Effect of sodium nitrite inhibition on intracellular thiol groups and on the activity of certain glycolytic enzymes in Clostridium perfringens.

Activities of glyceraldehyde-3-phosphate dehydrogenase (EC 1.2.1.12) (GAP-DH) and aldolase (EC 4.1.2.13) in cells of Clostridium perfringens that had been inhibited with sodium nitrite were investigated. A complete loss in GAP-DH activity and a 67% decrease in aldolase activity were observed when growth of C. perfringens was inhibited. There was also a 91% decrease in the concentration of free sulfhydryl groups of soluble cellular components. Dithiothreitol restored some activity to inactive GAP-DH from sodium nitrite-inhibited cells, indicating that a loss of reduced sulfhydryl groups was involved in the inactivation of the enzyme. The evidence presented suggests that sodium nitrite inhibition of C. perfringens may involve an interaction of sodium nitrite as nitrous acid with sulfhydryl-containing constituents of the bacterial cell.     

Re-evaluation of the role of thiol groups in rabbit muscle aldolase A

Exposed thiol groups of rabbit muscle aldolase A were modified by 5,5'-dithiobis(2-nitrobenzoic) acid with concomittant loss of enzyme activity. When 5-thio-2-nitrobenzoate residues bound to enzyme SH groups were replaced by small and uncharged cyanide residues the enzyme activity was restored by more than 50%. The removal of a bulky C-terminal tyrosine residue from the active site of aldolase A resulted in enzyme which was inhibited by 5,5'-dithiobis(2-nitrobenzoic) acid only by 50% and its activity was nearly unchanged after modification of its thiol groups with cyanide. The results obtained show directly that rabbit muscle aldolase A does not possess functional cysteine residues and that the inactivation of the enzyme caused by sulfhydryl group modification reported previously can be attributed most likely to steric hindrance of a catalytic site by modifying agents.     

Localization of nitrite and sulfite reductase in bundle sheath and mesophyll cells of maize leaves

The distribution of nitrite reductase (EC 1.7.7.1) and sulfite reductase (EC 1.8.7.1) between mesophyll ceils and bundle sheath cells of maize ( Zea mays L. cv. Seneca 60) leaves was examined. This examination was complicated by the fact that both of these enzymes can reduce both NO-₂ and SO^2-₃ In crude extracts from whole leaves, nitrite reductase activity was 6 to 10 times higher than sulfite reductase activity. Heat treatment (10 min at 55°C) caused a 55% decrease in salfite reductase activity in extracts from bundle sheath cells and mesophyll cells, whereas the loss in nitrite reductase activity was 58 and 82% in bundle sheath cells and mesophyll cell extracts, respectively. This result was explained, together with results from the literature, by the hypothesis that sulfite reductase is present in both bundle sheath cells and mesophyll cells, and that nitrite reductase is restricted to the mesophyll cells. This hypothesis was tested i) by comparing the distribution of nitrite reductase activity and sulfite reductase activity between bundle sheath and mesophyll cells with the presence of the marker enzymes ribulose-l, 5-bisphosphate carboxylase (EC 4.1.1.39) and phosphoe-nolpyruvate carboxylase (EC 4.1.1.32), ii) by examining the effect of cultivation of maize plants in the dark without a nitrogen source on nitrite reductase activity and sulfite reductase activity in the two types of cells, and iii) by studying the action of S^2-on the two enzyme activities in extracts from bundle sheath and mesophyll cells. The results from these experiments are consistent with the above hypothesis.     

Effects of sodium nitroprusside and nitrite on ouabaine-sensitive Na+, K(+)-ATPase activity of myometrium smooth muscle

Effective inhibiting effect of sodium nitroprusside and nitrite on Na+, K(+)-ATPase enzymatic activity of miometrium sarcolemma fraction was shown. Seeming Ki was of micromolar and submicromolar magnitudes. Investigations with sodium nitroprusside demonstrated an uncompetitive inhibition for ATP (growth of affinity for ATP and decrease of maximal velocity) and mixed inhibition for cations (decrease of maximal velocity and activation of constant for K+). Inhibitory effect of ouabain was reduced in the presence of sodium nitroprusside; ditiothreitol prevented enzyme inactivation by sodium nitroprusside. Kinetic analysis of experimental results using ouabain and ditiothreitol suggests chemical modification of enzyme sulfhydryl groups. Resistant component of Na+, K(+)-ATPase activity, which is sensitive to the action of detergent digitonine, was observed. In comparative investigations with postnucleus fraction stimulating actions of sodium nitroprusside, sodium nitrite, cGMP (more enhance) were shown. Methylene blue (soluble guanilate-cyclase inhibitor) prevented the activation of Na+, K(+)-ATPase activity by sodium nitrite. We suppose that the way of enzyme activation is prevalent in the condition of the moderate formation of nitric oxide and in the absence of hyper(over)production of reactive oxygen species.     

CDP-diacylglycerol synthase activity in Clostridium perfringens

CTP:phosphatidate cytidylyltransferase (CDP-diacylglycerol synthase; EC 2.7.7.41) was identified in the cell envelope fraction of the gram-positive anaerobe Clostridium perfringens. The association of this enzyme with the cell envelope fraction of cell extracts was demonstrated by glycerol density gradient centrifugation and by activity sedimenting with the 100,000 x g pellet. The enzyme exhibited a broad pH optimum between pH 6.5 and pH 7.5. Enzyme activity was dependent on magnesium (5 mM) or manganese (1 mM) ions. Activity was also dependent on the addition of the nonionic detergent Triton X-100 (5 mM). The apparent Km values for CTP and phosphatidic acid were 0.18 mM and 0.22 mM, respectively. Thioreactive agents inhibited activity, indicating that a sulfhydryl group is essential for activity. Maximal enzyme activity was observed at 50 degrees C.     

CDP-diacylglycerol synthase activity in Clostridium perfringens.

CTP:phosphatidate cytidylyltransferase (CDP-diacylglycerol synthase; EC 2.7.7.41) was identified in the cell envelope fraction of the gram-positive anaerobe Clostridium perfringens. The association of this enzyme with the cell envelope fraction of cell extracts was demonstrated by glycerol density gradient centrifugation and by activity sedimenting with the 100,000 x g pellet. The enzyme exhibited a broad pH optimum between pH 6.5 and pH 7.5. Enzyme activity was dependent on magnesium (5 mM) or manganese (1 mM) ions. Activity was also dependent on the addition of the nonionic detergent Triton X-100 (5 mM). The apparent Km values for CTP and phosphatidic acid were 0.18 mM and 0.22 mM, respectively. Thioreactive agents inhibited activity, indicating that a sulfhydryl group is essential for activity. Maximal enzyme activity was observed at 50 degrees C.     

The effect of food deprivation on enzyme activity in developing brain

Brain and body weights, contents of DNA and protein and activities of 1,6-diphosphofructoaldolase (aldolase, EC 4.1.2.13), creatine phosphokinase (CPK, EC 2.7.3.2), and isocitric dehydrogenase (ICD, EC 1.1.1.42) in brain (minus cerebellum and brain stem) were studied in control and food-deprived rats at 7, 14 and 21 days of postnatal age. Activities of all three enzymes per brain were less in the food-deprived animals. In both groups of rats the ratios of aldolase/DNA and CPK/DNA increased with maturation, indicating that increasing activity per brain during maturation was the result of both increased activity per cell and increased numbers of cells. The ratio of ICD/DNA decreased with maturation but was essentially the same in both the food-deprived and control groups. Increase of ICD activity per brain with maturation was attributable to increased numbers of cells. Food deprivation in immature animals resulted in lowered activities per brain for aldolase, CPK and ICD because of diminished cell multiplication.     

Effect of oxidizing agents and sulfhydryl group reagents on beta toxin from Clostridium perfringens type C

Purified beta toxin from Clostridium perfringens type C was inactivated by the oxidizing agents o-iodosobenzoate (OIBA), oxidized glutathione, and ferricyanide, and by the sulfhydryl group regents 5,5'-dithio-bis(2-nitrobenzoic acid) (DTNB) and N-ethylmaleimide, iodoacetamide, and iodoacetic acid, causing loss of activity in various degrees depending on the concentration used. The activity of the toxin was not influenced by exposure to 1.0 mM of p-chloromercuribenzoate. The toxin treated by OIBA or DTNB was reactivated by incubation with 2-mercaptoethanol and dithiothreitol. The data suggest that beta toxin contains thiol groups which are essential for the activity.     

Essential sulfhydryl groups of rat liver monoamine oxidase.

The inhibition by some thiol reagents of partly purified mitochondrial monoamine oxidase (MAO) (EC 1.4.3.4) from rat liver was studied, and the molar content of sulfhydryl groups in the enzyme determined. Sodium nitroprusside and 5,5'-dithiobis(2-nitrobenzoic acid) (DTNB) inhibited the enzyme, apparently reversibly, while sodium arsenite was not inhibitory. Concentrations of the respective inhibitors causing 50% inhibition after 15 min of preincubation with the enzyme at pH 7.0 and 37 degrees C are 5.80 times 10(-4) M and 4.35 times 10(-5) M. The thiol compounds cysteine, dithiothreitol, and 2-mercaptoethanol did not inhibit MAO. The average number of sulfhydryl groups per mole of enzyme, determined by reaction with DTNB, increased from 3.6 +/- 0.2 freely reacting sulfhydryl groups (n = 4) to 18.4 to total sulfhydryl groups (n = 2) on denaturation with 8 M urea.     

The effects of platinum complexes on seven enzymes.

The effects of K2PtCl4, cis-Pt(NH3)2Cl2, and trans-Pt(NH3)2Cl2 on the activities of glyceraldehyde-3-phosphate dehydrogenase, glucose-6-phosphate dehydrogenase, dihydrofolate reductase, fructose-1,6-bisphosphate aldolase, catalase, tyrosinase, and peroxidase have been investigated. All of the enzymes which are thought to have essential sulfhydryl groups (glyceraldehyde-3-phosphate dehydrogenase, aldolase, and glucose-6-phosphate dehydrogenase) were significantly inhibited by K2PtCl4. The other four enzymes studied are not known to have essential sulfhydryl groups, and were not significantly affected by the Pt compounds under the conditions employed. Glyceraldehyde-3-phosphate dehydrogenase was the only enzyme inhibited by all three Pt compounds tested, with K2PtCl4 being the most effective and cis-Pt(NH3)2Cl2 the least effective inhibitor. Semilogarithmic plots of residual activity versus inhibition time indicated that the inhibition reactions were not simple first-order processes, except for the inhibition of glucose-6-phosphate dehydrogenase by K2PtCl4 which appeared to be first-order with respect to enzyme concentration.     

Sites of cleavage of rabbit muscle aldolase by purified cathepsin M from rabbit liver

Rabbit liver cathepsin M, a sulfhydryl proteinase similar in catalytic properties to cathepsin B, causes a decrease in the activity of rabbit muscle aldolase assayed with fructose 1,6-bisphosphate but not with fructose 1-phosphate. Proteolytic modification of aldolase by cathepsin M is limited to the removal of small peptides from the COOH-terminus, including the COOH-terminal hexapeptide NH2-Ile-Ser-Asn-His-Ala-TyrOH. Correlation of loss of aldolase activity with COOH-terminal modification indicates that only three of the four subunits of muscle aldolase contribute to the catalytic activity of the tetrameric enzyme.     

Influence of very low doses of ionizing radiation on Synechococcus lividus metabolism during the initial growth phase

Previous results from this laboratory have shown that very low chronic doses of gamma radiation can stimulate proliferation of the Cyanobacterium Synechococcus lividus. This modification of cell proliferation occurred during the first doubling. In this paper, we have compared the metabolism of cells cultivated in a normal environment or under chronic irradiation. Incubation of the cells in a new medium induced a high superoxide dismutase (EC 1.15.1.1, SOD) activity at the 18th hour and a degradation of phycocyanin, thus demonstrating that cells were submitted to a photooxidative stress. This increase in superoxide dismutase activity was followed by concomittant peaks of glutathione reductase (EC 1.6.4.2, GR) and glucose-6-phosphate dehydrogenase (EC 1.1.1.49, G6P-DH) at the 24th hour. Irradiated cultures at a dose of 53.5 mGray/year show an earlier and higher peak of SOD, GR, and G6P-DH. In a second stage, cultures showed an earlier onset of photosynthesis under irradiation, as evidenced by an increase in pigment content and an enhancement of glyceraldehyde-3-phosphate dehydrogenase (EC 1.2.1.13, GAP-DH). These results show that the radiostimulation is related to the activation of enzymes protecting against peroxides that were induced under oxidative circumstances and to the activation of a glucose catabolism via the oxidative pentose phosphate pathway.Abbreviations mGy milli-Gray - SOD superoxide dismutase - G6P-DH glucose-6-phosphate dehydrogenase - GAP-DH glycer-aldehyde-3-phosphate dehydrogenase - GSSG oxidized glutathione     

Dimerization occurs during the reversible acid inactivation of 2-oxo-4-hydroxyglutarate aldolase from Escherichia coli

Exposure of Escherichia coli 2-oxo-4-hydroxyglutarate aldolase (4-hydroxy-2-oxoglutarate glyoxylate-lyase, EC 4.1.3.16) (molecular weight = 63 000) to phosphoric acid at pH 1.6 for 10 min at 4 degrees C causes 95% or greater inactivation. No significant effect on the rate or extent of inactivation is caused by varied aldolase concentrations or the presence of exogenous proteins. Chloride ion (50-100 mM) or 10 mM 2-oxo-4-hydroxyglutarate markedly decreases both the rate and extent of inactivation; good protection is also afforded by 10 mM pyruvate, glyoxylate, glyoxal, 2-oxoglutarate or 2-oxobutyrate. Whereas native aldolase has two free and three buried sulfhydryl groups, all five are exposed in the acid-inactivated enzyme and the molecular weight of this species at pH 1.6 is 126 000. Ultraviolet absorbance difference spectra, circular dichroism spectra and ultracentrifugation studies establish that the inactivation process is characterized by an alteration of secondary and tertiary structure as well as an aggregation to a dimer of the native molecule. Reactivation of enzyme activity to 60-80% of the original level is seen within 20 min at pH 6 to 8; examination of inactivation/reactivation as a function of pH indicates that these two processes occur via kinetically distinct pathways. Native and reactivated enzymes are identical in molecular weight, sulfhydryl titer, Km and alpha-helix content.     

Bacterial inhibitory effects of nitrite: inhibition of active transport, but not of group translocation, and of intracellular enzymes

Nitrite inhibited active transport of proline in Escherichia coli but not group translocation of sugar via the phosphoenolpyruvate:phosphotransferase system. These results were consistent with previous results that nitrite inhibits active transport, oxygen uptake, and oxidative phosphorylation in aerobic bacteria. Nitrite also inhibited aldolase (EC 4.1.2.13) from E. coli, Pseudomonas aeruginosa, Streptococcus faecalis, and rabbit muscle. Thus, these various data showed that nitrite has more than one site of attack in the bacterial cell. These data also indicated that nitrite is inhibitory to a wide range of physiological types of bacteria.     

Bacterial inhibitory effects of nitrite: inhibition of active transport, but not of group translocation, and of intracellular enzymes.

Nitrite inhibited active transport of proline in Escherichia coli but not group translocation of sugar via the phosphoenolpyruvate:phosphotransferase system. These results were consistent with previous results that nitrite inhibits active transport, oxygen uptake, and oxidative phosphorylation in aerobic bacteria. Nitrite also inhibited aldolase (EC 4.1.2.13) from E. coli, Pseudomonas aeruginosa, Streptococcus faecalis, and rabbit muscle. Thus, these various data showed that nitrite has more than one site of attack in the bacterial cell. These data also indicated that nitrite is inhibitory to a wide range of physiological types of bacteria.     

Effects of various compounds on the ascorbate-2-sulfate sulfohydrolase and arylsulfatase activities copurified from the liver of Charonia lampas.

The effects of various compounds on ascorbate-2-sulfate sulfohydrolase and arylsulfatase (EC 3.1.6.1) activities in the copurified preparation from the liver of Charonia lampas were investigated. The former activity was competively inhibited by inorganic phosphate and sulfate. The latter was not affected by sulfate. Higher concentrations of sodium chloride inhibited the former and activated the latter. Neither of the activities was inhibited by sulfhydryl reagents. Both activities were competively inhibited by the other substrate.     

High-level production and purification of Escherichia coli N-acetylneuraminic acid aldolase (EC 4.1.3.3).

The Escherichia coli gene which encodes N-acetylneuraminic acid aldolase was isolated by the polymerase chain reaction, cloned into the inducible expression vector pTTQ18, and overexpressed in E. coli. The high yield of aldolase was achieved through both optimum growth of cells and efficient expression of the aldolase gene (20-30% soluble cellular protein). The recombinant enzyme was purified to homogeneity with an activity of 1.2-2.2 U/mg, which compared favorably with that of commercial preparations of E. coli aldolase (1.1 U/mg) and Clostridium perfringens aldolase (0.4 U/mg). The cloning strategy, fermentation conditions, purification protocol, and activity assay are described.     

Studies on (Na+ + K+)-activated ATPase. XLII. Evidence for two classes of essential sulfhydryl groups.

1. Preincubation of purified (Na+ + K+)-ATPase (ATP phosphohydrolase, EC 3.6.1.3) preparations from rabbit kidney outer medulla with 5,5'-dithiobis-(2-nitrobenzoic acid) inhibits the (Na+ + 5+)-ATPase and K+-stimulated 4-nitro-phenylphosphatase activities. Phosphorylation of the enzyme by ATP and the Na+-stimulated ATPase activity are inhibited to the same extent as the (Na+ + K+)-ATPase activity, whereas the K+-stimulated 4-nitrophenylphosphatase activity is inhibited much less. 2. Titration with 5,5'-dithiobis-(2-nitrobenzoic acid) in sodium dodecyl sulphate shows the presence of 36 reactive sulfhydryl groups per molecule (Na+ + K+)-ATPase (Mr = 250 000). 3. Treatment with N-ethylmaleimide, resulting in complete inhibition of (Na+ + K+)-ATPase activity, leads to modification of 26 sulfhydryl groups, whereas treatment with 5,5'-dithiobis-(2-nitrobenzoic acid) results in modification of 12 sulfhydryl groups under the same conditions. 4. The reaction of N-ethylmaleimide with an essential SH-group is not prevented by previous blocking of sulfhydryl groups with 5,5'-dithiobis-(2-nitrobenzoic acid). 5. These findings indicate the existence of at least two classes of sulfhydryl groups on the enzyme, each containing at least one vital group. The difference between these classes consists in their different reactivity towards 5,5'-dithiobis-(2-nitrobenzoic acid) and N-ethylmaleimide.     

Purification and properties of nitrite reductase from Escherichia coli K12.

NADH-nitrite oxidoreductase (EC 1.6.4) was purified to better than 95% homogeneity from batch cultures of Escherichia coli strain OR75Ch15, which is partially constitutive for nitrite reductase synthesis. Yields of purified enzyme were low, mainly because of a large loss of activity during chromatography on DEAE-cellulose. The quantitative separation of cytochrome c-552 from nitrite reductase activity resulted in an increase in the specific activity of the enzyme: this cytochrome is not therefore an integral part of nitrite reductase. The subunit molecular weights of nitrite reductase and of a haemoprotein contaminant, as determined by sodium dodecyl sulphate/polyacrylamide-gel electrophoresis, were 88000 and 80000 respectively. The sedimentation coefficient was calculated to be in the range 8.5-9.5S, consistent with a mol.wt. of 190000. It is suggested therefore that the native enzyme is a dimer with two identical or similar-sized subunits. Purest samples contained 0.4 mol of flavin/mol of enzyme, but no detectable haem. Catalytic activity was totally inhibited by 20 micron-p-chloromercuribenzoate and 1 mM-cyanide, slightly inhibited by 1 micron-sulphite and 10mM-arsenite, but insensitive to 1 mM-2,2'-bipyridine, 4mM-1,10-phenanthroline and 10mM-NaN3. Three molecules of NADH were oxidized for each NO2-ion reduced: the product of the reaction is therefore assumed to be NH4+. The specific activity of hydroxylamine reductase increased at each step in the purification of nitrite reductase, and the elution profiles for these two activities during chromatography on DEAE-Sephadex were coincident. It is likely that a single enzyme is responsible for both activities.     

The interaction of FBPase with aldolase: a kinetic and fluorescence investigation on chicken muscle enzymes

Fructose-1,6-bisphosphatase (FBPase; EC 3.1.3.11) is strongly inhibited by AMP in vitro and, therefore, at physiological concentrations of substrate and AMP, FBPase should be completely inhibited. Desensitization of rabbit muscle FBPase against AMP inhibition was previously observed in the presence of rabbit muscle aldolase. In this study, we analysed the kinetics of an FBPase catalyzed reaction and interaction between chicken muscle FBPase and chicken muscle aldolase. The initial rate of FBPase reaction vs. substrate concentration shows a maximum activity at a concentration of 20 microM Fru-1,6P2 and then decreases. Assuming rapid equilibrium kinetics, the enzyme-catalyzed reaction was described by the substrate inhibition model, with Ks approximately 5 microM and Ksi approximately 39 microM and factor beta approximately 0.2, describing change in the rate constant (k) of product formation from the ES and ESSi complexes. Based on ultracentrifugation studies, aldolase and FBPase form a hetero-complex with approximately 1:1 stoichiometry with a dissociation constant (Kd) of 3.8 microM. The FBPase-aldolase interaction was confirmed via fluorescence investigation. The aldolase-FBPase interaction results in aldolase fluorescence quenching and its maximum emission spectrum shifting from 344 to 356 nm. The Kd of the FBPase-aldolase complex, determined on the basis of fluorescence changes, is 0.4 microM at 25 degrees C with almost 1:1 stoichiometry. This interaction increases the I(0.5) for the AMP inhibition of FBPase threefold, and slightly affects FBPase affinity to magnesium ions, increasing the Ka and Hill coefficient (n). No effect of aldolase on the FBPase pH optimum was observed. Thus, the decrease in FBPase sensitivity to AMP inhibition enables FBPase to function in vivo thanks to aldolase.