Purification and properties of the Pichia guilliermondii acid nucleotide pyrophosphatase hydrolyzing flavin adeninine dinucleotide

Acid nucleotide pyrophosphatase was isolated from the cell-free extracts of Pichia guilliermondii Wickerham ATCC 9058. The enzyme was 25-fold purified by saturation with ammonium sulphate, gel-filtration on Sephadex G-150 column and ion-exchange chromatography on DEAE-Sephadex A-50 column. The pH optimum was 5.9, temperature optimum--45 degrees C. The enzyme catalyzed the hydrolysis of FAD, NAD+ and NADH, displaying the highest activity with NAD+. The Km, values for FAD, NAD+ and NADH were 1.3 x 10(-5) and 2.9 x 10(-4) M, respectively. The hydrolysis of FAD was inhibited by AMP, ATP, GTP, NAD+ and NADP+. The K1 for AMP was 6.6 x 10(-5) M, for ATP--2.0 X 10(-5) M, for GTP--2.3 X 10(-6) M, for NAD+--1.7 X 10(-4) M. The molecular weight of the enzyme was 136 000 as estimated by gel-filtration on Sephadex G-150 and 142 000 as estimated by thin-layer gel-filtration chromatography on Sephadex G-200 (superfine). Protein-bound FAD of glucose oxidase was not hydrolyzed by acid nucleotide pyrophosphatase. The enzyme was stable at 2 degrees C in 0.05 M tris-maleate buffer, pH 6.2. Alkaline nucleotide pyrophosphatase hydrolyzing FAD was also detected in the cells of P. guilliermondii.     

Purification and properties of Pichia guilliermondii yeast alkaline nucleotide pyrophosphatase hydrolyzing flavin adenine dinucleotide

Alkaline nucleotide pyrophosphatase was isolated from the Pichia guilliermondii Wickerham ATCC 9058 cell-free extracts. The enzyme was 740-fold purified by saturation of ammonium sulphate, gel-chromatography on Sephadex G-150 and ion-exchange chromatography on DEAE-cellulose. Nucleotide pyrophosphatase is the most active at pH 8.3 and 49 degrees C. The enzyme catalyzes the hydrolysis of FAD, NAD+, NADH, NADPH, GTP. The Km value for FAD is 2.4 x 10(-4) M and for NAD+--5.7 x 10(-6) M. The hydrolysis of FAD was inhibited by NAD+, NADP+, ATP, AMP, GTP, PPi and Pi. The Ki for NAD+, AMP and Na4P2O7 was 1.7 x 10(-4) M, 1.1 x 10(-4) M and 5 x 10(-5) M, respectively. Metal chelating compounds, 8-oxyquinoline, o-phenanthroline and EDTA, inhibited completely the enzyme activity. The EDTA effect was irreversible. The molecular weight of the enzyme determined by gel-filtration on Sephadex G-150 and thin-layer gel-filtration chromatography was 78000 dalton. Protein-bound FAD of glucose oxidase is not hydrolyzed by the alkaline nucleotide pyrophosphatase. The enzyme is stable at 2 degrees C in 0.01 M tris-HCl-buffer (pH 7.5).     

Purification, properties and regulation of urocaninase from rat liver]

Urocaninase (EC 4.2.1.4.9) from rat liver homogenate has been purified, using protein precipitation at pH 4,8, ammonium sulfate fractionation, gel-filtration through Sephadex G-200 and chromatography on DEAE-cellulose. Upon DEAE-cellulose chromatography urocaninase is separated from the proteins possessing the activity of 3',5'-AMP-dependent protein kinase. The purified enzyme becomes activated after addition of ATP and exogenous protein kinase or one of the fractions resulting from DEAE-cellulose chromatography. Using [gamma-32P]ATP, it has been shown that such activation is accompanied by incorporation of at least one phosphate residue into the enzyme molecule. The mol. weight of urocaninase as determined by gel-filtration is about 110 000. The Km value for urocanate is 15 . 10(-6) M, the isoelectric point lies at 5,6. The mechanism of regulation of the urocaninase activity in rat liver is discussed.     

Regulation of urocaninase activity in the liver: role of 3',5'-AMP]

A dependence of rat liver urocaninase activity on the agents affecting the adenylate cyclase system was studied in vitro and in vivo. Urocaninase is considerably activated after the injection of glucagone, NaF, theophylline and 3',5'-AMP. Under conditions optimal for the protein kinase activity of phosphorylase the urocaninase of liver extracts was activated 7-fold on the average. The nezyme retains its activity after gel-filtration through Sephadex G-25 and is capable of inactivation in the presence of Mg2+ and of reactivation after addition of ATP and 3',5'-AMP. These data suggest a possibility of regulation of mammalian liver urocaninase activity by 3',5'-AMP-dependent phosphorylation of the enzyme. Derivatives of hypoxanthine (theophylline and caffeine) in concentration 10(-4) M activate urocaninase in liver extracts 2--3 and 1.5-fold respectively. The activation is probably not due to the 3',5'-AMP phosphodiesterase inhibition, since another phosphodiesterase inhibitor--papaverine--has no activating effect on urocaninase.     

NAD+-kinase from Neurospora crassa: isolation and properties

The NAD+ kinase (EC 2.7.1.23) of the filamentous fungus N. crassa is localized in cytosol. The activity in the dialyzed cell free extract has a pH optimum 8.3; it utilizes only ATP but not inorganic polyphosphates as a phosphoryl donor. A method for 200-fold purification of NAD+ kinase with a 20% yield has been developed. The procedure includes 105000 g centrifugation, fractionation with (NH4)2SO4, isoelectrofocusing in a Ultrodex layer and preparative electrophoresis in polyacrylamide gel. The molecular heterogeneity of NAD+ kinase was demonstrated by polyacrylamide gradient electrophoresis and by gel filtration through Sephadex G-200. The molecular weights of four individual forms of the enzyme are: 330000-338000, 305000-306000, 215000-229000 and 203000 Da. The Km values for the reaction catalyzed by purified NAD+ kinase for NAD+ and ATP are 3.0 X 10(-4) M and 0.9 X 10(-3) M, respectively.     

Protein kinase-mediated phosphorylation of a purified sterol ester hydrolase from bovine adrenal cortex.

Sterol ester hydrolase (cholesterol esterase, E.C. 3.1.1.13) of bovine adrenal cortex has been extensively purified by ammonium sulfate fractionation, acid precipitation, hydroxylapatite chromatography, and Sephadex G-200 chromatography. During the purification sequence, the hydrolase activity was purified free of endogenous protein kinase. With this purified preparation, activation by cyclic AMP and ATP-Mg2+ did not occur unless exogenous protein kinase was included in the activating system. Using [gamma-32P]ATP, the transfer of the terminal phosphate to the enzyme protein was demonstrated by three separate experimental approaches. With pooled fractions from Sephadex G-200 chromatography, significant binding of 32P by the enzyme protein was observed only in the presence of exogenous protein kinase. Time course studies disclosed a close concurrence between the extent of activation of the purified enzyme by cyclic AMP-dependent protein kinase and the level of 32P transfer from [gamma-32P]ATP to the enzyme protein. Finally, assays carried out during Sephadex G-200 chromatography showed a correspondence in the peaks for activated sterol ester hydrolase and for 32P binding by protein. The data confirm that activation of adrenal sterol ester hydrolase by cyclic AMP and ATP-Mg2+ involves protein kinase-catalyzed phosphorylation of the enzyme protein.     

Isolation and some properties of a deoxyribonuclease from Turbo cornutus.

1. Four DNases were found in the dried liver extract of a top shell, Turbo cornutus. The major one was purified 120-fold by phosphocellulose column chromatography, sulfoethylcellulose column chromatography and gel-filtration on Sephadex G-150. The yield was 2.7%. 2. The enzyme activity was not affected by Mg2+ (10(-3)--10(-2)M), EDTA (10(-3)--10(-2)M), or NaCl (10(-1)M). It showed a pH optimum of 4.7--4.8. Ionic strength was found to be critical for the maximal activity. The isoelectric point was 8.5--9.0. On heating at 50 degrees C C for 5 min the enzymic activity fell to half the initial value. 3. The enzyme preparation degraded native as well as heat-denatured DNA, but not RNA. It degraded heat-denatured DNA endonucleolytically to give oligonucleotides with 3'-phosphates. 4. The 3'-phosphate and 5'-hydroxy termini of oligonucleotides were investigated. At both the 3'- and 5'-terminal positions, purine nucleotides were predominant.     

Brain pyridoxal kinase. Purification, substrate specificities, and sensitized photodestruction of an essential histidine.

Pyridoxal kinase has been purified 2,000-fold from pig brain. The enzyme preparation migrates as a single protein and activity band on analytical gel electrophoresis. Pyridoxal kinase, 60,000 molecular weight, catalyzes the phosphorylation of pyridoxal (Km = 2.5 x 10(-5) M) and pyridoxine (Km = 1.7 x 10(-5) M). Pyridoxamine is not a substrate of the purified kinase. Irradiation of the kinase in the presence of riboflavin leads to irreversible loss of catalytic activity. Riboflavin binds to the kinase with a KD = 5 microM as shown by fluorometric titrations. Singlet excited oxygen, generated by energy transfer from the lowest triplet of riboflavin to oxygen, acts as the oxidizing agent of approximately one histidine residue per mol of enzyme. The amino acid residues tyrosine, tryptophan, and cysteine are not photooxidized by the sensitizer bound to the enzyme. It is postulated that histidine is involved in the binding of the substrate ATP to the catalytic site of pyridoxal kinase.     

Affinity chromatographic purification and properties of flavokinase (ATP:riboflavin 5'-phosphotransferase) from rat liver

Flavokinase (ATP:riboflavin 5'-phosphotransferase, EC 2.7.1.26) has been purified to apparent homogeneity from rat liver by affinity chromatography using flavinyl agarose beads (agarose-OCH2CONH(CH2)2NHCO(CH2)/N10-7,8-dimethylisoalloxazine). The specific activity of the pure enzyme is 9,900 units (nmol of FMN formed/h at 37 degrees C)/mg of protein, and reflects a one-step, 7000-fold purification. Flavokinase thus obtained, unlike previous preparations from mammalian sources, is free from contaminating phosphatase and FAD synthase. The purified enzyme rapidly loses activity upon storage but is stabilized by riboflavin and thiol-protecting reagents. The apparent molecular weight, estimated by gel filtration on Sephadex G-100 and sodium dodecyl sulfate-polyacrylamide gel electrophoresis, is 28,000 +/- 1,000. Flavokinase phosphorylates and/or is inhibited by a large number of riboflavin analogs; however, the physiologically important 8 alpha-(amino acid)riboflavins are poorly accommodated. The strongly preferred phosphate donors are ATP and dATP. Both Zn2+ and Mg2+, as well as several other divalent cations, activate flavokinase, but Zn2+ yields greatest activity (1.8 times that with Mg2+). The pH optimum for activity with either Zn2+ or Mg2+ is approximately 9.3; at pH 7.0, the activity is 40% of that at the pH optimum.     

Acid proteinase from the roe of the trout Salmo gairdneri R

Acidic proteinase from the trout spawn is 640 fold purified (yield 22%). Purification includes autolysis, acid treatment, ammonium sulphate fractionation, G-100 Sephadex gel-filtration, ion-exchange chromatography on DEAE-cellulose. Molecular mass of the enzyme under study is 70 kDa according to the data of gel-filtration. Acidic proteinase displays its greatest activity towards hemoglobin (pH 4.0, 37 degrees C) and is inhibited completely by EDTA, by 50%--by Pb2+ and soya inhibitor of trypsin and 2.8 times activated by Zn2+. Enzyme activity is not affected by dithiotreitol, iodine acetate, phenylmethylsulphonylfluoride parachloromercurybenzoate, Hg2+, Na+, Co2+, Ca2+.     

Deoxyguanosine kinase from human placenta

Deoxyguanosine kinase (ATP:deoxyguanosine 5'-phosphotransferase) has been purified up to a specific activity of 10.3 nmol/min per mg protein from human placenta. The enzyme appears to have a molecular weight of 58 000 from the results of Sephadex G-75 gel filtration. The enzyme catalyzed phosphorylation of deoxyguanosine and deoxyadenosine, but deoxycytidine was not phosphorylated. An apparent Km value for deoxyguanosine was 2.5 micro M. When ATP was used as a phosphate donor, the pH optimum was at pH 6.0, but the optimum was shifted to pH 6.8 by the addition of dTTP. At physiological pH, the activity was stimulated 3-4-fold by dTTP. dTTP was also an effective phosphate donor, but using dTTP as a phosphate donor, a broad pH optimum of 7.0 was observed. Two Km values of 0.13 and 2.2 mM were obtained for both MgATP2- and MgdTTP2-. The activity was strongly inhibited by dGTP and dGDP; 50% inhibition by 1.0 micro M dGTP and 2.1 micro M dGDP, respectively. The enzyme required the presence o Mg2+ or Mn2+.     

Partial purification and characterization of a soluble protein kinase from Leishmania donovani promastigotes

A soluble protein kinase from the promastigote form of the parasitic protozoon Leishmania donovani was partially purified using DEAE-cellulose, Sephadex G-200 and phosphocellulose columns. The enzyme preferentially utilized protamine as exogenous phosphate acceptor. The native molecular mass of the enzyme was about 85 kDa. Mg2+ ions were essential for enzyme activity; other metal ions, e.g. Ca2+, Co2+, Zn2+ and Mn2+, could not substitute for Mg2+. cAMP, cGMP, Ca2+/calmodulin and Ca2+/phospholipid did not stimulate enzyme activity. The pH optimum of the enzyme was 7.0-7.5, and the temperature optimum 37 degrees C. The apparent Km for ATP was 60 microM. Phosphoamino acid analysis revealed that the protein kinase transferred the gamma-phosphate of ATP to serine residues in protamine. The thiol reagents p-hydroxymercuribenzoic acid, 5-5'-dithio-bis(2-nitrobenzoic acid) and N-ethylmaleimide inhibited enzyme activity; the inhibition by p-hydroxymercuribenzoic acid and 5-5'-dithio-bis(2-nitrobenzoic acid) was reversed by dithiothreitol.     

Purification and properties of plasminogen activator from human blood plasma after sudden death]

Plasminogen activator from human blood plasma after sudden death was isolated and purified 60-90-fold by precipitation with ammonium sulfate, ZnSO4 and ethanol as well as by chromatography on DEAE-Sephadex A-50 and gel--filtration through Sephadex G-200. The resulting enzyme had specific activity of 110-210 units per mg of protein. The enzyme prepartion possessed no plasmin activity; total content of carbohydrates was 2.4-2.5%; that of syalic acids--1.2-1.3%. The enzyme was found heterogeneous during disc electrophoresis in 7.0% polyacrylamide gel and corresponded in its mobility to beta-globulins of blood plasma. Molecular weight of enzyme as determined by gel-filtration through Sephadex G-200 is 70000. The isoelectric point lies at pH 6.2.     

Biosynthesis of proline in Pseudomonas aeruginosa. Partial purification and characterization of gamma-glutamyl kinase.

A gamma-glutamyl kinase (ATP-L-glutamate 5-phosphotransferase) was purified about 85-fold from crude extracts of Pseudomonas aeruginosa strain PAO 1 by (NH4)2SO4 precipitation, molecular-sieving by Sephadex G-150 and DEAE-cellulose chromatography. The molecular weight of this enzyme was 84,000. The preparation catalysed formation of gamma-glutamyl hydroxamate from L-glutamate, ATP and Mg2+ or Mn2+ with concomitant hydrolysis of ATP to ADP + Pi. L-Proline inhibited the gamma-glutamyl kinase activity by 50% at 5 mM and almost completely at 30 mM. The inhibition of L-proline was non-competitive, wherease L-methionine-DL-sulphoximine inhibited the enzyme competitively. Proline was found to inhibit the gamma-glutamyl kinase activity of the wild-type strain and of representatives of two of the three transductional classes of proline-auxotrophic mutants. Strain PAO 879, a mutant representing the third transductional class of proline auxotrophs, lacked proline-inhibitible gamma-glutamyl kinase. Thiol-blocking reagents inhibited the gamma-glutamyl kinase and this effect was prevented by dithiothreitol.     

Isolation and characterization of a protein C activator from the moccasin snake venom

The protein C activator detectable in the venom of the Southern Copperhead snake (Agkistrodon contortrix contortrix) was isolated by a combination of chromatofocusing on PBE-94 in the range pH9-7 and gel-filtration on Sephadex G-100 column. The peak protein C activator from Sephadex G-100 column appeared as double diffuse bands with apparent molecular weight of 37,700 and 31,400 after electrophoresis in the presence of sodium dodecylsulfate and 2-mercaptoethanol. The isolated enzyme does not clot human fibrinogen and when mixed with normal plasma generates activity of Protein C. It can be used for the measurement of protein C functional activity.     

Isolation, purification and study of the properties of pyruvate kinase from the bovine adrenal cortex

Pyruvate kinase (ATP: pyruvate 2-0-phosphotransferase, EC 2. 7. 1. 40) from bovine adrenal cortex was purified 243 fold. The whole purification procedure included ammonium sulphate fractionation, heat treatment, Sephadex HW-55 chromatography and phosphocellulose chromatography. The specific activity of the preparation is 15.6 U/mg at 30 degrees C, the yield--36%. Pyruvate kinase showed only one protein band as judged by sodium dodecyl sulphate acrylamide gel electrophoresis. The enzyme displayed a hyperbolic saturation curve with respect to P-enolpyruvate. The apparent Km for this substrate was 0.55 X 10(-4) M, pH optimum--6.8-7.0. K+ concentrations above 0.1 M inhibit the enzyme.     

A galactokinase of Mycobacterium sp. 279 galactose mutant

A galactokinase and the other enzymes of a galactose catabolic pathway were found in Mycobacterium sp. 279 galactose mutant. The galactokinase was partially purified in a procedure involving ammonium sulfate precipitation, Sephadex G-100 filtration and DEAE-cellulose chromatography. The enzyme was 170-fold purified with 25% of recovery. It was most active at pH 7.8-8.0 in the presence of Mg2+, CO2+, Mn2+ or Fe2+ ions. The molecular weight of the enzyme as determined by Sephadex G-100 filtration amounted to 41,700. The apparent Michaelis constants for galactose and ATP in spectrophotometric test were 1.0 mM and 0.29 mM, respectively. Mercuric compounds at concentration of 0.4 mM completely blocked the enzyme. The galactokinase was quite stable during storage at moderatory temperatures and neutral pH but underwent rapid inactivation on heating above 50 degrees C.     

Preparation and properties of immobilized riboflavin kinase from Pichia guilliermondii

Riboflavin kinase (ATP: riboflavin-5'-phosphotransferase, EC 2.7.1.26) from n-alkane utilizing Pichia guilliermondii yeast has been immobilized by covalent attachment to CNBr-activated agarose beads. The enzyme activity yield during immobilization reached 71.6%. Immobilized riboflavin kinase showed no significant changes in temperature and pH optima as well as in specificity of the action in relation to synthetic substrate analogues with the substitution of methyl groups at positions 7 and 8 of the isoalloxazine ring. Immobilized riboflavin kinase was stable during FMN synthesis in the continuous-flow packed column enzyme reactor with half-life of 27 days.     

Purification and Characterization of β-d-Galactosidase and α-d-Mannosidase from Papaya (Carica papaya) Seeds

β-D-Galactosidase was purified 115-fold from a saline extract of papaya seeds by fractionation with ammonium sulfate, DEAE-Sephadex chromatography and gel-filtration on Sephadex G-75, G-150, and G-100. The purified β-D-galactosidase (MW, 56,000 daltons) had an isoelectric point (pI) at pH 8.4 and the optimal pH for its activity was 3.5 to 4.5. The enzyme activity was inhibited by Cu²⁺,Ag⁺,Hg²⁺,Pb²⁺,NaAsO₂ and р-chloromercuribenzoate at concentrations of 1x10^-3 M. Among the various mono- and oligosaccharides tested, D-galactose, D-galacturonic acid, D-galactono-γ-lactone and melibiose significantly inhibited the enzyme activities at concentrations of 2xl0^-3 to 1X10^-2M. The purified enzyme hydrolyzed β-nitrophenyl β-D-galactoside (Km = 1.0X10^-3M), methyl β-D-galactoside (Km=1.6x10^-2M), aminoethyl β-D-galactoside (Km =3.3X10^-2M) and lactose (Km = 9.1X10^-2M). β-(l→3)-Linked galactotetraosyl-eryth itol and asialo-glycopeptide isolated from fetuin were also hydrolyzed to the extent of 78 and 75%, 4respectively, on the basis of their galactose contents.

∝-D-Mannosidase from papaya seeds was also purified 130-fold by ammonium sulfate fractionation, DEAE-Sephadex chromatography, gel-filtration on Sephadex G-150 and hydroxylapatite chromatography. The purified enzyme (MW, 156,000 daltons), consisting of two subunits (78,000x2), was inhibited by Hg²⁺,Ag⁺,Cu²⁺, р-chloromercuribenzoate, D-glucose, D-glucosamine and D-mannose at concentrations of lx10^-3 to 1x10^-2M. The ∝-D-mannosidase hydrolyzed р-nitrophenyl ∝-D-mannoside (Km=5.6x10^-3M), methyl ∝-D-mannoside (Km=2.8X10^-2M), ∝-D-mannosyl-D-mannitol (Km=2.2X10^-2M), ∝-(l→2)linked D-mannobiosyl-D-mannitol (Km=6.3x10^-3M) and D-mannotriosyl-D-mannitol (Km=5.3x10^-3 M).     

Main physicochemical features of monofunctional flavokinase from Bacillus subtilis

The main properties of a monofunctional riboflavin kinase from B. subtilis have been studied for the first time; the enzyme is responsible for a key reaction in flavin biosynthesis—the ATP-dependent phosphorylation of riboflavin with production of flavin mononucleotide. The active form of the enzyme is a monomer with molecular weight of about 26 kD with a strict specificity for reduced riboflavin. To display its maximum activity, the enzyme needs ATP and Mg²⁺. During the phosphorylation of riboflavin, Mg²⁺ could be partially replaced by ions of other bivalent metals, the efficiencies of which decreased in the series Mg²⁺ > Mn²⁺ > Zn²⁺, whereas Co²⁺ and Ca²⁺ had inhibiting effects. The flavokinase activity was maximal at pH 8.5 and 52°C. ATP could be partially replaced by other triphosphates, their donor activity decreasing in the series: ATP > dATP > CTP > UTP. The Michaelis constants for riboflavin and ATP were 0.15 and 112 M, respectively. As compared to riboflavin, a tenfold excess of its analog 7,8-dimethyl-10-(O-methylacetoxime)-isoalloxazine decreased the enzyme activity by 30%. Other analogs of riboflavin failed to markedly affect the enzyme activity.