Nuclear phosphoprotein phosphatase from calf liver.

Calf liver nuclear phosphoprotein phosphatase (phosphoprotein phosphohydrolase, EC 3.1.3.16) has been purified approx. 850-fold. The enzyme has a mol. wt. of 34 000 as determined by SDS-polyacrylamide gel electrophoresis. The purified enzyme has a pH optimum between 7.0 and 7.5 with phosphophosphorylase, phosphohistones f1 and f2b, and phosphoprotamine as substrates. The enzyme activity towards these substrates follows the order, phosphophosphorylase greater than phosphohistone f1 greater than phosphohistone f2b greater than phosphoprotamine. The Km values toward phosphophospharylase and phosphohistone f1 are 17 and 28 micron phosphate, respectively. Dephosphorylated histone f1 and orthophosphate are competitive inhibitors of the enzyme with respective Ki values of 11 micron and 4.1 mM. NaCl and divalent metal ions inhibit the enzyme but CaCl2 is slightly stimulatory. It appears that metal ion inhibition occurs at two sites, one on the enzyme and the other on the substrate. The enzyme is also inhibited by NaF and EDTA. Nucleotides bearing the pyrophosphate structure are potent inhibitors of the enzyme while mononucleotides are slightly inhibitory. DNA and other polyions also inhibit the enzyme. The enzyme appears to require free sulfhydryl groups for activity since it is inhibited by N-ethylmaleimide and p-hydroxymercuribenzoate; the latter inhibition can be reversed by mercaptoethanol and dithiothreitol.     

Purification and characterization of chromatin-bound DNA-dependent RNA polymerase I from parsley (Petroselinum crispum). Influence of nucleoside triphosphates.

The isolation and purification of DNA-dependent RNA polymerase I (EC 2.7.7.6) from parsley (Petroselinum crispum) callus cells grown in suspension culture is described. The enzyme was solubilized from isolated chromatin. Purification was achieved by using DEAE- and phospho-cellulose in batches, followed by column chromatography on DEAE- and phospho-cellulose (two columns) and density-gradient centrifugation. The highly purified enzyme was stable over several months. The properties of purified parsley RNA polymerase I were investigated. Optimum concentration for Mn2+ was 1 mM, and for Mg2+ 4-6 mM, Mn2+ was slightly more stimulatory than Mg2+. The enzyme was most active at low ionic strengths [10-20 mM-(NH4)SO4]. The influence of various phosphates was tested: pyrophosphate inhibited RNA polymerase at low concentrations, whereas orthophosphate had no effect on the enzyme activity. ADP was slightly inhibitory, and AMP had no effect on the enzyme reaction. Nucleoside triphosphates and bivalent cations in equimolar concentrations in the range 4-11 mM did not influence the RNA synthesis in vitro. Free nucleoside triphosphates in excess of this 1:1 ratio inhibited the enzyme activity, unlike free bivalent cations, which stimulated RNA polymerase I.     

Metabolism of resorcinylic compounds by bacteria. Purification and properties of acetylpyruvate hydrolase from Pseudomonas putida 01.

Acetylpyruvate hydrolase, the terminal inducible enzyme of the pathway of orcinol catabolism in Pseudomonas putida, catalyzes the quantitative conversion of acetylpyruvate into acetate and pyruvate. The enzyme has been purified approximately 40-fold from extracts of Ps. putida grown on orcinol. Disc gel electrophoresis of the preparations show one major and one minor band of protein. The molecular weight of the enzyme is approximately 38,000 by sodium dodecyl sulfate electrophoresis. Acetylpyruvate is the only known substrate for the enzyme; maleylpyruvate, fumarylpyruvate, acetoacetate, oxalacetate, and acetylacetone are not hydrolyzed by acetylpyruvate hydrolase. Several divalent cations, includ-Mg2+, Mn2+, Co2+, Ca2+, and Zn2+, enhanced hydrolytic activity, but Cu2+ was inhibitory. The enzyme shows a sharp pH optimum at 7.4. Acetylpyruvate hydrolase has an apparent K-m of 0.1 mM for acetylpyruvate with a molecular activity of 36 min minus 1 at 25 degrees. Pyruvate, oxalacetate, and oxalate are competitive inhibitors of acetylpyruvate hydrolysis by the enzyme with K-i values of 6.0, 4.5, and 0.45 mM, respectively.     

Regulation of AMP deaminase from chicken erythrocytes. A kinetic study of the allosteric interactions.

The allosteric properties of AMP deaminase [EC 3.5.4.6] from chicken erythrocytes have been qualitatively and quantitatively accounted for by the concerted transition theory of Monod et al., on the assumption that this enzyme has different numbers of binding sites for each ligand. Theoretical curves yield a satisfactory fit for all experimental saturation functions with respect to activation by alkali metals and inhibition by Pi, assuming that the numbers of binding sites for AMP, alkali metals, and Pi are 4, 2, and 4, respectively. The enzyme was inhibited by concentrations of ATP and GTP below 0.1 and 0.25 mM, respectively, whereas activation of the enzyme was observed at ATP and GTP concentrations above 0.4 and 1.5 mM, respectively. These unusual kinetics with respect to ATP and GTP could be also accounted for by assuming 2 inhibitory and 4 activating sites for each ligand.     

Tyrosylprotein sulfotransferase in rat submandibular salivary glands.

1. The transfer of sulfate ester group from 3'-phosphoadenosine 5'-phosphosulfate (PAPS) to poly-(Glu6, Ala3, Tyr1) (EAY; Mr 47 kDa) in rat submandibular salivary gland has been investigated. The highest tyrosylprotein sulfotransferase activity was obtained in the Golgi-enriched fraction in the presence of 2 mM 5'AMP, 20 mM MnCl2 and 50 mM NaF at pH 6.2. 2. The apparent Km values for EAY and PAPS were 1.6 x 10(-6) and 1.9 x 10(-6) M, respectively. 3. Inclusion of NaCl, EDTA, NEM and DTT was inhibitory for the enzyme activity. The enzyme was 28 times less susceptible to 2,6-dichloro-4-nitrophenol inhibition than to phenol sulfotransferase inhibition. 4. This study is the first report characterizing a sulfotransferase activity specific for tyrosylprotein in rat submandibular salivary glands.     

Purification and some properties of uridine diphosphate N-acetylglucosamine pyrophosphorylase from Neurospora crassa.

Uridine diphosphate N-acetylglucosamine pyrophosphorylase (EC. 2.7.7.23) of Neurospora crassa has been purified approximately 210-fold with dithiothreitol as the stabilizing agent by use of chromatographic techniques. The enzyme preparation appeared to be homogeneous when subjected to electrophoresis. The molecular weight was estimated as approximately 37 000 by gel filtration. The enzyme had an isoelectric point around pH 4.4. Maximum activity of the enzyme was observed at pH 7.5. The enzyme required Mg2+, which may be replaced by other divalent cations such as Mn2+ and Co2+ for lesser degrees of effectiveness. The enzyme was strictly specific for UDP-N-acetylglucosamine as the substrate. The estimated values of Km were 2.2 mM for UDP-N-acetylglucosamine and 5.4 mM for inorganic pyrophosphate. The enzyme activity was highly stimulated by the addition of dithiothreitol or dithioerythritol but was lost by sulfhydryl inhibitory reagents.     

Purification and regulatory properties of pyruvate kinase from Veillonella parvula.

The nonglycolytic, anaerobic organism Veillonella parvula M4 has been shown to contain an active pyruvate kinase. The enzyme was purified 126-fold and was shown by disc-gel electrophoresis to contain only two faint contaminating bands. The purified enzyme had a pH optimum of 7.0 in the forward direction and exhibited sigmoidal kinetics at varying concentrations o-f phosphoenol pyruvate (PEP), adenosine 5'-monophosphate (AMP), and Mg-2+ ions with S0.5 values of 1.5, 2.0, and 2.4 mM, respectively. Substrate inhibition was observed above 4 m PEP. Hill plots gave slope values (n) of 4.4 (PEP), 2.8 (adenosine 5'-diphosphate), and 2.0 (Mg-2+), indicating a high degree of cooperativity. The enzyme was inhibited non-competitively by adenosine 5'-triphosphate (Ki = 3.4 mM), and this inhibition was only slightly affected by increasing concentration of Mg-2+ ions to 30 mM. Competitive inhibition was observed with 3-phosphoglycerate, malate, and 2,3-diphosphoglycerate but only at higher inhibitor concentrations. The enzyme was activated by glucose-6-phosphate (P), fructose-6-P, fructose-1,6-diphosphate (P2), dihydroxyacetone-P, and AMP; the Hill coefficients were 2.2, 1.8, 1.5, 2.1, and 2.0, respectively. The presence of each these metabolites caused substrate velocity curves to change from sigmoidal to hyperbolic curves, and each was accompanied by an increase in the maximum activity, e.g., AMP greater than fructose-1,6-P2 greater than dihydroxyacetone-P greater than glucose-6-P greater than fructose-6-P. The activation constants for fructose-1,6-P2, AMP, and glucose-6-P were 0.3, 1.1, and 5.3 mM, respectively. The effect of 5 mM fructose-1,6-P2 was significantly different from the other compounds in that this metabolite was inhibitory between 1.2 and 3 mM PEP. Above this concentration, fructose-1,6-P2 activated the enzyme and abolished substrate inhibition by PEP. The enzyme was not affected by glucose, glyceraldehyde-3-P, 2-phosphoglycerate, lactate, malate, fumerate, succinate, and cyclic AMP. The results suggest that the pyruvate kinase from V. parvula M4 plays a central role in the control of gluconeogenesis in this organism by regulating the concentration of PEP.     

Purification and properties of guanosine 5', 3'-polyphosphate synthetase from Bacillus brevis.

A ribosome-independent guanosine 5',3'-polyphosphate synthetase has been highly purified from Bacillus brevis (ATCC 8185). The enzyme has a molecular weight of 55,000, as measured by sucrose density gradient centrifugation. Like the ribosome-connected stringent factor of Escherichia coli, it catalyzes the synthesis of the guanosine 5', 3'-polyphosphates by a pyrophosphoryl transfer mechanism from adenosine triphosphate (ATP) to guanosine di- or triphosphates (GDP, GTP). It has an apparent Km of 0.14 mM for GDP and 0.77 mM for GTP, and is specific for the guanosine ribonucleotides as pyrophosphoryl acceptors. Several ATP analogues were tested for their ability to donate the pyrophosphoryl group. Mg2+ was required as a counter ion for the nucleotide substrate; however, an excess of Mg2+ was inhibitory. The property of the B. brevis enzyme is compared with the ribosome-linked enzyme of E. coli and an extracellular enzyme excreted by several types of Streptomyces reported upon recently.     

Purification of soluble guanylate cyclase from rat lung.

The soluble form of guanylate cyclase from rat lung has been purified approximately 23,000-fold to homogeneity by isoelectric precipitation, GTP-Sepharose chromatography, and preparative gel electrophoresis. A single protein-staining band is observed after analytical gel electrophoresis on either 4 or 7.5% polyacrylamide gels. The final purified enzyme has a specific activity of about 700 nmol of cyclic GMP formed/min/mg of protein at 37 degrees C in the presence of 4.8 mM MnCl2 and 100 micrometer GTP. Bovine serum albumin appears to slightly increase guanylate cyclase activity, but mainly stabilizes the purified enzyme; in its presence, specific activities in excess of 1 mumol of cyclic GMP formed/min/mg of enzyme protein can be obtained. When Mg2+ or Ca2+ are substituted for Mn2+, specific activities decrease to approximately 21 and 40 nmol of cyclic GMP formed/min/mg of protein, respectively. The apparent Michaelis constant for MnGTP in the presence of 4.8 mM MnCl2 is 10.2 micrometer. Kinetic patterns on double reciprocal plots as a function of free Mn2+ are concave downward. The native enzyme has a molecular weight of approximately 151,000 as determined on Sephacryl S-200; sodium dodecyl sulfate-polyacrylamide gel electrophoresis results in two protein-staining bands with approximate molecular weights of 79,400 and 74,000. Thus, it appears that the soluble form of guanylate cyclase from rat lung exists as a dimer.     

Staphylococcus aureus adenosine triphosphatase: inhibitor sensitivity and release from membrane.

Homogeneous preparations of cytoplasmic membrane isolated from Staphylococcus aureus 6538P exhibited membrane-associated adenosine triphosphatase (ATPase) activity. Membrane ATPase activity was activated by divalent cations (4.0 mM: Mg2+ greater than Mn2+ greater than Co2+ greater than Zn2+), and ATP was hydrolyzed more readily than other nucleoside triphosphates and phosphorylated substrates. The pH optimum for the membrane ATPase was 6.5. The ATPase could not be released from the membrane by differential osmotic treatments, but detergent treatment effectively solubilized active enzyme. The nonionic detergent Triton X-100 (1%) released a protein with ATPase activity, after substrate-dependent staining in polyacrylamide gels, that differed slightly in electrophoretic migration when compared to the active enzyme solubilized with sodium dodecyl sulfate (0.1%). Membrane-associated ATPase activity was inhibited by N,N'-dicyclohexylcarbodiimide (0.001 to 1 mM) and NaF (50% inhibition at 5 mM NaF). Azide and trypsin inhibited activity, whereas ouabain had a slight inhibitory effect. Diethylstilbestrol showed appreciable activation of the membrane ATPase over the range employed (0.001 to 1 mM).     

Polynucleotide kinase from rat-liver nuclei. Purification and properties.

A polynucleotide kinase, which catalyzes the phosphorylation of 5'-hydroxyl ends of deoxyribonucleic acid in the presence of adenosine triphosphate, has been purified 260-fold with a yield of 14% from 0.15 M NaCl extracts of rat liver nuclei. The purified enzyme has a pH optimum of 5.5. The enzyme is reversible inhibited by p-chloromercuribenzoate. The S0.5 value (ligand concentration required for a half-maximal activity) for ATP is 2.5 muM. A bivalent cation is essential for the reaction and S0.5 values for Mg2+, Ca2+ and Mn2+ are 3.3 mM, 4 mM and 0.05 mM respectively. Pyrophosphate remarkable inhibits the activity with I0.5 value (ligand concentration required for a half-maximal inhibition) of 0.2 mM, and sulfate, with I0.5 of 0.5 mM, whereas phosphate weakly inhibits the activity with I0.5 of about 20 mM. An apparent molecular weight of the purified enzyme is estimated to be 8 X 10(4) by gel filtration on a column of Sephadex G-150, and the Stokes radius of the enzyme molecule is shown to be about 0.36 nm. Sucrose density gradient centrifugation reveals that the enzyme has a sedimentation coefficient of about 4.4 S.     

Hydrolysis of 13-sophorosyloxydocosanoic acid esters by acetyl- and carboxylesterases isolated from Candida bororiensis.

Two enzymes have been isolated from Candida bogoriensis which catalyze the hydrolysis of 13-sophorosyloxydocosanoic acid (Glc2HDA) esters obtained from this organism. The 6',6"-diacetyl derivative of Glc2HDA (Ac2Glc2HDA) is hydrolyzed by an acetylesterase (EC 3.1.1.6) which has been purified 1300-fold. The acetylesterase has a molecular weight of 35,000 estimated from gel filtration, and is much more active with p-nitrophenyl acetate than with the acetylated glycolipid. The rate of hydrolysis increases with Ac2Glc2HDA concentration until a plateau is reached at a concentration of about 40 muM, near the critical micelle concentration of this glycolipid. These kinetic data are interpreted as an enzyme specificity for the monomeric, but not the micellar form of the glycolipid. The acetylesterase is inhibited by 0.1 to 10 mM diisopropyl fluorophosphate, 5 mM p-hydroxymercuribenzoate, and 5 mM N-ethylmaleimide, but only slightly by 5 mM iodoacetamide. The methyl ester of Ac2Glc2HDA is hydrolyzed by at least two carboxylesterases (EC 3.1.1) which differ in size according to gel filtration. Their molecular weights are estimated as 140,000 for carboxyesterase A and 40,000 for carboxyesterase B. Both carboxylesterases were purified over 20-fold, and carboxylesterase A was characterized further. Carboxylesterase A activity was inhibited completely by 0.1 to 10 mM diisopropyl fluorophosphate and by 10 mM p-hydroxymercuribenzoate, but only slightly by lower concentrations of p-hydroxymercuribenzoate or by N-ethylmaleimide or iodoacetamide. The carboxylesterase A preparation also acted as a thioesterase with palmityl-CoA (palmityl-CoA hydrolase, EC 3.1.2.2), showing the following approximate relative activities: palmityl-CoA, 100; octanoyl-CoA, 90; methyl Glc2HD, 22; butyryl-CoA, 18; methyl AcGlc2HD, 15; methyl Ac2Glc2HD, 10; and acetyl-CoA, O. Methyl Ac2Glc2HD showed some substrate inhibition at higher concentrations, but neither methyl Ac2Glc2HD nor palmityl-CoA approached enzyme saturation until well above their critical micelle concentrations, indicating hydrolysis of the micellar substrate was occurring. The carboxylesterase and palmityl-CoA hydrolase activities were destroyed in a parallel fashion by heat denaturation, and each substrate inhibited the action of the preparation on the other substrate, but the preparation has not been purified sufficiently to establish with certainty that both activities reside in the same protein.     

Some regulation profiles of ornithine transcarbamylase synthesis in vitro.

The regulation profiles of OTCase (argF, argI) synthesis in vitro were investigated by using the in vitro system described in the accompanying paper. Addition of 2.6 mM arginine, crude repressor and partially purified repressor to the in vitro system demonstrated that lambdadargF-DNA-directed OTCase-FFF synthesis is more sensitive to the repressor than lambdapargI-DNA-directed OTCase-III synthesis. The effects of some low-molecular substances on FFF and III syntheses were investigated; guanosine 3'-diphosphate 5'-diphosphate (ppGpp) stimulated both syntheses while cAMP and guanosine 5'-tetraphosphate (Gpppp) were not effective on III synthesis and were slightly inhibitory for FFF synthesis. The substances had no effect on the maturation of the enzyme or on the activity of the enzyme, FFF or III, synthesized. We suggest that argF- and argI-genes are regulated in a slightly different fashion and that the operator-promotor regions are not completely identical for these two genes.     

Purification and characterization of transfer RNA (guanine-1)methyltransferase from Escherichia coli

The tRNA modifying enzyme, tRNA (guanine-1)methyltransferase has been purified to near homogeneity from an overproducing Escherichia coli strain harboring a multicopy plasmid carrying the structural gene of the enzyme. The preparation gives a single major band on sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The enzyme is probably a single polypeptide chain of molecular weight 32,000. The amino acid composition is presented and the NH2-terminal amino acid sequence was established to be H2N-Met-Trp-Ile-Gly-Ile-Ile-Ser-Leu-Phe-Pro. The enzyme has a pI of 5.2. The tRNA (guanine-1)-methyltransferase has a pH optimum of 8.0-8.5, an apparent Km of 5 microM for S-adenosylmethionine. S-adenosylhomocysteine is a competitive inhibitor for the enzyme with an apparent Ki of 6 microM. Spermidine or putrescine are not required for activity, but they stimulate the rate of methylation 1.2-fold with optima at 2 and 6 mM, respectively. Ammonium ion is not required and is inhibitory at concentrations above 0.15 M. Magnesium ion inhibited the activity at a concentration as low as 2 mM. Sodium and potassium ions were inhibitory at concentrations above 0.1 M. The molecular activity of tRNA (guanine-1)-methyltransferase was calculated to 10.0 min-1. It was estimated that the enzyme is present at 80 molecules/genome in cells growing with a specific growth rate of 1.0.     

Inhibitory effects of ofloxacin and cefepime on enzyme activity of 6-phosphogluconate dehydrogenase from chicken liver

In this study, effects of some antibiotics, namely, ofloxacin, cefepime, cefazolin, and ampicillin on the in vitro enzyme activity of 6-phosphogluconate dehydrogenase have been investigated. For this purpose, 6-phosphogluconate dehydrogenase was purified from chicken liver 535-fold with a yield of 18% by using ammonium sulphate precipitation, 2',5'-ADP Sepharose 4B affinity chromatography, and Sephadex G-200 gel filtration chromatography. In order to check the purity of the enzyme, SDS polyacylamide gel electrophoresis (SDS-PAGE) was performed. This analysis revealed a highly pure enzyme band on the gel. Among the antibiotics, ofloxacin and cefepime exhibited inhibitory effects, but cefazolin and ampicillin showed neither important inhibitory nor activatory effects on the enzyme activity. The measured I(50) values by plotting activity percent vs. inhibitor concentration, [I(50)] were 0.1713 mM for ofloxacin and 6.0028 mM for cefepime. Inhibition constants, K(i), for ofloxacin and cefepime were also calculated as 0.2740 +/- 0.1080 mM and 12.869 +/- 16.6540 mM by means of Lineweaver-Burk graphs, and inhibition types of the antibiotics were found out to be non-competitive and competitive, respectively. It has been understood from the calculated inhibitory parameters that the purified chicken enzyme has been quite inhibited by these two antimicrobials.     

Purification and biochemical characterization of two soluble alpha-mannosidases from Candida albicans.

Two soluble alpha-mannosidases, E-I and E-II, were purified from C. albicans yeast cells by a three-step procedure consisting of size exclusion and ion exchange chromatographies in Sepharose CL6B and Mono Q columns, respectively, and preparative nondenaturing electrophoresis. E-I and E-II migrated as monomeric polypeptides of 54.3 and 93.3 kDa in SDS-PAGE, respectively. Some biochemical properties of purified enzymes were investigated by using 4-methylumbelliferyl-alpha-D-mannopyranoside and p-nitrophenyl-alpha-D-mannopyranoside as substrates. Hydrolysis of both substrates by either enzyme was optimum at pH 6.0 with 50 mM Mes-Tris buffer and at 42 degrees C. Apparent Kmvalues for hydrolysis of 4-methylumbelliferyl-alpha-D-mannopyranoside and p-nitrophenyl-alpha-D-mannopyranoside by E-I were 0.83 microM and 2. 4 mM, respectively. Corresponding values for E-II were 0.25 microM and 1.86 mM. Swansonine and deoxymannojirimicin strongly inhibited the hydrolysis of 4-methylumbelliferyl-alpha-D-mannopyranoside by both enzymes. On the contrary, hydrolysis of p-nitrophenyl-alpha-D-mannopyranoside by E-I and E-II was slightly stimulated or not affected, respectively, by both inhibitors. E-I and E-II did not depend on metal ions although activity of the latter was slightly stimulated by Mn2+and Ca2+in the range of 0.5-2 mM. At the same concentrations, Mg2+was slightly inhibitory of both enzymes. Substrate specificity experiments revealed that both E-I and E-II preferentially cleaved alpha-1,6 and alpha-1,3 linkages, respectively.     

Purification and properties of NAD-dependent glutamate dehydrogenase from Phycomyces spores

The NAD-dependent glutamate dehydrogenase from Phycomyces spores was purified more than 300-fold. Estimation of Mr by gel filtration gave a value of 98,000 whereas after SDS-PAGE one major band of Mr 54,000 was found, suggesting that the enzyme is a dimer. The enzyme was virtually dependent on the presence of AMP for activity and showed half-maximal activation at 9.5 and 43 microM-AMP in the direction of animation and deamination respectively. ADP was nearly as effective at 20-fold higher concentrations. Other nucleotide monophosphates were ineffective and nucleoside triphosphates were slightly inhibitory. Hyperbolic kinetics were found for all substrates yielding Km values of about 10 mM for ammonium, 1 mM for 2-oxoglutarate and 0.1 mM for NADH in the direction of amination, and 10 mM for glutamate and 0.7 mM for NAD in the direction of deamination.     

Polyadenylate polymerase from cytoplasm and nuclei of N.I.H.-Swiss mouse embryos.

Poly (A) polymerase activity from cytoplasm and nuclei of 12-16-day-old mouse embryos has been partially purified by (NH4)2SO4 fractionation, DEAE-cellulose, phosphocellulose and tRNA-Sepharose affinity chromatography, and their properties have been compared. The nuclear and cytoplasmic enzymes exhibit similar chromatographic elution profiles, and similar biochemical and physical properties. Poly(A) polymerase has an absolute requirement for a divalent cation, ATP and an oligo- or polyribonucleotide primer. With tRNA, the divalent salt concentrations for optimum enzyme activity are 1 mM MnCl2 or 10 mM MgCl2. The enzyme activity with MnCl2 is 10-15-fold higher than that with MgCl2. The molecular weight of the native enzyme is about 65 000 and its sedimentation coefficient is around 4.5 S. The average chain length synthesized by the enzyme is between 10 and 13 nucleotides. The inhibitors of RNA polymerase do not affect poly (A) polymerase activity; however, some synthetic rifamycin SV derivatives are potent inhibitors of this enzyme.     

Ribulose diphosphate carboxylase/oxygenase. III. Isolation and properties.

Similarities in properties of ribulose diphosphate carboxylase and oxygenase activities further substantiate the hypothesis that the same protein catalyzes both reactions. The Km (ribulose diphosphate) is 0.33 mM for the ribulose diphosphate oxygenase, when assayed in air with an oxygen electrode. Maximum activity is obtained with 10 to 35 mM MgCl2. Higher MgCl2 concentrations are inhibitory, but they shift the pH optimum from 9.3 or 9.4 to 8.7 or 9.0. MnCl2 is an effective cofactor of the oxygenase and some activity is obtained with CoCl2. Both the ribulose diphosphate carboxylase and oxygenase activity of the purified protein from spinach leaves are slowly inactivated by storage at 0 degrees and reactivated in 10 min at 50 degrees, provided both 25 mM MgCl2 and 1 mM dithiothreitol are present. The sulfhydryl groups of the enzyme which react rapidly with 5,5'-dithiobis(2-nitrobenzoic acid) are approximately 4 at pH 7.8 and 11 at pH 9.4. At both pH values ribulose diphosphate prevents two of these sulfhydryl groups from reacting with this reagent. About 50% inhibition of the oxygenase activity at pH 9.0 occurs with 50 mM bicarbonate in the presence of 3 mM ribulose diphosphate, and from variations in these parameters the inhibition is attributed to the CO2 species. The purified enzyme of acrylamide gels prevented the reduction of nitroblue tetrazolium in the presence of the superoxide radical, but the enzyme in solution did not react as a superoxide dismutase.     

Effects of some metals on paraoxonase activity from shark Scyliorhinus canicula

Paraoxonase (PON) is an organophosphate hydrolyser enzyme which also has antioxidant properties in metabolism. Due to its crucial functions, the inhibition of the enzyme is undesirable and very dangerous. PON enzyme activity should not be altered in any case. Inhibitory investigations of this enzyme are therefore important and useful. Metal toxicology of enzymes has become popular in the recent years. Here, we report the in vitro inhibitory effects of some metal ions, including Ni(2+), Cd(2+), Cu(2+) and Hg(2+), on the activity of shark serum PON (SPON). For this purpose, we first purified the enzyme from shark Scyliorhinus canicula (LINNAEUS, 1758) serum and analysed the alterations in the enzyme activity in the presence of metal ions. The K(M) and V(max) is 0.227 mM and 454.545 U/mL, respectively. The results show that metal ions exhibit inhibitory effects on SPON1 at low concentrations with IC(50) values ranging from 0.29 to 2.00 mM. Copper was determined to be the most effective inhibitor with IC(50) of 0.29 mM.