On the Purification and some Properties of 5′-Adenylic Acid-Deaminating Enzyme from Microsporum audouini

From culture broth of Microsporum audouini, 5′-adenylic acid-deaminating enzyme has been purified to about 600-fold. The pH optimum was found to be 5.0 in acetate, 5.5 in succinate, 5.7 in citrate buffer. Velocity constant was 1.83×10^?1 per minute. The optimal temperature was 40°C and activation energy was 15,000 calories. Michaelis-Menten constant was 6×10^?4 m. This enzyme preparation removes amino groups of 5′- AMP, ADP and ATP quickly, of adenosine, 3′-AMP, 5′-deoxyAMP and NAD slowly, but adenine, 2,6-diaminopurine, 2′-AMP and NADP were not deaminated. The enzyme activity was inhibited with F^?, pCMB, Fe^{+ + +}, Cu^{+ +} and Zn^{+ +}     

Purification and properties of 5′-nucleotidase from alkalophilic Bacillus No. C-3

5′-Nucleotidase (EC 3. 1. 3. 5) from alkalophilic Bacillus no. C-3 was purified to homogeneity. The molecular weight of the enzyme was 80,000 by gel filtration. The optimum pH for the activity was 9.5, and the enzyme was stable at pH 9.5–10.5 in a buffer containing 10 mM 2-mercaptoethanol. Substrate specificity study revealed that the enzyme acted on 5′-AMP strongly, on several 5′-nucleotides and ADP to a certain extent, but not on 3′-nucleotides, 2′-nucleotides, p-nitrophenyl phosphate, or ATP. The K_m value for 5′-AMP was 3.0 × 10⁻⁴ M. The enzyme required no divalent cation for its activity. The enzyme was inhibited by borate and arsenite ions but not by 1 mM EDTA.     

Stimulation by ATP of [3H]dopamine binding to synaptic membrane fractions of canine caudate nucleus

The rate of [³H]dopamine binding to crude synaptic membranes from canine caudate nucleus was considerably increased by 2 mM ATP, 5′-adenylylimidodiphosphate and GTP or by 1 mM 5′-guanylyl-imidodiphosphate, while strongly inhibited by 2 mM ADP and GDP. Half maximal concentrations of [³H]dopamine to bind to the membranes were 1.11 × 10^?7M and 8.75 × 10^?6M in the absence of 4 mM ATP, indicating a negative cooperativity of the dopamine receptor, and 9.25 × 10^?7 M in its presence. Hill coefficient was increased from 0.70 to 1.04 by addition of 4 mM ATP. The optimal concentration of ATP for [³H]dopamine binding was in the range of 0.5 to 5 mM.     

A Thermostable phosphofructokinase from the extreme thermophile Thermus X-1.

Two relatively simple procedures are described for the purification of phosphofructokinase from the extreme thermophile, Thermus X-1. The native enzyme has a molecular weight of 1.32 × 10⁵ and contains four apparently identical polypeptide chains. One substrate, fructose-6-phosphate, induces a cooperative protein transition while the other substrate, ATP, does not. Phosphoenolpyruvate functions as an avid negative effector and ADP is a positive effector. The enzyme has an optimum temperature for catalysis of 80 °C. Persistence of the catalytic and allosteric properties over the temperature range 20–80 °C suggests that the same protein structure is retained throughout this temperature range. Thermus X-1 phosphofructokinase is more stable to inactivation by heat, urea, guanidine hydrochloride or acidification than the phosphofructokinases obtained from the mesophilic organisms Escherichia coli and Clostridium pasteurianum. Comparison of the amino acid compositions of the three enzymes indicates no substantive differences in their hydrophobicity, hydrogen bonding potential or average residue size. The markedly elevated optimum temperature for catalysis exhibited by the Thermus enzyme appears to result from stabilization of its catalytically functional conformational to a reversible thermal inactivation above 40 °C and to ligation of the substrate fructose-6-phosphate.     

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.     

Purification and Properties of Pantothenate Kinase from Brevibacterium ammoniagenes IFO 12071

Pantothenate kinase (ATP: pantothenate 4′-phosphotransferase, EC 2.7.1.33) was purified about 200-fold from the cell extract of Brevibacterium ammoniagenes IFO 12071 by ammonium sulfate fractionation, DEAE-cellulose chromatography, and Sephadex G-150 gel filtration. The purified enzyme gave a single band on polyacrylamide gel electrophoresis. The molecular weight was calculated approximately 45,000. The enzyme catalyzed the formation of pantothenic acid 4′-phosphate and ADP from pantothenate and ATP in the presence of Mg²⁺ ATP could be substituted for, partly, by ITP, GTP, and UTP. The enzyme phosphorylated not only pantothenate, but also pantothenoylcysteine, pantetheine, and pantothenyl alcohol. Apparent Km values were 6.7×10^?5 m for pantothenate, 3.5×10^?5 m for ATP, and 10^?3 m for Mg²⁺. The reaction was inhibited by the intermediates of CoA biosynthesis, of which CoA itself was a most effective inhibitor. Other properties of the enzyme were also investigated.     

Cyclic AMP and citric acid accumulation by Aspergillus niger

Aspergillus niger accumulated citric acid in the medium under certain conditions. Cyclic AMP concentrations of the order of 10^?6M and higher caused an increase in the rate of citrate synthesis. Adenosine, ATP, and cyclic GMP at 10^?3M also stimulated, but were ineffective at 10^?4M. 5′-AMP had no effect while 5′-GMP and guanosine inhibited slightly. ADP showed a 42% inhibition. Theophylline enhanced the cyclic AMP effect. It is proposed that citric acid accumulation by Aspergillus niger may result from abnormal cyclic AMP metabolism.     

Inactivation of E. coli RNA polymerase by pyridoxal 5'-phosphate: identificationof a low pKa lysine as the modified residue.

The inactivation of E. coli RNA polymerase (3.3 × 10^?7M) by pyridoxal 5′-phosphate (1 × 10^?4M to 5 × 10^?4M) is a first order process with respect to the remaining active enzyme. Studies of the variation of the first order rate constant with the concentration of pyridoxal 5′-phosphate show that the inactivation reaction follows saturation kinetics. The formation of a reversible enzyme-inhibitor intermediate is postulated. Kinetic studies at different pH values indicate that the inactivation rate constant depends on the mole fraction of one conjugate base with pK_a 7.9. The apparent equilibrium constant (association) for the inactivation reaction is independent of the pH and is 1.8 × 10⁴ M^?1. By electrophoretic and chromatographic analysis of enzyme hydrolyzates after pyridoxal 5′-phosphate and NaBH₄ treatment only N-ε-pyridoxyllysine was found. It is postulated that a lysine ε-amino group with a low pK_a is critical for the activity of the enzyme.     

Correlations among the responses of suspensions of Dictyostelium Discoideum to pulses of 3′,5′-cyclic AMP: Transient turbidity decrease,the cyclic AMP signal,and variation in adenine mononucleotide levels

Aggregation-competent myxamoebae of the cellular slime mold Dictyostellium discoideum are known to exhibit two responses to extracellular pulses of 3′5′-cyclic AMP: an immediate chemotactic movement; and a delayed generation of intracellular cyclic AMP which is subsequently released into the medium. The mechanism of the latter, the so-called signalling response, may depend on alterations in intracellular metabolite levels and is the subject of this communication.Myxamoebae of the wild-type strain NC-4 of D. discoideum were suspended in an aerated, stirred 17 mM potassium phosphate buffer. pH 6.0, at a concentration of approx. 6 · 10^?7 cells/ml (8%, v/v) at 25°C and were pulsed with 1. 10^?8—1 · 10^?7 M cyclic AMP at 10–20-min intervals for periods of 3–5 h over incubation of 4–9 h. Suspensions were monitored continuously for transient turbidity decreases following the cyclic AMP pulses as an indication of the magnitude and duration of the cellular response to cyclic AMP. When the pattern of turbidity decrease indicated that a signalling response had developed, samples were withdrawn at 10–15-s intervals from the suspension, inactivated with perchloric acid, and analyzed for cyclic AMP, ATP, ADP, AMP, pyruvate, and glucose 6-phosphate. In separate experiments, steady-state oxygen tension was monitored along with turbidity to detect possible changes in respiratory rate.The following consistent patterns were observed after the added cyclic AMP pulse: a transient increase in the ADP level which reaches maximum between 0.7 and 1.7 min; transient decreases in ATP and pyruvate which concide with and approximately equal the magnitude of the increase in ADP; a later increase in glucose 6-phosphate which reaches maximum approx. 2 min after the ADP     

Affinity chromatography of phosphofructokinase.

The behavior of mammalian phosphofructokinase on immobilized adenine nucleotides was investigated. Three different insolubilized ligands were compared using a pure rabbit muscle phosphofructokinase. N⁶-[(6-aminohexyl)-carbamoyl-methyl]-ATP-Sepharose bound at least 90 times more enzyme than either N⁶-(6-aminohexyl)-AMP-agarose or ATP-adipic acid hydrazide-Sepharose. The elution of phosphofructokinase from the ATP-Sepharose with various metabolites and combinations of metabolites was investigated. The enzyme is eluted specifically from N⁶-[(6-aminohexyl)-carbamoyl]-ATP-Sepharose with a mixture of 25 μm each of fructose 6-phosphate and ADP (±Mg²⁺). The enzyme is not eluted either with ATP (25 μm), fructose 1,6-diphosphate (1 mm), ADP (25 μm), fructose 6-phosphate (1 mm) alone, or with a mixture of fructose 1,6-diphosphate (25 μm) and ATP (25 μm). The recovery of bound enzyme was usually greater than 90%. A mixture of glucose 6-phosphate and ADP or a mixture of IDP and fructose 6-phosphate also elutes the enzyme, but the recovery with these eluants was only about 40%. It was concluded that the “dead-end” complex is the most effective in the elution. Using this method, phosphofructokinase has been prepared in an essentially homogeneous form from muscle and brain of rabbit and rat. The overall isolation procedure involves a high speed centrifugation of crude extracts which sediments phosphofructokinase as a pellet, followed with adsorption on N⁶-[(6-aminohexyl)-carbamoyl-methyl]-ATP-Sepharose and specific elution with the mixture of fructose 6-phosphate and ADP.     

A cAMP receptor from mouse liver cytosol whose binding capacity is enhanced by Mg++-ATP.

A receptor with a dissociation constant of 2·10^?6M for cyclic 3′,5′-AMP (cAMP) has been found in mouse liver cytosol. This cAMP binding activity can be differentiated from the cAMP-dependent protein kinase holoenzymes and the free regulatory subunits also found in the cytosol. Mg⁺⁺-ATP increases the number of binding sites for cAMP several fold. This increased capacity for cAMP binding persists after Sephadex G-25 filtration, and incubation for 14 hours in the presence of 5 mM EDTA. Among several adenosine- and guanosine-derivatives tested, only AMP, ADP and ATP compete efficiently with [³H] cAMP for the cAMP binding site.     

Effects of AMP and Cibacron blue F3G-A on the fructose 6-phosphate binding of yeast phosphofructokinase.

The positive effector 5′-AMP of yeast phosphofructokinase does not influence the binding of fructose 6-phosphate to the enzyme. Cibacron blue F3G-A considered an ATP analogue decreases the affinity of the enzyme to fructose 6-phosphate without exerting an effect on the cooperativity of fructose 6-phosphate binding. The peculiarities of the interactions of AMP and Cibacron blue with fructose 6-phosphate binding demonstrate compatibility of the allosteric kinetics with the binding behavior of the enzyme.     

Characterization and Regulatory Properties of Glucose-6-Phosphate Dehydrogenase from Black Gram (Phaseolus mungo)

Glucose-6-phosphate dehydrogenase (E.C. 1.1.1.49) was partially purified by fractionation with ammonium sulfate and phosphocellulose chromatography. The K_m value for glucose-6-phosphate is 1.6 × 10^?4 and 6.3 × 10^?4M at low (1.0–6.0 × 10^?4M) and high (6.0–30.0 × 10^?4M) concentrations of the substrate, respectively. The K_m value for NADP⁺ is 1.4 × 10^?5M. The enzyme is inhibited by NADPH, 5-phosphoribosyl-1-pyrophosphate, and ATP, and it is activated by Mg²⁺, and Mn²⁺. In the presence of NADPH, the plot of activity vs. NADP⁺ concentration gave a sigmoidal curve. Inhibition of 5-phosphoribosyl-1-pyrophosphate and ATP is reversed by Mg²⁺ or a high pH. It is suggested that black gram glucose-6-phosphate dehydrogenase is a regulatory enzyme of the pentose phosphate pathway.     

Manganese substrate complexes of phosphofructokinase studies by pulsed magnetic resonance

The formation of binary, ternary, and quaternary complexes between phosphofructokinase, manganese, and substrates has been demonstrated by use of pulsed nuclear magnetic resonance techniques. A Scatchard plot of the interaction of manganese with phosphofructokinase as determined by electron paramagnetic resonance shows two types of manganese binding sites. Phosphofructokinase seems to contain one or two of the metal binding sites with K_d = 20 μm and ?_b ≦ 4, and perhaps, as many as 14 binding sites with K_d ~ 0.8 mm and ?_b ≦ 12 ± 2 per enzyme. Addition of ATP or ADP results in a further enhancement of the relaxation rate indicating ternary complex formation. The concentration of ATP and ADP which results in half maximal change of enhancement is 30–100 μm and 80 μm, respectively. No change in the water proton relaxation rate was detected upon addition of fructose-6-P or fructose-1,6-bisphosphate. A quaternary complex was detected by proton relaxation measurements upon addition of fructose-6-P to a reaction mixture containing β, γ-methylene ATP, manganese, and enzyme with 50 μm fructose-6-P required to obtain the half maximal observed effect. This evidence for a quaternary complex is consistent with a sequential reaction mechanism for phosphofructokinase.     

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 of regulatory metabolism in Moniezia expansa: the role of phosphofructokinase (with a note on pyruvate kinase).

Some of the properties of a partially purified preparation of phosphofructokinase (PFK) from Moniezia expansa are described. PFK has a pH optimum between 7·4 and 8·0, and is activated by magnesium and divalent manganese ions. It exhibits sigmoid kinetics with fructose-6-phosphate, and ATP decreases the affinity of the enzyme for F6P. This inhibition is partially relieved by F6P, AMP and ammonium ions. GTP and ITP act as substrates for the PFK reaction but do not exert the same inhibitory effects. The effect of ATP on pyruvate kinase was also examined, and was found to inhibit both the activated and inactivated enzyme. Apparent K_m's for both enzymes are presented.Generally, PFK and pyruvate kinase from M. expansa show properties similar to the enzymes from mammalian sources. The presence of sigmoid kinetics for F6P and ATP at pH8 is, however, a significant departure from what is observed in PFK from mammalian sources. Possibilities exist in M. expansa for controls of metabolism similar to those found in mammalian tissues.     

Affinity labeling of AMP-ADP sites in heart phosphofructokinase by 5-p-fluorosulfonylbenzoyl adenosine.

The purine nucleotide derivative, 5′-p-fluorosulfonylbenzoyl adenosine (5′-FSO₂B_ZAdo) functions as an affinity label for the allosteric sites of phosphofructokinase. The modified enzyme at pH 6.9 is insensitive to allosteric inhibition by ATP, activation by AMP, c-AMP, ADP and shows no sigmoidal kinetics for fructose-6-P. The reaction does not appear to occur at the catalytic site since modification of the enzyme does not significantly affect its specific activity nor its Michaelis constant at pH 8.2. ADP, and to a much lesser degree AMP and ATP, protects the enzyme from modification by the adenosine reagent. The modified enzyme essentially does not bind significant amounts of AMP, c-AMP, ADP, but still binds an analog of ATP, AppNHp. The adenosine affinity label will be of value in studies on the nature of the AMP-ADP allosteric sites.     

Cyclic 3′,5′-AMP phosphodiesterase in Physarum polycephalum I. Chemotaxis toward inhibitors and cyclic nucleotides

The effect of several inhibitors of the enzyme cyclic 3′,5′-AMP phosphodiesterase as chemoattractants in Physarum polycephalum was examined. Of the compounds tested, 4-(3-butoxy-4-methoxybenzyl)-2-imidazolidinone (Roche 20-1724/001) and 1-ethyl-4-(isopropylidinehydrazino)-1H-pyrazolo-(3,4-b)-pyridine-5-carboxylic acid ethyl ester, hydrochloride (Squibb 20009) were the most potent attractants. 3-Isobutyl-1-methyl xanthine, theophylline, and morin (a flavanoid) were moderate attractants and sometimes gave negative chemotaxis at high concentrations. Cyclic 3′,5′-AMP was an effective, but not potent attractant. A repellent effect following the positive chemotactic action was sometimes observed with cyclic 3′,5′-AMP at concentrations as high as 1 · 10^?2 M. Dibutyryl cyclic AMP appeared to be a somewhat more potent attractant than cyclic 3′,5′-AMP. The 8-thiomethyl and 8-bromoderivatives of cyclic AMP, which are poorly hydrolyzed by the phosphodiesterase, were not attractants in Physarum. Possible participation of cyclic 3′,5′-AMP in the directional movement in P. polycephalum is discussed.     

Purification and Some Properties of Adenosine (Phosphate) Deaminase from the Liver of the Squid Todarodes pacificus

An enzyme that catalyzed the deamination of adenosine 3′-phenylphosphonate was purified from squid liver to homogeneity as judged by SDS-PAGE. The molecular weight of the enzyme was estimated to be 60,000 by SDS-PAGE and 140,000 by Sephadex G-150 gel filtration. The enzyme deaminated adenosine, 2′-deoxyadenosine, 3′-AMP, and 2′,3′-cyclic AMP, but not adenine, 5′-AMP, 3′,5′-cyclic AMP, ADP, or ATP. The apparent K_m and V_max at pH 4.0 for these substrates were comparable (0.11-0.34mM and 179-295 μmol min^?1 mg^?1, respectively). The enzyme had maximum activity at pH 3.5-4.0 for adenosine 3′-phenylphosphonate, at pH 5.5 for adenosine and 2′-deoxyadenosine, and at pH 4.0 for 2′,3′-cyclic AMP and 3′-AMP when the compounds were at concentration of 0.1 mM. The K_m at 4.0 and 5.5 for each substrate varied, but the Vmax were invariant. These results indicated that the squid enzyme was a novel adenosine (phosphate) deaminase with a unique substrate specificity.     

Pathways of Chlorophenol Formation in Oxidative Biodegradation of BHC

Hexose 1-phosphate uridylyltransferase (EC 2.7.7.12) was present constitutively in Bifidobacterium bifidum. The enzyme was purified to a homogeneous state from B. bifidum grown on a glucose medium and characterized. The molecular weight of the enzyme is about 110,000.The pH optimum of the enzyme was 7.5. The enzyme was very labile on the acidic side below pH 4.5. Thymidine diphosphate glucose could serve as a substrate with about 60% efficiency of UDP-glucose. The Km values for UDP-gtucose, galactose 1-phosphate (Gal-l-P), UDP-galactose and glucose 1-phosphate (Glc-1-P) were estimated to be 2.3×10^?5M, 5.0 × 10^?4M, 3.1 × 10^?5 M and 1.4 × 10^?4M, respectively. From these results the physiological roles of the enzyme were considered in relation to galactose metabolism in B. bifidum.