 nm, and shoulders at 343 and 450 nm.

The enzyme catalyzes the oxidation of various nucleosides, and the Km value for inosine was 4.4 × 10^-5 M. The enzyme was most active at pH 5 ~ 6, and was most stable between pH 5.0 ~ 6.0 and at temperatures below 60°C. The activity was strongly inhibited by N-bromosuccinimide and potassium cyanide.     

Hydrolysis of 3′,4′-dichloropropionanilide by an aryl acylamidase from Taraxacum officinale

An aryl acylamidase (aryl-acylamine amidohydrolase, E.C. 3.5. 1.a) which hydrolyses the herbicide propanil (3′,4′-dichloropropionanilide), was isolated from dandelion roots and partially purified and characterized. Specificity tests on the enzyme revealed that it could hydrolyse various chlorine ring-substituted propionanilides and 3,4-dichloroanilide alkyl compounds. The partially purified enzyme was inhibited by several sulfhydryl reagents and metal ions. The pH optimum was broad, between 7·4 and 7·8. The apparent activation energy, determined from an Arrhenius plot, was 9·0 kcal/mol (37 700 J/mol) for the hydrolysis of 3′,4′-dichloropropionanilide. The apparent K_m was 1·7 × 10⁻⁴ M with propanil as substrate.     

Properties of a Mg2+ independent isocitrate lyase from gamma irradiated preclimacteric banana (Musa cavendishii)

Isocitrate lyase (Ee 4·1,3·1) was purified seventy fold from gamma irradiated banana pulp tissue acetone powder. It showed an optimum pH of 6·0, and the Km value for DL-isocitrate was 0·8 mM. Among the various metabolic inhibitors, oxaloacetate was found to be the most potent and its inhibition was competitive. The enzyme activity was not dependent on externally added Mg²⁺. The Mg²⁺ content of the purified enzyme was 10–12 ng/rng protein. A method for the detection of the two multiple forms of isocitrate lyase present in this preparation was developed using 2,4-dinitrophenylhydrazine as detecting agent for glyoxylate formed during the isocitrate lyase reaction.     

Further studies on double-stranded RNA species from raspberry plants infected with aphid-borne viruses

Analysis by electrophoresis in polyacrylamide gels, followed by silver staining, of dsRNA extracted from many samples of raspberry leaves infected with raspberry leaf mottle virus (RLMV) and/or raspberry leaf spot virus (RLSV) failed to detect reliably any significant quantities of dsRNA species in excess of 1·0 × 10⁶mol. wt. This contrasts with results reported from Canada where three dsRNA species of estimated mol. wt 2·6 × 10⁶1·6 × 10⁶and 1·1 × 10⁶were consistently associated with infection with RLSV but none were associated with RLMV. However, in Scotland, four dsRNA species of estimated mol. wt 2·4 × 10⁶1·6 × 10⁶0·7 × 10⁶and 0·3 × 10⁶were detected in raspberry infected with apple mosaic ilarvirus. These results suggest that the dsRNA species reported from Canada are not those of RLSV but are probably those of a second virus, possibly an ilarvirus, which occurs together with RLSV and/or induces similar symptoms. A few samples from plants infected with RLMV and RLSV contained very small amounts of two dsRNA species of estimated mol. wt 4·7 × 10⁶and 4·5 × 10⁶. It is not known whether these species are those of RLMV and RLSV.     

Micrococcus lysodeikticus ATPase: Purification by preparative gel electrophoresis and subunit structure studied by urea and sodium dodecylsulfate gel electrophoresis

Micrococcus lysodeikticus ATPase was purified by preparative gel electrophoresis after its “shock wash” release from the membrane. The method afforded the highest yield of pure protein in the minimum time as compared with former purification procedures. The pure protein had a specific activity of 7 μmol P_i·min^?1·mg^?1 with incubation times not longer than 3 min, 345 000 mol. wt and was not stimulated by trypsin. By gel electrophoresis at alkaline pH (8.5) in 8 M urea or in sodium dodecylsulfate, the ATPase revealed a complex pattern with two major subunits (α and β) and two minor ones (γ and δ). The non-identity between the major subunits was demonstrated.     

Purification and properties of benzoate-4-hydroxylase from a soil pseudomonad.

Benzoate-4-hydroxylase from a soil pseudomonad was isolated and purified about 50-fold. Polyacrylamide gel electrophoresis of this enzyme preparation showed one major band and one minor band. The approximate molecular weight of the enzyme was found to be 120,000. Benzoate-4-hydroxylase was most active around pH 7.2. The enzyme showed requirements for tetrahydropteridine as the cofactor and molecular oxygen as the electron acceptor. NADPH, NADH, dithiothreitol, β-mercaptoethanol, and ascorbic acid when added alone to the reaction mixture did not support the hydroxylation reaction to any significant extent. However, when these compounds were added together with tetrahydropteridine, they stimulated the hydroxylation. This stimulation is probably due to the reduction of the oxidized pteridine back to the reduced form. This enzyme was activated by Fe²⁺ and benzoate. It was observed that benzoate-4-hydroxylase could catalyze the oxidation of NADPH in the presence of benzoate,p-aminobenzoate, p-nitrobenzoate, p-chlorobenzoate, and p-methylbenzoate, with only benzoate showing maximum hydroxylation. Inhibition studies with substrate analogs and their kinetic analysis revealed that the carboxyl group is involved in binding the substrate to the enzyme at the active center. The enzyme catalyzed the conversion of 1 mol of benzoate to 1 mol of p-hydroxybenzoate with the consumption of slightly more than 1 mol of NADPH and oxygen.     

Purification and Characterization of S-adenosylmethionine Synthetase from Wheat Embryos: Subunit Structure and Molecular Properties

S-adenosylmethionine synthetase from wheat embryos was purified to electrophoretic homogeneity. The mol wt of the enzyme was 174,000 as determined by molecular sieve chromatography on Sephacryl S-200. A single subunit of purified AdoMet synthetase was observed on SOS-PAGE with a mol wt of 84,000 suggesting that the enzyme is a homodimer. The apparent Km of purified enzyme with ATP and methionine is 80 μM and 100 μM, respectively. The pH optimum of the enzyme is 7.75. The enzyme requires MgCb, KCI and reduced glutathione for optimum activity. The ³H-labelled putative S-adenosylmethionine reaction product was converted into ³H-labelled 5′-methyl-thioadenosine by heat treatment (100°C, 10 min, pH 7.0). This proved the authenticity of the reaction product of the AdoMet synthetase in wheat embryos.     

Electron transport systems of Candida utilis. Purification and properties of the respiratory chain-linked external NADH dehydrogenase+

The respiratory chain-linked external NADH dehydrogenase has been isolated from Candida utilis in highly purified form. The enzyme is soluble and has a molecular weight of approx. 1.5 · 10⁶. The enzyme contains two moles of FMN per mole of enzyme and is composed of two large subunits of mol. wt. 270 000 and eight smaller subunits of mol. wt. 135 000. Iron and copper are present in the preparations, but appear to be contaminants. The enzyme catalyzes the oxidation of NADH and NADPH at nearly equal rates and reacts readily with 2,6-dichlorophenolindophenol, CoQ₆ and CoQ₁ derivatives as acceptors. Rotenone (10^?5 M) and seconal (10^?3 M) do not inhibit enzymatic activity.     

Further properties of black raspberry latent virus, and evidence for its relationship to tobacco streak virus

Purified preparations of an isolate of black raspberry latent virus (BRLV) contained quasispherical particles with a mean diameter of 28·5 nm; these particles were resolved into three sedimenting components (s_{20, w}= 82S, 95S and 104S), but when centrifuged to equilibrium in caesium chloride solution they formed a single infective band (σ= 1·35 g/cm³). During electrophoresis in polyacrylamide gels, virus particles separated into three classes, and virus RNA was resolved into three major (mol. wt 1·35, 1·10 and 0·85 × 10⁶) and one minor (mol. wt 0·4 × 10⁶) component. The protein from virus particles had an estimated mol. wt of 28000. Isolates of BRLV were found to be serologically related but not identical to some strains of tobacco streak virus. No symptoms developed in black raspberry seedlings infected with BRLV by mechanical inoculation, nor in eight red raspberry cultivars infected by graft inoculation. However, graft inoculation of BRLV to Rubus henryi, R. phoenicolasius and Himalaya blackberry induced symptoms typical of necrotic shock disease.     

棉铃虫N-乙酰-β-D-氨基葡萄糖苷酶的分离纯化及酶学性质

以棉铃虫Helicoverpa armigera蛹为材料,通过硫酸铵沉淀分级分离、Sephadex G-200分子筛柱层析和DEAE-32离子交换柱层析纯化,获得聚丙烯酰胺凝胶电泳纯的N-乙酰- β-D-氨基葡萄糖苷酶酶制剂。纯酶的比活力为2 678.79 U/mg。以对硝基苯-N-乙酰-β-D-氨基葡萄糖苷(pNP-β-D-GlcNAc)为底物,研究酶催化底物水解的反应动力学。结果表明：酶的最适pH为5.63,最适温度为55℃。该酶在pH 4～8区域较稳定,而在pH>8时能迅速失去活力;在50℃以下处理30 min,酶活力仍保持稳定,高于50℃,酶很快失去活力。酶促反应动力学符合米氏双曲线方程,测得米氏常数Km为0.16 mmol/L,最大反应速度Vm为10.73 μmol·L^-1·min^-1。酶催化pNP-β-D-GlcNAc反应的活化能为66.24 kJ/mol。     

Characterization of an ethanol‐tolerant 1,4‐β‐xylosidase produced by Pichia membranifaciens

Aims: The purification and biochemical properties of the 1,4‐β‐xylosidase of an oenological yeast were investigated. Methods and Results: An ethanol‐tolerant 1,4‐β‐xylosidase was purified from cultures of a strain of Pichia membranifaciens grown on xylan at 28°C. The enzyme was purified by sequential chromatography on DEAE cellulose and Sephadex G‐100. The relative molecular mass of the enzyme was determined to be 50 kDa by SDS‐PAGE. The activity of 1,4‐β‐xylosidase was optimum at pH 6·0 and at 35°C. The activity had a K_m of 0·48 ± 0·06 mmol l^?1 and a V_max of 7·4 ± 0·1 μmol min^?1 mg^?1 protein for p‐nitrophenyl‐β‐d ‐xylopyranoside. Conclusions: The enzyme characteristics (pH and thermal stability, low inhibition rate by glucose and ethanol tolerance) make this enzyme a good candidate to be used in enzymatic production of xylose and improvement of hemicellulose saccharification for production of bioethanol. Significance and Impact of the Study: This study may be useful for assessing the ability of the 1,4‐β‐xylosidase from P. membranifaciens to be used in the bioethanol production process.     

Affinity interaction of hydroxypyruvate reductase from Methylophilus spp. with Cibacron blue F3GA-derived poly(HEMA EGDMA) microspheres: partial purification and characterization

A methylotrophic hydroxypyruvate reductase was partially purified and characterized from Methylophilus spp. using the biomimetic dye, Cibacron Blue F3FA attached to poly(HEMA-EGDMA) microspheres. The absorption capacities of the dye-affinity microspheres were determined by changing pH and the concentration of the proteins in the adsorption medium. Hydroxypyruvate reductase was desorbed from the dye-affinity support specifically with 2 mM NADH solution. The enzyme was purified 10·4-fold with 47% yield. The molecular mass and subunit molecular mass of the enzyme was estimated to be 75 kDa and 37 kDa on the basis of its mobility in polyacrylamide and SDS-polyacrylamide gels, respectively. This suggested a homogeneous dimer structure. The optimal pH was between 5·0 and 7·0, and the maximum enzyme activity was obtained at 50°C. The K_m values of hydroxpyruvate reductase were 0·222 mM for hydroxpyruvate and 0·067 mM for NADH.     

Purification and characterisation of 1F-fructosyltransferase from the roots of asparagus (asparagus officinalis L.)

A fructosyltransferase that transfers a terminal d-fructosyl group from a (2→1)-β-linked fructosaccharide to HO-1 of another d-fructosyl group has been purified from an extract of asparagus roots by successive chromatography with DEAE-cellulose, octyl-Sepharose, Sephadex G-200, and raffinose-coupled Sepharose 6B. The disc-electrophoretically homogeneous enzyme was free from β-d-fructofuranosidase, sucrose:sucrose 1-fructosyltransferase, and 6^G-frutosyltransferase activity, and catalysed the d-fructosyl transfer from 1-kestose more rapidly to saccharides of the neokestose series [1^F(1-β-d-fructofuranosyl)_m-6^G(1-β-d-fructofuranosyl)_nsucrose] than to those of the 1-kestose series [1^F(1-β-d-fructofuranosyl)_nsucrose]. The enzyme was tentatively termed 1^F-fructosyltransferase. The general properties of the enzyme were as follows: mol. wt., ~64,000; optimum pH, ~5.0; stable at pH 5.0–5.5 at 45° for 20 min; stable at 30–45° for 10 min; inhibited by Hg²⁺, p-chloromercuribenzoate, and Ag⁺.     

Evidence for conversion of N-Tyr-MIF-1 into MIF-1 by a specific brain aminopeptidase

N-Tyr-MIF-1 (Tyr-Pro-Leu-Gly·NH₂), an immunoreactive neuropeptide exhibiting saturable high affinity binding in rat brain was found to be converted into MIF-1 (Pro-Leu-Gly·NH₂) by a specific brain aminopeptidase present in rat brain homogenates or cytosol, but with low activity associated with synaptosomal plasma membranes and microsomes. Conversion occurred at a rate of 16 μmol per g w/wt per h and was unaffected by puromycin but inhibited by bestatin (I₅₀, 5 × 10^?5 M). Aminopeptidases purified from cytosolic fractions of rat brain (arylamidase), mouse brain (Mn²⁺-activated aminopeptidase) or porcine kidney (leucine aminopeptidase) were inactive towards N-Tyr-MIF-1 but degraded MIF-1 with release of Leu-Gly·NH₂ as detected by RP-HPLC procedures. Morphiceptin (Tyr-Pro-Phe-Pro·NH₂), a μ opioid agonist, also acted as a substrate for the N-Tyr-MIF-1 converting enzyme with cleavage of the Tyr-Pro bond. These tetrapeptides, but not MIF-1 or its N-blocked analogs, were degraded in vitro by a metalloendopeptidase purified from kidney membranes. Since dipeptide products were not detected for crude extracts, a significant role for brain metalloendopeptidase on turnover can be excluded. Thus the results point to the presence of a specific (X-Pro-degrading) aminopeptidase in brain cytosol as an enzyme responsible for converting N-Tyr-MIF-1 and inactivating morphiceptin.     

Characterization of endo-β-mannanase from <Emphasis Type="Italic">Enterobacter ludwigii</Emphasis> MY271 and application in pulp industry

β-Mannanases are the second most important enzymes for the hydrolysis of hemicelluloses. An endo-β-mannanase from Enterobacter ludwigii MY271 was purified at 11.7 ± 0.2-fold to homogeneity with a final recovery of 15.2 ± 0.2 %. Using purified β-mannanase protein and SDS-PAGE, the molecular mass was found to be 43.16 kDa. The optimal pH and temperature of the enzyme was found to be 7.0 and 55 °C, respectively. The β-mannanase activity was stable over a broad pH range of pH 2.0–10.0. In addition, the purified enzyme was highly activated by several metal ions and chemical reagents, such as Mg²⁺, l-cysteine, glutathione (GSH) and β-mercaptoethanol. Whereas the enzyme was strongly inhibited by Hg²⁺, Cu²⁺, N-bromosuccinimide (NBS), 1-ethyl-3-(3-dimethyl-amino-propyl)-carbodiimide (EDC), phenylmethanesulfonyl fluoride (PMSF), and sodium dodecyl sulfate (SDS). The β-mannanase was highly active towards glucomannan, and showed endo-activity by producing a mixture of oligosaccharides. Moreover, the enzyme displayed a classical endo-type mode on mannooligosaccharides. The β-mannanase coupled with xylanase significantly improved the brightness of kraft pulp, whereas it has no remarkable effect on the tensile strength of the pulp. Our functional studies of the purified β-mannanase indicate that the enzyme is beneficial to industrial applications, in particular, biotechnological processes, such as food, feed and pulp industry.     

Synthesis and properties of some O-(2,2-dialkoxyethyl)glycolaldehydes

β-d-Mannosidase (β-d-mannoside mannohydrolase EC 3.2.1.25) was purified 160-fold from crude gut-solution of Helix pomatia by three chromatographic steps and then gave a single protein band (mol. wt. 94,000) on SDS-gel electrophoresis, and three protein bands (of almost identical isoelectric points) on thin-layer iso-electric focusing. Each of these protein bands had enzyme activity. The specific activity of the purified enzyme on p-nitrophenyl β-d-mannopyranoside was 1694 nkat/mg at 40° and it was devoid of α-d-mannosidase, β-d-galactosidase, 2-acet-amido-2-deoxy-d-glucosidase, (1→4)-β-d-mannanase, and (1→4)-β-d-glucanase activities, almost devoid of α-d-galactosidase activity, and contaminated with <0.02% of β-d-glucosidase activity. The purified enzyme had the same K_m for borohydride-reduced β-d-manno-oligosaccharides of d.p. 3–5 (12.5mm). The initial rate of hydrolysis of (1→4)-linked β-d-manno-oligosaccharides of d.p. 2–5 and of reduced β-d-manno-oligosaccharides of d.p. 3–5 was the same, and o-nitrophenyl, methylumbelliferyl, and naphthyl β-d-mannopyranosides were readily hydrolysed. β-d-Mannobiose was hydrolysed at a rate ～25 times that of 6¹-α-d-galactosyl-β-d-mannobiose and 6³-α-d-galactosyl-β-d-mannotetraose, and at ～90 times the rate for β-d-mannobi-itol.     

Biological and biochemical properties of an isolate of cherry rasp leaf virus from red raspberry

A mechanically transmissible virus obtained from symptomless plants of a red raspberry selection imported into Scotland from Quebec, Canada was indistinguishable serologically from a cherry isolate of cherry rasp leaf virus (CRLV). The raspberry isolate, CRLV-R, was graft transmitted to several virus indicator species and cultivars of Rubus without inducing noticeable symptoms. In Chenopodium quinoa sap, CRLV-R lost infectivity after dilution to 10^-5 or heating for 10 min at 60°C but was infective after 16 days (the longest period tested) at 18°, 4° or - 15°C. The virus particles are isometric, c. 28 nm in diameter, and were purified with difficulty from infected C. murale and C. quinoa plants. The particles comprise two nucleoprotein components with sedimentation coefficients of 89 and 115 S and are prone to aggregate during purification. When centrifuged to equilibrium in CS₂SO₄ solution, purified virus preparations formed two major components with p= 1·28 and 1·36 g/cm³. Virus particles contained two RNA species which, when denatured in glyoxal and electrophoresed in agarose gels, had estimated mol. wt of 2·56 × 10⁶ (RNA-1) and 1·26 × 10⁶ (RNA–2). Infectivity of CRLV-R RNA was abolished by treatment with proteinase K, suggesting that the RNA is linked to protein necessary for infectivity; RNA molecules contained polyadenylate. In reticulocyte lysates, CRLV-R RNA stimulated the incorporation of ³H-leucine, mainly into two polypeptides of estimated mol. wt 200 000 and 102 000. When electrophoresed in polyacrylamide gels, protein obtained from CRLV-R particles purified by centrifugation to equilibrium in Cs₂SO₄ separated into three bands with estimated mol. wt 26 000 , 23 000 and 21 000.     

Purification and properties of Bacillus coagulans cyclomaltodextrin glucanotransferase

Cyclomaltodextrin glucanotransferase (CGTase), produced in a culture filtrate by Bacillus coagulans, was purified to a single, homogeneous protein. It has a monomeric structure with a molecular weight of 65,000, isoelectric point of 4.6, and contains 2 mol of Ca²⁺ per mol of the enzyme. The enzyme was most active at pH 6.0 and at 70°C. It did not lose its activity by heat treatment at 70°C for 10 min in the presence of CaCl₂ in the pH range of 5.5∼9.5, and by incubation in the pH range of 5.0∼10.5 at 4°C for one month. The enzyme converted about 60% of potato starch to cyclodextrins for 20 h at 50°C, and the ratio of α-: β-: γ-cyclodextrin produced was 8.1:8.9:1.0 B. coagulans CGTase was compared with B. macerans CGTase which was purified by the same method.