TYROSINE HYDROXYLASE IN BOVINE CAUDATE NUCLEUS

Approximately 80 per cent of tyrosine hydroxylase activity in bovine caudate nucleus was particle-bound. The rest of the activity was found in the soluble fraction. The enzyme activity in crude tissue preparations was inhibited, probably by the presence of endogenous inhibitors. Dilution of crude tissue preparations such as the crude mitochondrial fraction caused an increase in the specific activity. The particle-bound enzyme was solubilized by incubation with trypsin. The presence of deoxycholate increased the degree of solubilization. The activity of the solubilized enzyme from the washed particles was also inhibited, but the subsequent purification by ammonium sulphate could eliminate the inhibition. The solubilized enzyme was partially purified by ammonium sulphate fractionation and Sephadex G-150 chromatography. A tetrahydropteridine and ferrous ion were required as cofactors for the partially purified enzyme. Among various divalent cations, only ferrous ion could activate the partially purified enzyme. The enzyme was inhibited by L-α-methyl-p-tyrosine and catecholamines such as dopamine. The optimum pH was found between 5.5 and 6.0. K_m values toward tyrosine, 2-amino-4-hydroxy-6,7-dimethyltetrahydropteridine and Fe²⁺, were approximately 5 × 10^?5 M, 1 × 10^?4 M and 4 × 10^?4 M, respectively.     

Activation of snail (Helix pomatia) nervous tissue tyrosine monooxygenase by calcium in vitro.

Addition of calcium chloride to soluble preparations of tyrosine monooxygenase from snail brain appears to produce an activation of the enzyme when assayed with subsaturating concentrations of the pteridine cofactor 6 MPH4 (2-amino-4-hydroxy-6-methyltetrahydropteridine). While some increase in the activity occurs with calcium chloride at a concentration of 0.01 mM, activation is increased by about 100% at 1mM and reaches a maximum at 5mM (144%) where it remains more or less constant up to 10mM. Barium chloride also produces an activating effect although it is much less pronounced while magnesium chloride is without effect. EGTA has no direct effect on the enzyme but antagonises the activation produced by calcium chloride. The activation of tyrosine monooxygenase by calcium is reflected in changes in the kinetic properties of the enzyme, decreasing the Km from 43 muM to 19 muM for tyrosine and from 670muM to 230muM for the pteridine cofactor. No change was observed with V values for either tyrosine or pteridine cofactor. It is suggested that calcium, which enters the nerve terminal during nerve stimulation, regulates the transmitter dopamine by activating the rate-limiting enzyme tyrosine monooxygenase.     

Flow-injection chemiluminescent determination of glycerol-3-phosphate and glycerophosphorylcholine using immobilized enzymes

Flow injection procedures with immobilized enzyme mini-columns are described for the determination of glycerol-3-phosphate, and glycerophosphorylcholine with chemiluminescent detection. The hydrogen peroxide produced on-line is coupled with a luminol (5-amino-2,3-dihydro-1,4-phthalazinedione) peroxidation chemiluminescent system. The detection limits for glycerol-3-phosphate and glycerophosphorylcholine are 5×10⁻⁷ M and 1×10⁻⁶ M respectively with r.s.d. <2%. The sample throughput is 40/h. The immobilized enzyme columns did not show any deterioration in activity after usage for 3 months. © 1997 John Wiley & Sons, Ltd.     

INFLUX OF GLUTAMIC ACID IN PERIPHERAL NERVE—CHARACTERISTICS OF INFLUX1

Abstract— —The influx of glutamic acid in frog sciatic nerve has been studied by monitoring the disappearance of ¹⁴C labelled compound from the bathing medium. After 5hr of incubation in 10 ⁻⁶m non-labelled l -glutamic acid and 0·01, μc/ml labelled isotope, the intracellular concentration of labelled glutamic acid is about 15 times the concentration in the bathing medium; however, there appears to be a net loss of non-labelled compound with incubation. Uptake of L,-glutamic acid is accompanied by conversion of significant amounts of labelled E-glutamic acid to carbon dioxide and glutamine; small amounts of γ-aminobutyric acid and aspartic acid are also formed. The rate of disappearance of labelled l -glutamic acid decreases with increasing concentration of non-labelled isotope in the bathing medium. Construction of a Lineweaver-Burk plot from initial velocities of influx yields an average V_m of 4·02 × 10⁻⁹ moles/g/min and an average K_m. of 3·23 × 10 ⁻⁵ moles/l. The influx of glutamic acid is highly specific with regard to molecular structure; of the compounds tested, only l -glutamine, l -glutamic acid, GABA, l -lysine, and l -aspartic acid are taken up, and only l -aspartic acid will compete with l -glutamic acid for uptake.     

Highly sensitive enzyme thermistor determination of ADP and ATP by multiple recycling enzyme systems

An enzyme thermistor method for the determination of ADP and/or ATP with signal amplification by recycling procedures is described. Pyruvate kinase (PK) and hexokinase (HK) coimmobilised on aminopropyl-controlled pore glass were applied in a column reactor. Addition of an excess of phosphoenolpyruvate (PEP) and glucose leads to cofactor recycling and production of glucose-6-phosphate and pyruvate. In presence of PEP an amplification of the sensitivity up to 30 times was reached as compared with the HK-catalysed reaction alone. An additional signal amplification was accomplished by recycling the pyruvate leaving the first enzyme reactor in a second reactor containing L-lactate dehydrogenase, lactate oxidase and catalase. In the presence of NADH an overall amplification of the sensitivity for ATP or ADP up to 1700 times was found. The limits of detection were 6 × 10⁻⁵ M cofactor without recycling at all, 2 × 10⁻⁶M with recycling in the kinase bienzyme reactor and 1 × 10⁻⁸M with the dual recycling system.     

Studies on the Guanosine Degrading System in Bacterial Cell

A highly purified preparation of the enzyme, guanosine deaminase, has been obtained by a four-step purification procedure from the cells of Pseudomonas convexa No. 149. The enzyme deaminates guanosine, deoxyguanosine and 8-azaguanosine, and the deamination of guanosine is inhibited by Hg²⁺ and 4-amino-5-imidazole carboxamide ribonucleoside.

The enzyme has the optimum pH at pH range from 6.0 to 6.5 and the optimum temperature at 45°C. Metal ions are not required for the enzyme activity.

The molecular weight of the enzyme is 1 × 10⁵ to 2 × 10⁵.     

Immobilized glucose dehydrogenase for cofactor regeneration in heme-catalyzed demethylation and hydroxylation reactions

Glucose dehydrogenase (E.C. 1.1.1.47) from B. megaterium M 1286 was immobilized together with mutarotase (E.C. 5.1.3.3) on several organic carriers and by different methods. The storage stability of the enzyme at pH-values > 6 is slightly improved by immobilization and the pH-optimum is shifted from 8.3 to 8.0. Kinetic constants of the immobilized enzyme are: K′_M(NAD⁺) = 5.36 × 10^?4 mol/l K′_M(glucose) = 3.76 · 10^?2 mol/l and V′_max = 5.54 · 10^?5 mol/(l min g carrier) for the most active preparation (2.16 mg enzyme/g carrier). In reactor experiments the immobilized glucose dehydrogenase was used with glucose to regenerate NADPH in NADPH-dependent iron-III-protoporphyrin-IX-imidazole catalyzed hydroxylation and demethylation of model substrates of cytochrome P-450. The advantages of the coupling of both reactions with cofactor recycling are shown and discussed.     

MEMBRANE THIAMINE TRTPHOSPHATASE FROM RAT BRAIN: INHIBITION BY ATP AND ADP

—The hydrolysis of ThTP by rat brain membrane-bound ThTPase is inhibited by nucleoside diphosphates and triphosphates. ATP and ADP are most effective, reducing hydrolysis by 50% at concentrations of 2 × 10^?5m and 7·5 × 10^?5m respectively. Nucleoside monophosphates and free nuclcosides as well as P_i have no effect on enzyme activity. ThMP and ThDP also fail to inhibit hydrolysis in concentrations up to 5 × 10^?3m . Non-hydrolysable methylene phosphate analogs of ATP and ADP were used in further kinetic studies with the ThTPase. The mechanism of inhibition by these analogs is shown to be of mixed non-competitive nature for both compounds. An observed K_i, of 4 × 10^?5m for the ATP analog adenosine-PPCP and 9 × 10^?5m for the ADP analog adenosine-PCP is calculated at pH 6·5. Formation of the true enzyme substrate, the [Mg²⁺. ThTP] complex, is not significantly affected by concentrations of analogs producing maximal (>95%) inhibition of enzyme activity. Likewise the relationships between pH and observed K_m and pH and V_max are not shifted by the presence of similar concentrations of inhibitor.     

THE MARINE MIXOTROPH DINOBRYON BALTICUM (CHRYSOPHYCEAE): PHAGOTROPHY AND SURVIVAL IN A COLD OCEAN1

The marine chrysophyte Dinobryon balticum (Schzütt) Lemm. was one of the dominant members of the phytoplankton community (1.8×10³ cells-L⁻¹) in June and July in Conception Bay, Newfoundland. Dinobryon balticum colonies were common only in samples from June and July. The cells were concentrated at 5 m (X±SD=1.11±4 × 10⁵ cells.L⁻¹) and at 40 m (3.32±2×10⁴.L⁻¹) depths. Colonies were composed of up to 560 cells with a mean (±SD) colony size of 10 ± 1 cells at 5 m and 40 ± 8 cells at 40 m. Fluorescent latex bead-uptake experiments conducted with field samples indicated that this marine species was capable of phagotrophy and that twice as many Dinobryon cells were ingesting beads at 40 m than at 5 m, although the ingestion rates for those cells actively ingesting beads were similar at both depths. This chrysophyte was found in association with bacteria-and nutrient-rich microhabitats of microaggregates and fecal pellets. The cells and colonies observed in this study appeared to be healthy, as demonstrated by their appearance and their ability to ingest beads.     

Purification and Properties of 5-Ketogluconate Reductase from Gluconobacter liquefaciens

5-Ketogluconate reductase (5KGR) from the cell free extract of Gluconobacter liquefaciens (IFO 12388) was partially purified about 120-fold by a procedure employing ammonium sulfate fractionation, and DEAE-cellulose-, hydroxylapatite- and DEAE-Sephadex A-50-column chromatographies. NADP was specifically required for the oxidative reaction of gluconic acid. The optimum pH for the oxidation of gluconic acid (GA) to 5-ketogluconic acid (5KGA) by the enzyme was 10.0 and for the reduction of 5KGA was 7.5. The optimum temperature of the enzyme was 50°C for both reactions of oxidation and reduction. The enzyme was considerably unstable and lost all of its activity within 3 days. The enzyme activity was strongly inhibited with p-chloromercuribenzoate and mercury ion, but remarkably stimulated by EDTA (1 × 10^?3m). Apparent Km values were 1.8 × 10^?2m for GA, 0.9 × 10^?3m for 5KGA, 1.6 × 10^?5 m for NADP, and 1.1 × 10^?5 m for NADPH₂.     

Effect of prostaglandin F2α on propulsive activity of the isolated segmental colon of the guinea-pig

The effects of prostaglandin F_2α (PGF_2α) on propulsive activity in segments of isolated colon and on isolated strips of guinea-pig colon were investigated.Using experimental conditions under which spontaneous propulsive activity was negligible, PGF_2α (5×10⁻⁸×1×10⁻⁶M), added to the bathing medium, increased propulsive activity in a concentration dependent manner. This increase of propulsive activity was abolished in the presence of atropine or tetrodotoxin (1×10⁻⁷g/ml).The contractions produced by PGF_2α(5×10⁻⁷ − 1×10⁻⁵M) in isolated longitudinal and circular smooth muscle strips of guinea-pig colon were unaffected in the presence of atropine or tetrodotoxin (1×10⁻⁷g/ml).From these results it is concluded that under the conditions employed in this study propulsive activity stimulated by PGF_2α may depend on the contractions of both muscle layers and stimulation of the peristalic reflex.     

Purification and Characterization of 6-Phosphogluconate Dehydrogenase from Phormidium sp.

the native enzyme was 104,000 by gel filtration, and SDS-polyacrylamide gel electrophoresis showed that the enzyme consisted of two subunits with an identical molecular weight of 52,000. The optimum pH of the reaction was 8.0. The Km values for 6-phosphogluconate and NADP were 3.6×10^?5m and 1.3 × 10^?5m, respectively. The enzyme showed no Mg^2𠀫 requirement for the activity, but was activated by Mn^2𠀫 and Ca^2𠀫. The enzyme was inhibited by sulfhydryl reagents, indicating that a sulfhydryl group may be involved in the active site of the enzyme. The enzyme was also inhibited by NADPH₂, ATP, and the intermediates formed during photosynthesis. The substrate 6-phosphogluconate and cofactor NADP partially protected the enzyme from inactivation. The enzyme had enzymological and physicochemical properties similar to enzymes isolated from other sources.     

Rabbit brain purine nucleoside phosphorylase. Physical and chemical properties. Inhibition studies with aminopterin, folic acid and structurally related compounds.

Rabbit brain purine nucleoside phosphorylase used in this study was purified 6000-fold to apparent homogeneity and a specific activity or 50 μmol min^?1 mg ^?1 protein. A molecular weight of 70.000 daltons was determined for the native enzyme by gel filtration on Sephadex. Electrophoresis on polyacrylamide gel, in presence of sodium dodecyl sulfate, gave a subunit molecular weight of 34,500 daltons, suggesting that the enzyme is dimeric with, probably, identical subunits. The relationship of the structure of certain biologically active substances to their inhibitory action on the enzyme was examined. Folic acid and the compound d,l-6-methyl 5,6,7,8-tetrahydropterine, with similar substituents on their primary ring structure, were competitive inhibitors of the enzyme. The inhibition constants calculated were 3.37 × 10^?5M for folic acid and 3.80 × 10^?5m for d,l-6-methyl 5,6,7,8-tetrahydropterine. Aminopterin and the purine analog 8-aza-2,6-diaminopurine, with similar substituents on their primary ring structure, were noncompetitive inhibitors of the enzyme. Their respective inhibition constants were 1.50 × 10^?4 and 1.95 × 10^?4m. Erythro-9-(2-hydroxy-3-nonyl) adenine, an adenosine deaminase inhibitor, was also examined for inhibitory potency with mammalian purine nucleoside phosphorylase, and was observed to be a competitive inhibitor of this enzyme, with an inhibition constant of 1.90 × 10^?4m. The Michaelis constant for the substrate guanosine was near 6.0 × 10^?5m. Physical probe of the nature of the functional groups which participate in enzymic catalysis implicated both histidine and cysteine as the essential catalytic species. Photooxidation studies suggested a pH-dependent sensitivity of an essential catalytic group, and its probable location at the active site.     

Determination of pioglitazone hydrochloride by flow injection chemiluminescence tris(2,2′-bipyridyl)ruthenium(II)–silver(III) complex system

A novel flow injection-chemiluminescence (FI–CL) approach is proposed for the assay of pioglitazone hydrochloride (PG-HCl) based on its enhancing influence on the tris(2,2′-bipyridyl)ruthenium(II)–silver(III) complex (Ru(bipy)₃²⁺-DPA) CL system in sulfuric acid medium. The possible CL reaction mechanism is discussed with CL and ultraviolet (UV) spectra. The optimum experimental conditions were found as: Ru(bipy)₃²⁺, 5.0 × 10⁻⁵ M; sulfuric acid, 1.0 × 10⁻³ M; diperiodatoargentate(III) (DPA), 1.0 × 10⁻⁴ M; potassium hydroxide, 1.0 × 10⁻³ M; flow rate 4.0 ml min⁻¹ for each flow stream and sample loop volume, 180 μl. The CL intensity of PG-HCl was linear in the range of 1.0 × 10⁻³ to 5.0 mg L⁻¹ (R² = 0.9998, n = 10) with limit of detection [LOD, signal-to-noise ratio (S/N) = 3] of 2.2 × 10⁻⁴ mg L⁻¹, limit of quantification (LOQ, S/N = 10) of 6.7 × 10⁻⁴ mg L⁻¹, relative standard deviation (RSD) of 1.0 to 3.3% and sampling rate of 106 h⁻¹. The methodology was satisfactorily used to quantify PG-HCl in pharmaceutical tablets with recoveries ranging from 93.17 to 102.77 and RSD from 1.9 to 2.8%.     

Changes in Hypocholesterolemic Activity in Rats by Various Konnyaku Powder Treatments

Galactosylsucroses contained in soybeans are not digestible. Thus we wished to detect α-galactosidase (EC 3.2.1.22) in intestinal bacteria. The strain of E. coli in the title was found to produce considerably this enzyme adaptively. We could prepare rather pure solution of the enzyme from the sonicate of the strain. It was purified about 142-fold. It showed optimum pH and temperature at 6.8 and 37°C, respectively, with the substrate p-nitrophenyl-α-d-galactoside (PNPG). Dilute enzyme solutions were very unstable even at 0–5°C. However, concentrated solutions were considerably stable. The Michaelis constant (m) was 1.07 × 10^?4, 2.33 × 10^?3, and 3.65 × 10^?2 for PNPG, melibiose, and raffinose, respectively. The maximum velocity (mole/min/mg protein) was 2.72 × 10^?5, 2.67 × 10^?5, and 2.04×l0^?5, respectively for the same three substrates. This enzyme had a weak transferase action.     

Lack of dependence of amine or prostaglandin biosynthesis on absolute cerebral level of pteridine cofactor

Using 2-amino-6-(5'-2'-deoxyphosphoribosyl)-amino-5- or -6-formamido-6-hydroxypyrimidine (dFPyd-P₃), a specific inhibitor of tetrahydrobiopterin (BH₄) synthesis, cerebral pools of BH₄ were reduced to half of that of controls; while, simultaneously, the biosynthesis $\text{de novo}$ of L-erythrodihydroniopterin (BH₂) from GTP was inhibited by about 98%. Nevertheless, there was no effect on the cerebral levels of serotonin, dopamine, norepinephrine or on the biosynthesis of prostaglandin E₂ or F₁. The data are presented in evidence that the absolute level of the cofactor (BH₄) is not regulatory of amine or protaglandin biosynthesis $\text{in vivo}$. Amine and prostaglandin biosynthesis proceeded even at cofactor concentrations of 9×10⁷ M nsuggesting that their biosynthesis is dependent on the rate of H⁺ + e shuttle between BH₂ and BH₄.     

FORMAMIDASE IN RAT BRAIN

Kynurenine formamidase (aryl-formylamine amidohydrolase, EC 3.5.1.9) was found to be present in rat brain and was partially purified and characterized. The partially purified enzyme catalysed the hydrolysis of 5-hydroxyformyl-dl -kynurenine to 5-hydroxy-dl -kynurenine and that of formyl-l -kynurenine to l -kynurenine at similar rates. The apparent K_m values of the enzyme for 5-hydroxyformyl-dl -kynurenine and formyl-l -kynurenine were 4.0 ± 10^?4 and 1.8 ± 10^?4m , respectively. The enzyme was active over a wide pH range (5.5–8.5). The activity was inhibited by low concentrations of Ag⁺ and Hg²⁺. The physiological significance of the enzyme is discussed.     

Ecdysone 20-hydroxylation in Manduca sexta midgut: Kinetic parameters of mitochondrial and microsomal ecdysone 20-monooxygenases

The larval midgut of the tobacco hornworm, Manduca sexta, has high ecdysone 20-monooxygenase (E20MO) activity, located both in the mitochondria and in the microsomes. The apparent kinetic parameters for E20MO in mitochondria and microsomes were determined. The K_m⁵ (for ecdysone) of the mitochondrial and microsomal enzymes were 1.63 × 10⁻⁵ and 3.67 × 10⁻⁷ M, respectively. The V_max was 82.7 pmol/min/mg protein for mitochondria and 32.0 pmol/min/mg protein for microsomes. Although the mitochondrial E20MO has the higher V_max, at physiological ecdysone concentrations (10⁻⁷ − 10⁻⁸ M) it is only one-eighth to one-tenth as active as the microsomal enzyme. It is concluded that the microsomal E20MO is the primary, if not the only, enzyme involved in ecdysone 20-hydroxylation in M. sexta midgut. © 1996 Wiley-Liss, Inc. This article is a US Government work and, as such, is in the public domain in the United States of America.     

Novel nonclassical inhibitors of glycinamide ribonucleotide formyltransferase: 10-Formyl and 10-hydroxymethyl derivatives of 5,8,10-trideazapteroic acid

Several new 10-formyl and 10-hydroxymethyl derivatives of 5,8,10-trideazapteroic acid have been synthesized by a novel and convenient enamine alkylation procedure. Two of these compounds (10a and 10b) were shown to be very powerful inhibitors of L. casei (10a, IC₅₀ = 8 × 10⁻⁶ M ; 10b, IC₅₀ = 7 × 10⁻⁶ M ) and recombinant mouse (10a, IC₅₀ = 3.4 × 10⁻⁵ M ; 10b, IC₅₀ = 2.8 × 10⁻⁵ M ) glycinamide ribonucleotide formyltransferase (GARFT). These IC₅₀ values are comparable to the classical GARFT inhibitor (6R)-DDATHF (IC₅₀, L. casei 2.3 × 10⁻⁶M ; recombinant mouse 2.3 × 10⁻⁵ M ) under identical assay conditions. For both compounds, the inhibition of L. casei GARFT increased with time of incubation, but not markedly with the recombinant mouse enzyme. Due to their potential ability to interfere with purine biosynthesis and to penetrate microbial cells the new nonclassical GARFT inhibitors reported here may be useful for the treatment of infections caused by microorganisms that are sensitive and resistant to conventional antimicrobial agents.     

Inactivation of tyrosine hydroxylase by reduced pterins

Tyrosine hydroxylase [E.C. 1.14.16.2] is inactivated by incubation with its reduced pterin cofactors L-erythro-tetrahydrobiopterin, 2-amino-4-hydroxy-6-methyl-5,6,7,8-tetrahydropterin and 2-amino-4-hydroxy-6,7-dimethyl-5,6,7,8-tetrahydropterin. Each of the two diastereoisomers of L-erythro-tetrahydrobiopterin inactivates tyrosine hydroxylase but the natural (6R) form is much more potent than the unnatural (6S) form at equimolar concentrations. The pterin analog 6-methyl-5-deazatetrahydropterin, which has no cofactor activity, also inactivates the enzyme whereas the oxidized pterins 7,8 dihydrobiopterin and biopterin do not. The inactivation process is both temperature and time dependent and results in a reduction of the Vmax for both tetrahydrobiopterin and tyrosine. Neither tyrosine nor oxygen inactivates tyrosine hydroxylase.