Catalytic properties of three lactate dehydrogenases from potato tubers (Solanum tuberosum)

Two l-lactate dehydrogenase isoenzymes and one dl-lactate dehydrogenase could be separated from potato tubers by polyacrylamide-gel electrophoresis. The enzymes are specific for lactate, while β-hydroxybutyric acid, glycolic acid, and glyoxylic acid are not oxidized. Their pH optima are pH 6.9 for the oxidation and 8.0 for the reduction reaction.The K_m values for l-lactate for the two isoenzymes are 2.00 × 10^?2 and 1.82 × 10^?2, m. In the reverse reaction the affinities for pyruvate are 3.24 × 10^?4 and 3.34 × 10^?4, m. Both enzymes have similar affinities for NAD and NADH (3.00 × 10^?4; 4.00 × 10^?4, and 8.35 × 10^?4; 5.25 × 10^?4, m).The dl-lactate oxidoreductase may transfer electrons either to NAD or N-methyl-phenazinemethosulfate. The K_m values of this enzyme for l-lactate are 4.5 × 10^?2, m and for d-lactate 3.34 × 10^?2, m. Its affinity for pyruvate is 4.75 × 10^?4, m. The enzyme is inhibited by excess NAD (K_m = 1.54 × 10^?4, M) and has an affinity toward NADH (K_m = 5.00 × 10^?3, M) which is about one tenth of that of the two isoenzymes of l-lactate dehydrogenase.     

Identity of aliphatic L- -hydroxyacid oxidase and glycolate oxidase from rat livers

l-α-Hydroxyacid oxidase and glycolate oxidase have been partially purified from rat livers and found to be identical, judging by substrate specificities, K_m values for certain substrates and coenzyme (FMN), activation energy, inhibition rates by various reagents and pH optimum. K_m values are as follows; glycolate, 2.4 × 10^?4m; l-α-hydroxyisocaproate, 1.26 × 10^?3; glyoxylate, 1.41 × 10^?4m; and FMN, 1.13 × 10^?6m. K_m values for glycolate and FMN are one-tenth and one-twentieth the literature values for hepatic glycolate oxidase. Sucrose density gradient centrifugation establishes that this enzyme is located in hepatic peroxisomes.     

Uridine-cytidine kinase. Kinetic studies and reaction mechanism.

The initial velocity pattern has been determined for uridine-cytidine kinase purified from the murine mast cell neoplasm P815. With either uridine or cytidine as phosphate acceptor, and ATP as phosphate donor, the pattern observed was one of intersecting lines, ruling out a ping-pong reaction mechanism, and suggesting that the reaction probably proceeds by the sequential addition of both substrates to the enzyme to form a ternary complex, followed by the sequential release of the two products. This pattern was obtained whether the reaction was run in 0.01 m potassium phosphate buffer, pH 7.5, or in 0.1 m Tris-HCl, pH 7.2. When analyzed by the Sequen computer program, the data indicated an apparent K_m of the enzyme for uridine of 1.5 × 10^?4m, an apparent K_m for cytidine of 4.5 × 10^?5m, and a K_m for ATP, with uridine or cytidine as phosphate acceptor, of 3.6 × 10^?3m or 2.1 × 10^?3m, respectively. The V was 1.83 μmol phosphorylated/min/mg enzyme protein for the uridine kinase reaction and 0.91 μmol for the cytidine kinase reaction.     

S-adenosylmethionine: Protein-carboxyl methyltransferase from erythrocyte

Protein methylase II (S-adenosylmethionine:protein—carboxyl methyltrans-ferase), which modifies free carboxyl residues of protein, was purified from both rat and human blood, and properties of the enzymes were studied. The pH optima for the reaction were dependent on the substrate proteins used; pH 7.0 was found with endogenous substrate, 6.1 with plasma, 6.5 with γ-globulin, and 6.0 with fibrinogen. The molecular weight of the enzymes from both rat and human erythrocytes were identical (25,000 daltons) determined by Sephadex G-75 chromatography. Partially purified enzyme from rat erythrocytes showed three peaks on electrofocusing column at pH 4.9, 5.5 and 6.0. The K_m values of the enzymes from rat and human erythrocytes showed 3.1 × 10^?6m and 1.92 × 10^?6m at pH 6.0, 1.96 × 10^?6m and 1.78 × 10^?6m at pH 7.2, respectively, for S-adenosyl-l-methionine. It is also found that S-adenosyl-l-homocysteine is a competitive inhibitor for protein methylase II with K_i value of 1.6 × 10^?6m.     

Phosphatidohydrolase and base-exchange activity of commercial phospholipase D

Base-exchange activity was contrasted to the usual phosphatidohydrolase activity of commercial phospholipase D preparation from cabbage. The former activity was assayed by measuring the incorporation of labeled ethanolamine and choline into phospholipids. The latter activity was assayed by measuring the formation of phosphatidic acid with radioactive phosphatidylcholine microdispersion as substrate. The pH optimum for the base-exchange activity was about 9.0, whereas the phosphatidohydrolase activity had a pH optimum around 5.6. The incorporation of ethanolamine and choline into phospholipid was dependent upon the amount of acceptor asolectin microdispersion present. The optimum concentration of Ca²⁺ in the base-exchange reaction was about 4 mm, whereas the optimum concentration for the phosphatidohydrolase activity was greater than 28 mm. The incorporation of ethanolamine into phospholipid was decreased 50% by heating the enzyme preparation at 50°C for about 10 min, whereas the choline incorporation decreased approximately 20% and the phosphatidohydrolase activity decreased by about 10% under these conditions.Hemicholinium-3 was found to be a noncompetitive inhibitor for the incorporation of both ethanolamine and choline into phospholipid with respective K_i, values of 1.25 × 10^?3 and 2.50 × 10^?3m. The K_m values for ethanolamine and choline in the base-exchange reaction were 1.25 × 10^?3 and 2.50 × 10^?3m, respectively. The apparent K_m for phosphatidylcholine for the phosphatidohydrolase activity was about 1.5 × 10^?3m, and there was no inhibition by hemicholinium-3.     

The hydrolysis of 3-(2-furylacryloyl)-glycyl-l-leucine amide by thermolysin

The kinetics of the hydrolysis of 3-(2-furylacryloyl)-glycycl-l-leucine amide by thermolysin has been reinvestigated. It was found that the K_m for the enzyme substrate interaction is 2.5 × 10^?3m at pH 7.2. This K_m is an order of magnitude less than what has been previously assumed to be the K_m for the enzyme-substrate interaction. The normally recommended assay has 1–3 × 10^?3m substrate and is based on the assumption that the substrate concentration is much less than the K_m. Our data indicate that this assumption appears to be invalid. The hydrolysis of 3-(2-furylacryloyl)-glycyl-l-leucine amide results in a maximum decrease in absorbance at 322 nm. The change in absorbance is nearly 10-fold greater at 322 nm than the change in absorbance at 345 nm where the hydrolysis has been customarily followed. By following the hydrolysis of the substrate at 10^?4m at 322 nm it is possible to work under conditions where the substrate concentration is much less than the K_m.     

Arsenazo I and tetramethylmurexide as optical calcium indicators

Calcium-binding stoichiometry, dissociation equilibrium constants at zero ionic strength (K⁰), and molar extinction difference coefficients (Δ?_λ) at the wavelength λ of the metallochromic indicators arsenazo I (ArsI) and tetramethylmurexide (TMX) were reevaluated with a computerized method based on mass conservation and thermodynamic consistency checks. This new method is shown to provide a more critical assessment of the assumed calcium-dye complexing model than is afforded by the commonly used reciprocal-plot method. The analyses of spectrophotometric Ca titrations confirm that both dyes form only 1:1 complexes in aqueous solution. For TMX, K⁰ = 1.3 × 10^?3m and Δ?₄₈₀ = 1.5 × 10⁴m^?1 cm^?1; for ArsI, K⁰ = 5.8 × 10^?3m and Δ?₅₆₂ = 1.8 × 10⁴m^?1 cm^?1 at pH 7.0 and T = 293°K. The discriminatory power of the analytical method is demonstrated by comparison of these results with those found for a different dye, arsenazo III, which complexes Ca in 1:1, 1:2, and 2:1 forms.     

Soybean alcohol dehydrogenase

Alcohol dehydrogenase was prepared from germinating soybean seeds. Specific activity was increased from 511 to 31316 units. The coenzyme is NAD with a K_m of 10^?4M. Allyl alcohol is oxidized faster than ethanol; with the latter substrate, the K_m is 1.3 × 10^?2M, and the pH optimum 8.7. The enzyme catalyses acetaldehyde reduction, with a K_m of 10^?2M and a pH opt of 7.1. The MW is 53(±5) × 10^?3.     

Esteroproteolytic enzymes from the submaxillary gland. Kinetics and other physicochemical properties.

Two esteroproteolytic enzymes (A and D) have been isolated from the mouse submaxillary gland and shown to be pure by ultracentrifugation, immunoelectrophoresis, acrylamide-gel electrophoresis, and amino acid analyses. The enzymes have molecular weights of approximately 30,000 and are structurally and antigenically related. Narrow pH optima between 7.5 and 8.0 are exhibited by both enzymes. The “pK₁'s” are between 6.0 and 6.5 and the “pK₂'s” are near 9.0. A marked preference for arginine-containing esters is shown by both enzymes. The maximum specific activity of enzyme A on p-tosylarginine methyl ester (TAME) at pH 8 was 2500–3000 μm min^?1 mg^?1 and for enzyme D, 400–600 μm min^?1 mg^?1. With TAME as substrate, the K_m for enzyme A was 8 × 10^?4m at 25 °C and 6 × 10^?4m at 37 °C. For D, K_m was 3 × 10^?4 at 25 °C and 2 × 10^?4m at 37 °C.An apparent activation of enzyme D by tosylarginine (TA), a product of TAME hydrolysis, and all α-amino acids examined was due to removal of an inhibitor by chelation. This effect could be duplicated by 8-hydroxyquinoline and diethyldithiocarbamate but not by EDTA. Enzyme A was not affected by these substances to any remarkable extent. Several divalent ions proved to be potent inhibitors of enzyme D. Both enzymes are inactivated by the active site reagents diisopropyl phosphofluoridate and tosyllysine chloromethylketone but much less rapidly than is trypsin. Nitrophenyl-4-guanidionobenzoate reacts with a burst of nitrophenol liberation but with a rapid continuing hydrolysis. One active site per molecule is indicated. Enzyme D is inactivated by urea, reversibly at 10 m and with maximal permanent losses at 6 m. Autolysis of the unfolded form by the native enzyme when they coexist at intermediate urea concentrations appears to occur.Identity of enzyme D and the epithelial growth factor binding protein is demonstrated.     

Mechanism of action of thrombin on fibrinogen. Reaction of the N-terminal CNBr fragment from the Aalpha chain of human fibrinogen with bovine thrombin

The 51-residue N-terminal cyanogen bromide fragment from the Aα chain of human fibrinogen was isolated, and the Michaelis-Menten constants, K_m and k_cat, for its hydrolysis by bovine thrombin were determined. The measured values of K_m and k_cat are 4.7 × 10^?5m and 4.8 × 10^?10m [(NIH U/liter) sec]^?1, respectively. Since these values are similar to those for fibrinogen, it appears that the N-terminal CNBr fragment contains all amino acid residues whose interactions with thrombin account for the high specificity of this enzyme for fibrinogen.     

The interaction of substrate-related ketals with bacterial and viral neuraminidases

Arthrobacter sialophilus neuraminidase catalyzes the hydration of 5-acetamido-2,6-anhydro-3, 5-dideoxy-d-glycero-d-galacto-non-2-enonic acid (2,3-dehydro-AcNeu) with K_m and k_cat values of 8.9 × 10^?4m and 6.40 × 10^?4 s^?1, respectively. The methyl ester of 2,3-dehydro-AcNeu as well as 2,3-dehydro-4-epi-AcNeu are also hydrated by the enzyme. The product resulting from the enzymatic hydration of 2,3-dehydro-AcNeu is N-acetylneuraminic acid. A series of derivatives of 2,3-dehydro-AcNeu (K_I 1.60 × 10^?6m) including 2,3-dehydro-4-epi-AcNeu (2.10 × 10^?4m) and 2,3-dehydro-4-keto-AcNeu (K_I = 6.10 × 10^?5 m) were each competitive inhibitors of the enzyme. The methyl esters of these ketal derivatives were also competitive enzyme inhibitors. Dissociation constants for these ketals were determined independently by fluorescence enzyme titrations which gave values similar to those found kinetically. These six relatives of 2,3-dehydro-AcNeu were also competitive inhibitors for the influenza viral neuraminidases. For the viral neuraminidases, the dissociation constant for 2,3-dehydro-AcNeu and its methyl ester were 2.40 × 10^?6 and 1.17 × 10^?3m, respectively. The interpretation placed upon the K_I values determined for these ketals against the Arthrobacter versus influenza neuraminidases is that the bacterial enzyme has a more flexible glycone binding site.     

Purification and properties of UDP-glucose: coniferyl alcohol glucosyltransferase from suspension culturesof Paul's scarlet rose.

UDP-glucose:coniferyl alcohol glucosyltransferase was isolated from 10-day-old, darkgrown cell suspension cultures of Paul's scarlet rose. The enzyme was purified 120-fold by (NH₄)₂SO₄ fractionation and chromatography on DEAE-cellulose, hydroxyapatite, and Sephadex G-100. The enzyme has a pH optimum of 7.5 in Tris-HCl buffer, required an -SH group for activity, and is inhibited by ?-chloromercuribenzoate and EDTA. Its molecular weight is estimated to be 52,000. The enzyme is specific for the glucosylation of coniferyl alcohol (K_m 3.3 × 10^?6 M) and sinapyl alcohol (K_m 5.6 × 10^?6 M). With coniferyl alcohol as substrate the apparent K_m value for UDP-glucose is 2 × 10^?6m. The enzyme activity can be detected in a number of callus-tissue and cell-suspension cultures. The role of this enzyme is believed to be to catalyze the transfer of glucose from UDPG to coniferyl (or sinapyl) alcohol as storage intermediates in lignin biosynthesis.     

Purification and properties of a D-fructose 1,6-bisphosphatase from Saccharomyces cerevisiae.

Fructose 1,6-bisphosphatase (EC 3.1.3.11) from Saccharomyces cerevisiae has been purified to homogeneity. A molecular weight of 115,000 has been obtained by gel filtration. The enzyme appears to be a dimer with identical subunits. The apparent K_m for fructose bisphosphatase varies with the Mg²⁺ concentration of the enzyme, being 1 × 10^?6m at 10 mm Mg²⁺ and 1 × 10^?5m at 2 mm Mg²⁺. Other phosphorylated compounds are not significantly hydrolyzed by the enzyme. An optimum pH of 8.0 is exhibited by the enzyme. This optimum is not changed by addition of EDTA. AMP inhibits the enzyme with a K_i of 8.0 × 10^?5m at 25 °C. The inhibition is temperature dependent, the value of K_i increasing with raising temperature. 2-Deoxy-AMP is also inhibitory with a K_i value at 25 °C of 1.6 × 10^?4m. An ordered uni-bi mechanism has been deduced for the reaction with phosphate leaving the enzyme as the first product and the fructose 6-phosphate as the second one.     

Purifications and properties of L-mandelate- 4-hydroxylase from Pseudomonas convexa.

An inducible l-mandelate-4-hydroxylase has been partially purified from crude extracts of Pseudomonas convexa. This enzyme catalyzed the hydroxylation of l-mandelic acid to 4-hydroxymandelic acid. It required tetrahydropteridine, NADPH, Fe²⁺, and O₂ for its activity. The approximate molecular weight of the enzyme was assessed as 91,000 by gel filtration on Sephadex G-150. The enzyme was optimally active at pH 5.4 and 38 °C. A classical Michaelis-Menten kinetic pattern was observed with l-mandelate, NADPH, and ferrous sulfate and K_m values for these substrates were found to be 1 × 10^?4, 1.9 × 10^?4, and 4.7 × 10^?5m, respectively. The enzyme is very specific for l-mandelate as substrate. Thiol inhibitors inhibited the enzyme reaction, indicating that the sulfhydryl groups may be essential for the enzyme action. Treatment of the partially purified enzyme with denaturing agents inactivated the enzyme.     

The Interaction of Chromium(VI) with Urease in Solution

The interaction between K₂Cr₂O₇ and urease was investigated using fluorescence, UV-vis absorption, and circular dichroism (CD) spectroscopy. The experimental results showed that the fluorescence quenching of urease by K₂Cr₂O₇ was a result of the formation of K₂Cr₂O₇–urease complex. The apparent binding constant K _A between K₂Cr₂O₇ and urease at 295, 302, and 309 K were obtained to be 2.14?×?10⁴, 1.96?×?10⁴, and 1.92?×?10⁴ L mol^?1, respectively. The thermodynamic parameters, ΔH° and ΔS° were estimated to be ?5.90 kJ mol^?1, 43.67 J mol^?1 K^?1 according to the Van’t Hoff equation. The electrostatic interaction played a major role in stabilizing the complex. The distance r between donor (urease) and acceptor (K₂Cr₂O₇) was 5.08 nm. The effect of K₂Cr₂O₇ on the conformation of urease was analyzed using UV-vis absorption, CD, synchronous fluorescence spectroscopy, and three-dimensional fluorescence spectra, the environment around Trp and Tyr residues were altered.     

Purification and properties of agmatine iminohydrolase from groundnut cotyledons

Agmatine iminohydrolase was purified ca 375-fold from groundnut cotyledons. The enzyme exhibited an optimum pH between 5.5 and 8.5 and the energy of activation was 22 kcal/mol. The K_m for agmatine was (7.57 ± 0.77) × 10^?4 M. The enzyme was inhibited by tryptamine, putrescine, cadaverine, spermidine and spermine. Inhibition by cadaverine and spermidine was competitive. The K_i values for cadaverine and spermidine were 4.1 × 10^?3 and 7.5 × 10^?4 M, respectively.     

Properties of phenylalanine ammonia-lyase extracted from Cucumis sativus hypocotyls

Some of the in vitro properties of PAL from gherkin hypocotyls were investigated. No metal ion requirement for this enzyme could be demonstrated but there were indications that PAL was a sulphydryl enzyme. Kinetic analysis suggested that PAL exhibited negative homotropic cooperativity. Two K_m values were determined, these were K_m^H, 2·9 × 10^?4 M and K_m^L, 4·3 × 10^?5 M. Strong inhibition of the enzyme was exerted by d-phenylalanine, trans-cinnamic acid, o-coumaric acid, gallic acid, quercetin and kaempferol. Kinetic studies on the inhibition patterns of these compounds established that d-phenylalanine linearized the curvilinear kinetics, trans-cinnamic acid and o-coumaric acid acted as competitive inhibitors whilst gallic acid, quercetin and kaempferol acted as mixed inhibitors. Using a number of desensitization techniques PAL was partially desensitized to inhibition by the mixed inhibitors. These results led to the conclusion that PAL may have a regulatory role in phenol, coumarin and flavonoid biosynthesis.     

Lectin-carbohydrate interactions studied by a competitive enzyme inhibition assay

The principle of competitive binding assay in combination with an immobilized lectin (concanavalin A), in close proximity to an oxygen sensor, has been used to quantify carbohydrates and to determine association constants for lectin-carbohydrate interactions. Methyl α-d-mannopyranoside was determined down to 0.5 μg/ml. K_a (maltose) and K_a (maltotriose) was found to be 2.1 × 10³ and 1.7 × 10³m^?1, respectively, which are comparable to values quoted in the literature of approximately 2.8 × 10³m^?1 for both maltose and maltotriose. Furthermore, the estimation of the bonus effect, due to multipoint attachment, for a low-molecular-weight dextran is discussed.     

Evaluation of Bacterial Community Structure and Its Influence on Sulfide Oxidation in a Bio-Leaching Environment

The mechanism of sulfide oxidation by adhering bacteria (direct oxidation mechanism) and by ferric ion in the aqueous phase was studied by quantitative assessment of bacterial activity on the sulfide surface. To probe for the principal bacterial species on the surface and in the supernatant, a library of DNA genes encoding portions of bacterial 16S rRNA was constructed. The PCR-amplified DNA from the bacterial populations was cloned employing PROMEGA's pGEM-T Easy Vector system. The clone frequency indicated that iron-oxidizing bacteria were dominant in the liquid phase, while Acidithiobacillus ferroixdans, which is both sulfur and iron oxidizer was the most prevalent on the sulfide surface. Samples of crystalline pyrite were exposed to the bacterial consortium to evaluate surface alterations caused by bacteria. Chemical (abiotic) oxidation experiments with ferric ion as the oxidant were carried out in parallel with the biological oxidation tests. Changes in the surface topography were monitored by atomic force microscopy (AFM) while changes in surface chemistry were examined by Raman spectroscopy. Bacterial attachment resulted in a 53% increase in the specific surface area in comparison to a 13% increase caused by chemical (ferric ion) oxidation. The oxidation rate was assessed by evaluating the iron release. After corrections for surface area changes, the specific abiotic (oxidation by Fe^{3 +}) and biotic oxidation rates with adhering bacteria were nearly the same (2.6 × 10^{? 9} mol O₂/s/m² versus 3.3 × 10^{? 9} mol O₂/s/m²) at pH = 2 and a temperature of 25°C. The equality of rates implies that the availability of ferric ion as the oxidant is rate limiting.     

Analysis of copper corrosion in compacted bentonite clay as a function of clay density and growth conditions for sulfate‐reducing bacteria

Aims: To investigate the relationships between sulfate‐reducing bacteria (SRB), growth conditions, bentonite densities and copper sulfide generation under circumstances relevant to underground, high‐level radioactive waste repositories. Methods and Results: Experiments took place 450 m underground, connected under in situ pressure to groundwater containing SRB. The microbial reduction of sulfate to sulfide and subsequent corrosion of copper test plates buried in compacted bentonite were analysed using radioactive sulfur (³⁵SO₄^2?) as tracer. Mass distribution of copper sulfide on the plates indicated a diffusive process. The relationship between average diffusion coefficients (D_s) and tested density (ρ) was linear. D_s (m² s^?1) = ?0·004 × ρ (kg m^?3) + 8·2, decreasing by 0·2 D_s units per 50 kg m^?3 increase in density, from 1·2 × 10^?11 m² s^?1 at 1750 kg m^?3 to 0·2 × 10^?11 m² s^?1 at 2000 kg m^?3. Conclusions: It is possible that sulfide corrosion of waste canisters in future radioactive waste repositories depends mainly on sulfide concentration at the boundary between groundwater and the buffer, which in turn depends on SRB growth conditions (e.g., sulfate accessibility, carbon availability and electron donors) and geochemical parameters (e.g., presence of ferrous iron, which immobilizes sulfide). Maintaining high bentonite density is also important in mitigating canister corrosion. Significance and Impact of the Study: The sulfide diffusion coefficients can be used in safety calculations regarding waste canister corrosion. The work supports findings that microbial activity in compacted bentonite will be restricted. The study emphasizes the importance of growth conditions for sulfate reduction at the groundwater boundary of the bentonite buffer and linked sulfide production.