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.     

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.     

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.     

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.     

Mathematical modeling and analysis of the flocculation process in chambers in series

In this study, the flocculation process in continuous systems with chambers in series was analyzed using the classical kinetic model of aggregation and break-up proposed by Argaman and Kaufman, which incorporates two main parameters: K _a and K _b. Typical values for these parameters were used, i. e., K _a = 3.68 × 10^?5–1.83 × 10^?4 and K _b = 1.83 × 10^?7–2.30 × 10^?7 s^?1. The analysis consisted of performing simulations of system behavior under different operating conditions, including variations in the number of chambers used and the utilization of fixed or scaled velocity gradients in the units. The response variable analyzed in all simulations was the total retention time necessary to achieve a given flocculation efficiency, which was determined by means of conventional solution methods of nonlinear algebraic equations, corresponding to the material balances on the system. Values for the number of chambers ranging from 1 to 5, velocity gradients of 20–60 s^?1 and flocculation efficiencies of 50–90 % were adopted.     

Kinetics of phosphate uptake in excised maize root

The short term uptake of phosphate involving 10 min absorption followed by 5 min desorption, both at 30 °C, in the concentration range 1.0×10^?9 to 7.5×10^?2 M KH₂PO₄ by fresh and washed maize (Zea mays L. cv. Ganga Safed-2) roots can be described by a single isotherm having five phases (0 and I–IV) with regularly spaced kinetic constants. Almost identical kinetics were observed in both fresh and washed maize roots. The kinetics of phase 0 in the concentration range 1.0×10^?9–3.0×10^?5 M. was sigmoidal in fresh maize roots, however, in washed tissue exhibited 2 phases termed here as 0a and 0b. 0a covered the concentration range 1.0×10^?9–5.0×10^?6 M and 0b 6.0×10^?6–3.0×10^?5 M. In the concentration range 1.0×10^?4–7.5×10^?2 M four distinct phases, termed as I, II, III and IV were evident in both fresh and washed maize roots. Each phase obeyed Michaelis—Menten kinetics. The values of K_m and V_max have been estimated for each phase. The uptake isotherm was accompanied by discontinuous transitions.     

The effect of pH on the structure and activity of yeast RNA polymerase I

The analysis of the effect of pH upon the rate of polymerization indicates that the activity of yeast RNA polymerase I is optimal between pH 7.5 and 9 and depends on the ionization state of two groups with apparent pK_a values of 6.5 and 10. Yeast RNA polymerase I is extremely labile at acid pH. Below pH 5 the enzyme is irreversibly inactivated by [H⁺], with a second-order rate constant of 1.6 × 10^?4m^?1 min^?1. Sucrose gradient sedimentation and gel electrophoresis analysis of the enzyme inactivated at acid pH indicates the sequential dissociation of several enzyme subunits. The polypeptides of 44,000 and 24,000 daltons dissociate first from the enzyme core followed by the dissociation of the polypeptides of 48,000 and 36,000 daltons.     

Swainsonine,a novel fungal metabolite: optimization of fermentative production and bioreactor operations using evolutionary programming

The optimization of bioreactor operations towards swainsonine production was performed using an artificial neural network coupled evolutionary program (EP)-based optimization algorithm fitted with experimental one-factor-at-a-time (OFAT) results. The effects of varying agitation (300–500 rpm) and aeration (0.5–2.0 vvm) rates for different incubation hours (72–108 h) were evaluated in bench top bioreactor. Prominent scale-up parameters, gassed power per unit volume (P _g/V _L, W/m³) and volumetric oxygen mass transfer coefficient (K _L a, s^?1) were correlated with optimized conditions. A maximum of 6.59 ± 0.10 μg/mL of swainsonine production was observed at 400 rpm-1.5 vvm at 84 h in OFAT experiments with corresponding P _g/V_L and K _L a values of 91.66 W/m³ and 341.48 × 10^?4 s^?1, respectively. The EP optimization algorithm predicted a maximum of 10.08 μg/mL of swainsonine at 325.47 rpm, 1.99 vvm and 80.75 h against the experimental production of 7.93 ± 0.52 μg/mL at constant K _L a (349.25 × 10^?4 s^?1) and significantly reduced P _g/V _L (33.33 W/m³) drawn by the impellers.     

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.     

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.     

Bioreduction of nitroxides by Staphylococcus aureus.

Two nitroxide radicals (TEMPO, I; OXAN, II) and a spin labeled penicillin (III) were reduced by $\text{Staphylococcus aureus}$. A short induction period preceded zero order reduction of these substrates leading to a K_m of 8 × 10^?4M, 6.67 × 10^?5M and 5.7 × 10^?4M and V_max of 106, 26 and 11 μ mole/min mg bacteria for I, II and III, respectively.     

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.     

Interaction of the regulatory gene product with the operator site in the l-arabinose operon of Escherichia coli

The DNA binding properties of the araC protein in the absence of l-arabinose have been studied in Escherichia coli using the nitrocellulose membrane filter technique. Equilibrium competition experiments demonstrate that the araC protein binds specifically to the ara operator. The apparent K_m of the interaction is 1 × 10^?12m at 20 °C. The rates of association and dissociation of the complex have also been determined. A k_a of 2 × 10⁹m^?1 s^?1at 20 °C is calculated assuming binding to a single site. The half-life of the complex is three minutes. The equilibrium constant calculated from the ratio of k_a to k_d is 2.8 × 10^?12m at 20 °C. The good agreement between the equilibrium and kinetic determinations of the equilibrium constant suggest that the kinetic studies are providing true rate constants. It is calculated that about 1% of the purified araC protein is active with respect to operator binding activity.     

Noncompetitive Amine Uptake Inhibition by the New Antidepressant Pridefine

Abstract: Pridefine (AHR-1118) is a pyrrolidine derivative with clinically established antidepressant efficacy. Previous work from this laboratory indicates that pridefine is a reuptake blocker of catecholamines and serotonin with weak releasing activity. This study characterized the mode of amine uptake inhibition by pridefine as noncompetitive. The uptake experiments were performed utilizing ouabain instead of zero-degree controls to differentiate between the passive and active components of uptake. Furthermore, the passive component was resolved into diffusion and binding of substrate. Correction was made for the effects of ouabain on binding. Kinetic constants determined from Lineweaver-Burk plots were: K_m= 3 × 10^?7 M for NE, K_m= 9 × 10^?8 M for DA, and K_m= 3 × 10^?8 M for 5-HT. Dixon analyses of uptake at various pridefine concentrations indicated noncompetitive inhibition with K_i= 2.5 × 10^?6 M for NE uptake, K_i= 2.0 × 10^?6 M for DA uptake, and K_i= 1 × 10^?5 M for 5-HT uptake. These constants compare well with IC₅₀ values for the same transmitters: NE, IC₅₀= 2.4 × 10^?6 M; DA, IC₅₀= 2.8 × 10^?6 M; 5-HT, IC₅₀= 1.0 × 10^?5 M. The in vitro results indicate that pridefine is relatively specific as a catecholamine uptake blocker. It differs from tricyclic antidepressants which are reportedly competitive inhibitors of monoamine uptake. The possible mechanisms by which pridefine acts as a noncompetitive inhibitor are discussed.     

Binding of heparin to antithrombin III: The use of dansyl and rhodamine labels

Low molecular weight heparin of low-anticoagulant activity and high molecular weight heparin of correspondingly high activity were prepared by chromatography on protamine-Sepharose; preparations subjected to limited N-desulfation (5–10% free amino groups) by solvolysis were labeled with 5-dimethylaminonaphthalene-1-sulfonyl chloride (dansyl chloride) or rhodamine B isothiocyanate (RITC). The fluorescent heparins retained approximately 50% of the original anticoagulant activities. Dansyl-heparin on binding to antithrombin III (ATIII) exhibited a 2.5-fold enhancement of dansyl fluorescence intensity. This effect could be prevented by excess unlabeled heparin. A 7900 molecular weight dansyl-heparin preparation bound to ATIII with a stoichiometry of close to 2:1 and with an apparent association constant for binding (K_a) of 4.9 × 10⁵, m^?1, whereas a 21,600 molecular weight fraction bound at 0.7:1 with the protein and with an apparent K_a = 7.9 × 10⁵, m^?1. When ATIII reacted with a mixture of low molecular weight dansyl-heparin and low molecular weight RITC-heparin, there was enhancement of RITC fluorescence emission when excited at the dansyl excitation maximum; this effect was not observed when either of the labeled heparin species was prepared from high molecular weight material. The results are consistent with the proposal that a single molecule of high molecular weight, high-activity heparin occupies two sites when it binds to ATIII, whereas low molecular weight, low-activity heparin binds to the two sites separately.     

Class II α-mannosidase from Aspergillus fischeri: Energetics of catalysis and inhibition

Energetics of the catalysis of Class II α-mannosidase (E.C.3.2.1.24) from Aspergillus fischeri was studied. The enzyme showed K_cat/K_m for Man (α1-3) Man, Man (α1-2) Man and Man (α1-6) Man as 7488, 5376 and 3690 M^?1 min^?1, respectively. The activation energy, E_a was 15.14, 47.43 and 71.21 kJ/mol for α1-3, α1-2 and α1-6 linked mannobioses, respectively, reflecting the energy barrier in the hydrolysis of latter two substrates. The enzyme showed K_cat/K_m as 3.56 × 10⁵ and 4.61 × 10⁵ M^?1 min^?1 and E_a as 38.7 and 8.92 kJ/mol, towards pNPαMan and 4-MeUmbαMan, respectively. Binding of Swainsonine to the enzyme is stronger than that of 1-deoxymannojirimycin.     

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.     

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.     

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.     

Cerebral microvessels contain a beta 2-adrenergic receptor

Cerebral microvessels isolated from cat forebrain contain a specific β-adrenergic-sensitive adenylate cyclase. Among various compounds tested, the most potent activator of enzyme activity is isoproterenol (k_a = 1.4 × 10^?7M), followed in order by epinephrine (k_a= 1.5 × 10^?6M), norepinephrine (k_a= 1.4 × 10^?5M) and phenylephrine (k_a> 3 × 10^?4M). Isoproterenol-stimulated enzyme activity is blocked by propranolol (k_i= 2.4 × 10^?9M, IPS 339 (k_i= 4 × 10^?9M), H35/25 (k_i = 1.2 × 10^?7M), atenolol (k_i= 5.9 × 10^?6M) and practolol (k_i= 1.8 × 10^?5M). These agonist and antagonist properties are quite similar to those demonstrated by β₂-adrenergic receptors and β₂-stimulated adenylate cyclase present in other tissues and indicate that the majority of adenylate cyclase-associated adrenergic receptors in cerebral microvessels are β₂. The findings are relevant to physiological studies of cerebral blood flow and vascular permeability.