Linear Double-reciprocal Plots Research Articles

Efficient electrocatalytic oxidation and selective determination of isoniazid by Fe(tmphen) 3 2+ -exchanged Nafion®-modified electrode

Cyclic voltammetry and electrochemical impedance studies of Fe(tmphen)32+ (where tmphen = 3,4,7,8-tetramethyl-1,10-phenanthroline)-immobilized Nafion®-modified glassy carbon electrode (GC/Nf/Fe(tmphen)32+) are carried out in 0.1 M Na2SO4 solution. Nafion–Fe(tmphen)32+ complex exhibits efficient electrocatalytic oxidation of isoniazid. The linear double reciprocal plot of current and concentration of isoniazid shows a Michaelis–Menten-type catalytic process. The catalytic oxidation currents are proportional to the concentration of isoniazid and show a wide linear calibration range for the quantitative determination of isoniazid. Detection limit and sensitivity are found to be 13 μM and 2.5 μA mM−1, respectively.

Losac, the First Hemolin that Exhibits Procogulant Activity through Selective Factor X Proteolytic Activation

Envenoming by the contact of human skin with Lonomia obliqua caterpillars promotes a hemorrhagic syndrome characterized by a consumptive coagulopathy. Losac (Lonomia obliqua Stuart factor activator) is a component of the bristle of L. obliqua that is probably partially responsible for the observed syndrome because it activates factor X and is recognized by an effective antilonomic serum. Here we unveil the proteolytic activity of Losac and demonstrate the feasibility of its recombinant production. On the other hand, Losac has no homology to known proteases, but it can be inhibited by PMSF, a serine protease inhibitor. Instead, it shows closer homology to members of the hemolin family of proteins, a group of cell adhesion molecules. The recombinant protein (rLosac) shortened the coagulation time of normal and deficient plasmas, whereas it was ineffective in factor X-deficient plasma unless reconstituted with this protein. rLosac was able to activate factor X in a dose- and time-dependent manner but not γ-carboxyglutamic acid domainless factor X. Moreover, phospholipids and calcium ions increased rLosac activity. Also, rLosac had no effect on fibrin or fibrinogen, indicating its specificity for blood coagulation activation. Linear double reciprocal plots indicate that rLosac follows a Michaelis-Menten kinetics. Cleavage of factor X by rLosac resulted in fragments that are compatible with those generated by RVV-X (a well known factor X activator). Together, our results validate Losac as the first protein from the hemolin family exhibiting procoagulant activity through selective proteolysis on coagulation factor X.

Giardia intestinalis:Volume Recovery in Response to Cell Swelling

The trophozoite form of the protozoan parasiteGiardia intestinalisis subjected to a changing osmotic environment in the small intestine of the host, and consequently effective osmoregulation and control of cell volume are essential to its survival. As a first step toward investigating the mechanism by which hypoosmotically-activated transport is controlled in this organism, we used a light scattering technique to monitor continuously changes in cell volume after osmotic challenge. There was a hyperbolic relationship betweenA550and giardial protein concentration, resulting in linear double reciprocal plots and allowing the calculation of relativeGiardiacell volumes fromA550values. The initial rate of cell swelling was directly proportional to the hypoosmotic gradient when the hypoosmotic difference was greater than 50 mOsm kg−1. However, a hypoosmotic challenge of <30 mOsm kg−1had little effect on cell swelling, suggesting that giardial cell rigidity can resist small changes in medium osmolarity. The use of light scattering as a measure of giardial cell volume changes was validated using a rapidly penetrating solute, ethylene glycol, to induce isoosmotic cell swelling. We have previously shown that trophozoites swelled initially when subjected to a hypoosmotic challenge and that a subsequent regulatory volume decrease was accompanied by rapid alanine efflux and activation of the uptake of an alanine analog, 2-aminoisobutyrate. The ethylene glycol-induced isoosmotic cell swelling was also followed by a regulatory volume decrease, accompanied by a similar rapid release of intracellular alanine and activation of 2-aminoisobutyric acid uptake. This suggests that an increase in cell volume is the primary stimulus for the rapid alanine efflux after hypoosmotic challenge.

Kinetics of a Thermolysin-Catalyzed Peptide Formation Reaction in Acetonitrile–Water

Abstract Thermolysin-catalyzed peptide condensation reactions from acylamino acids and amino acid amides were studied in an acetonitrile (MeCN)–water mixed solvent in a homogeneous system. A kinetic analysis of the condensation reaction was performed by taking into account the simultaneous, solvent-induced, gradual inactivation of the enzyme. Although the dependence on the concentration of the carboxyl component gave linear double-reciprocal plots, that on the amine component at 70% MeCN showed an apparent substrate inhibition profile. An analysis of the apparent Km and kcat parameters for both components at 40% MeCN indicated that the condensation reaction proceeded by a random Bi–Bi mechanism. The Km values were estimated to be 5.5 mM for Cbz–Phe and 110 mM for LeuNH2, and kcat was 4—5 s−1. The Km values for Cbz–Phe and LeuNH2 were also evaluated at 70% MeCN to be 5 and 100—150 mM, respectively. This indicates that the catalytic property of this enzyme is not significantly influenced by the existence of a higher concentration of such an organic solvent, except for the second nonproductive binding of the amine component.

Photochemistry of Locally Excited and Intramolecular Charge Transfer States of a Disilane Bearing a Cyclic Arylethenyl Electron Acceptor Substituent

1-(p-cyanophenyl)-2-(pentamethyldisilanyl)cyclopentene (1) exhibits a fluorescence at λ>400 nm, which strongly shifts to the red with increasing polarity of the solvent. The corresponding solvatochromic plot is indicative of an emissive, lowest energy charge-transfer (CT) state. Stern-Volmer quenching of the fluorescence by dilute methanol in pentane gives a linear plot with a unit intercept and a slope k Q τ ct =0.70. Quenching is accompanied by formation of (arylcyclopentenyl)dimethylsilane 7, the major product observed upon photolysis of 1 in the presence of methanol. This is evidenced by the linear double-reciprocal plot of quantum yields of 7 versus methanol concentration, which gives a k Q τ ct value in agreement with fluorescence quenching

Mathematical model for the luminol chemiluminescence reaction catalyzed by peroxidase

A kinetic model that accurately describes intensity vs. time reaction profiles for the chemiluminescence reaction between luminol and hydrogen peroxide, as catalyzed by horseradish peroxidase, is derived and evaluated. A set of three differential equations is derived and solved to provide intensity time information for the first 200 seconds of the reaction. The model accurately predicts intensity-time profiles when literature values are used for all but one of the reaction rate constants. Furthermore, the model predicts a nonlinear curve for plots of light intensity versus the initial hydrogen peroxide concentration. Experimental data confirm that such plots are nonlinear. Finally, a linear double-reciprocal plot is predicted by the model and the experimental data verify this relationship.

The hydrogen-tritium exchange activity of Megasphaera elsdenii hydrogenase.

The hydrogenase of Megasphaera elsdenii was purified to a specific activity of 350 units/mg. The hydrogen-tritium exchange assay of Hallahan et al. [Hallahan, D.L., Fernandez, V. M., Hatchikian, E. C. and Hall, D. O. (1986) Biochimie (Paris) 68, 49-54] was adapted to allow its use in the study of the M. elsdenii hydrogenase preparation. Under the assay conditions routinely employed, the enzyme's exchange activity was inhibited by Tris/HCl and MgCl2; it was stimulated by ethylene glycol. Maximal activity in this standard assay occurred at pH 7.1. The effect of the concentration of molecular hydrogen (1H2 plus 3H1H) on the exchange activity was studied. The resulting double-reciprocal plot was linear; its slope and its intercepts on the ordinate and abscissa were pH-dependent. The rate equations for a number of models of the exchange activity were derived. Each model gave rise to a linear double-reciprocal plot at constant pH, but none could explain fully the observed effects of varying pH. The experimental data corresponded most closely to the predictions of models in which protons were treated both as substrates and as regulators of the enzyme's activity.

Kinetic analysis of Escherichia coli RNase H using DNA-RNA-DNA/DNA substrates.

The kinetic properties of Escherichia coli ribonuclease H (RNase H) were investigated using oligonucleotide substrates that consist of a short stretch of RNA, flanked on either side by DNA (DNA-RNA-DNA). In the presence of a complementary DNA strand, RNase H cleavage is restricted to the short ribonucleotide stretch of the DNA/RNA heteroduplex. The DNA-RNA-DNA substrate utilized for kinetic studies: (formula; see text) is cleaved at a single site (decreases) in the presence of a complementary DNA strand, to generate (dT)7-(rA)2-OH and p-(rA)2-(dT)9. Anion exchange high performance liquid chromatography was used to separate and quantitate the cleavage products. Under these conditions, RNase H-specific and nonspecific degradation products could be resolved. Kinetic parameters were measured under conditions of 100% hybrid formation (1.2-1.5 molar excess of complementary DNA, T much less than Tm). A linear double reciprocal plot was obtained, yielding a Km of 4.2 microM and a turnover number of 7.1 cleavages per s per RNase H monomer. The kinetic properties of substrate analogs containing varying lengths of RNA (n = 3-5) and 2'-O-methyl modifications were also investigated. Maximal turnover was observed with DNA-RNA-DNA substrates containing a minimum of four RNA residues. Kcat for the rA3 derivative was decreased by more than 100-fold. The Km appeared to decrease with the size of the internal RNA stretch (n = 3-5). No significant difference in turnover number of Km was observed when the flanking DNA was replaced with 2'-O-methyl RNA, suggesting that RNase H does not interact with this region of the heteroduplex.

Chromatographic separation of activated and unactivated forms of aldose reductase

Chromatography of bovine kidney aldose reductase using Matrex Orange A affinity gel results in the separation of the unactivated and activated enzyme forms. The former washes through the column, while the latter is eluted with an NADPH step-gradient. The separated enzyme forms display V max and K m glycolaldehyde values, and relative sensitivities to inhibition by the aldose reductase inhibitor AL-1576 (spiro[2,7-difluorofluorene-9, 4′-imidazolidine]-2′,5′-dione), that are similar to those reported previously for the individual forms. However, because V max is 17-fold lower for the unactivated enzyme, the purification of aldose reductase via NADP(H) elution from a dye-ligand affinity matrix can result in the selective purification of only the activated enzyme form. These results have direct implications for the study of potential aldose reductase inhibitors, and may explain why linear double-reciprocal plots are commonly observed for enzyme prepared in this manner, while nonlinear plots are seen in other cases.

Kinetics of human erythrocyte glucose-6-phosphate dehydrogenase dimers.

The steady-state kinetics of human erythrocyte glucose-6-phosphate dehydrogenase (D-glucose-6-phosphate: NADP+ 1-oxidoreductase, EC 1.1.1.49) dimers were studied by initial rate measurement. These experiments gave intersecting double-reciprocal plots suggesting a ternary complex mechanism with a Km for NADP and glucose 6-phosphate of 11 microM and 43 microM, respectively. These studies were combined with rate measurements in the presence of one product (NADPH), dead-end inhibitors, as well as alternative substrates. The inhibition by NADPH was found to be competitive with respect to both substrates. Alternate substrates experiments gave linear double-reciprocal plots over a wide range of substrate concentrations. The results suggest that the dimeric enzyme follows either a random or a Theorell-Chance mechanism.

Inhibition of catechol estrogen formation in rat liver microsomes by hormonal steroids and related compounds

The inhibitory action of a number of different hormonal steroids and related compounds on the 2-hydroxylation of estradiol by male rat liver microsomes was examined by a radiometric assay. Progesterone, Diethylstilbestrol, testosterone and 4-androstenedione were found to be the most potent of the compounds tested but inhibition was also observed with other steroids and a group of androgen analogs which are aromatization inhibitors. The kinetic constant K i for those steroids which gave linear double reciprocal plots when added to [2- 3H]estradiol was determined and the products from [ 14C]estradiol in the presence of the inhibitors were examined by TLC and autoradiography. The addition of steroids with a 17-hydroxyl group such as testosterone or dihydroequilin resulted in the formation of mainly 2-hydroxyestradiol with smaller amounts of other metabolites while those with a reducible ketonic group such as progesterone, 4-androstenedione, equilin or equilenin gave rise to considerable amounts of estrone in addition to the catechol estrogens. Further purification of the liver microsomes did not alter this effect. The possible role of progesterone and the catechol estrogens in the control of estrogen hydroxylation in liver as well as other aspects of steroid interaction are discussed.

Lactate Uptake by Toadfish Hepatocytes: Passive Diffusion is Sufficient

ABSTRACT Mechanisms of hepatic lactate uptake were studied in the gulf toadfish, Opsanus beta by following the accumulation of [l4C]lactate by isolated hepatocytes in vitro. Uptake rates were linear for up to 5 s at external lactate concentrations of 1 and 20 mmol 1−1. 5-s uptake rates for several concentrations in the physiological range (i.e. 0 2-20 mmol 1−1) yielded a linear double reciprocal plot (1/v VS l/[lactate]) that passed through the origin. Lactate uptake was not inhibited by known inhibitors of carrier-mediated lactate transport (e.g. 5 mmol 1−1 α-cyano-4-hydroxycinnamate; 10 mmol 1−1 pyruvate), and was not stereospecific for L-( +)-lactate. These observations indicate that lactate uptake is by passive diffusion in toadfish hepatocytes. Lactate uptake by toadfish hepatocytes further differed from lactate uptake by mammalian tissues in that rates were not altered by changes in either pHe (6·5−8·0) or [Na+]e (3−151 mmol 1−1). Rates of lactate conversion to glucose and CO2 were measured and compared to uptake rates, and it appears that rates of lactate metabolism are not limited by passive diffusion. These results are discussed in relation to rates of lactate uptake and metabolism in mammalian hepatocytes.

Accurate, quantitative assays for the hydrolysis of soluble type I, II, and III 3H-acetylated collagens by bacterial and tissue collagenases

Accurate and quantitative assays for the hydrolysis of soluble 3H-acetylated rat tendon type I, bovine cartilage type II, and human amnion type III collagens by both bacterial and tissue collagenases have been developed. The assays are carried out at any temperature in the 1–30°C range in a single reaction tube and the progress of the reaction is monitored by withdrawing aliquots as a function of time, quenching with 1,10-phenanthroline, and quantitation of the concentration of hydrolysis fragments. The latter is achieved by selective denaturation of these fragments by incubation under conditions described in the previous paper of this issue. The assays give percentages of hydrolysis of all three collagen types by neutrophil collagenase that agree well with the results of gel electrophoresis experiments. The initial rates of hydrolysis of all three collagens are proportional to the concentration of both neutrophil or Clostridial collagenases over a 10-fold range of enzyme concentrations. All three assays can be carried out at collagen concentrations that range from 0.06 to 2 mg/ml and give linear double reciprocal plots for both tissue and bacterial collagenases that can be used to evaluate the kinetic parameters K m and k cat or V max. The assay developed for the hydrolysis of rat type I collagen by neutrophil collagenase is shown to be more sensitive by at least one order of magnitude than comparable assays that use rat type. I collagen fibrils or gels as substrate.

Cobra venom phospholipase A2 inhibition by manoalide. A novel type of phospholipase inhibitor.

Manoalide, an unusual nonsteroidal sesterterpenoid recently isolated from sponge, antagonizes phorbol-induced inflammation but not that induced by arachidonic acid, suggesting that manoalide acts prior to the cyclooxygenase step in prostaglandin synthesis, possibly by inhibiting phospholipase A2. We have now studied the inhibitory effect of manoalide on a homogeneous preparation of phospholipase A2 from cobra venom. For a given concentration of manoalide, the inhibition of phospholipase A2 activity toward dipalmitoylphosphatidylcholine/Triton X-100 mixed micelles is time-dependent and plateaus at about 85% inhibition of the initial velocity even after extensive preincubation. Metal ions (Ca2+, Ba2+, Mn2+) increase the inhibition, while lysophosphatidylcholine and substrate micelles protect. Increasing manoalide concentration shows increasing inhibition of the initial velocity until a plateau is reached, giving a typical saturation curve with a linear double-reciprocal plot. Under typical conditions (20-min preincubation, 40 degrees C, pH 7.1), 50% inhibition is achieved at a manoalide concentration of about 2 X 10(-6) M. The data indicate that manoalide is a potent inhibitor of the cobra venom phospholipase A2. Manoalide is now shown to react irreversibly with lysine residues in the enzyme. Surprisingly, the cobra venom phospholipase normally acts poorly on phosphatidylethanolamine as substrate, but after reaction with manoalide, the enzyme is somewhat more active toward this substrate rather than being inhibited. This suggests that a lysine residue may be important in understanding the substrate specificity of phospholipase A2.

Glucose-6-phosphate Dehydrogenase from Agaricus bisporus: Purification and Properties

Agaricus bisporus glucose-6-phosphate dehydrogenase (EC 1.1.1.49) was purified to homogeneity. The subunit molecular weight was 55300 and the molecular weight of the native protein was 138000 by gel filtration and 131 800 by rate zonal ultracentrifugation on sucrose gradients. There was some evidence for a small population of higher molecular weight oligomers during rate zonal centrifugation. A sedimentation coefficient of 8·5S was determined by analytical ultracentrifugation (suggesting the presence of the tetramer). Three bands of enzyme activity were separated on polyacrylamide gels. Digests of the proteins in the three bands gave peptide patterns with no detectable difference after SDS electrophoresis. Linear double reciprocal plots were obtained with glucose 6-phosphate as a variable substrate. The apparent Km for glucose 6-phosphate was 214 μm and the V max was 520 μmol (mg protein)–1 min–1. A sigmoidal saturation curve and a non-linear double reciprocal plot were observed for NADP, giving a Hill constant of 1·5 and [S]0·5 for NADP of 89 μm. The enzyme was inhibited by NADPH, with 50% inhibition at a NADP: NADPH ratio of 0·4 when total NADP(H) concentration was 0·2 mm. ATP, ADP, AMP and cAMP had no effect on enzyme activity. Palmitoyl CoA inhibited the initial velocity of the reaction by 95% at a concentration of 1·5 μm when incubated with enzyme prior to NADP addition.

Fatty acid chain elongation by microsomal enzymes from the bovine meibomian gland

The composition of meibomian gland lipids suggested that fatty acid chain elongation might play a major role in the synthesis of such lipids. A fatty acid synthase preparation from the bovine meibomian gland catalyzed the formation of C 16 acid and the enzyme was immunologically quite similar to that in the mammary gland. The microsomal fraction from the gland, on the other hand, catalyzed elongation of endogenous fatty acids in the presence of ATP and Mg 2+ and of exogenous C 18-CoA using malonyl-CoA and NADPH as the preferred reductant. The elongated products, ranging up to C 28 in chain length, were found mainly as CoA esters and products derived from them. With C 18-CoA as the exogenous primer, the elongation rate was linear with incubation time up to 20 min but the rate changed in a sigmoidal manner with increasing protein concentration. The elongation rate was maximal at a pH around 7.0. Typical Michaelis-Menten-type substrate saturation patterns were observed with both malonyl-CoA and NADPH. From linear double-reciprocal plots, the K m values for the two substrates were calculated to be 52 and 11 μ m, respectively, with a V of about 340 pmol min −1 mg protein −1 with respect to malonyl-CoA. Exogenous CoA esters of C 16 to C 22 fatty acids were elongated to give products up to C 28 without exhibiting any preference for the primer. The present elongation system could account for the formation of most of the very long chains found in meibomian lipids.

Kinetic studies of Ox-liver glutamate dehydrogenase oxidative deamination of two glutamate analogues, L- threo-γ-methylglutamate and L-α-amino-γ-nitraminobutyrate, in the presence of the allosteric effector ADP

1. 1. Ox-liver glutamate dehydrogenase is known to utilise a wide range of amino acid substrates. Kinetic studies are presented here for L- threo-γ-methylglutamate and L-α-amino-γ-nitraminobutyrate in the presence of the allosteric effector ADP. 2. 2. The results presented are considered in the light of similar studies presented elsewhere in which the cofactor was systematically replaced by a variety of analogues. 3. 3. These amino acid analogues share the same pH optimum as glutamate, unlike the monocarboxylic amino acids including alanine and norvaline, and give linear double-reciprocal plots under the conditions used here. 4. 4. Studies with the alternative coenzymes have suggested an ordered addition of glutamate before coenzyme in the presence of ADP. The present results obtained under identical conditions support this conclusion.

A kinetic study of the oxidative deamination of L-glutamate by Peptostreptococcus asaccharolyticus glutamate dehydrogenase using a variety of coenzymes.

The NAD+-specific glutamate dehydrogenase from Peptostreptococcus asaccharolyticus follows Michaelis-Menten kinetics in contrast to the enzyme from several other sources, and thus gives linear double-reciprocal plots of initial-rate data. The initial-rate parameters have been determined for this bacterial dehyrogenase in the direction of oxidative deamination. The use of alternative coenzymes leads to some conclusions about the order of substrate addition. An investigation of the pH dependence of this reaction reveals that the binding of oxidised coenzyme is independent of pH over the range 6-9. The kinetic data are consistent with an ordered addition of coenzyme prior to glutamate, the reverse of the mechanism derived with ox glutamate dehydrogenase in the presence of ADP.

Kinetic properties of spermine synthase from bovine brain.

A kinetic analysis including initial-velocity and product-inhibition studies were performed with spermine synthase purified from bovine brain. The enzyme activity was assayed in the presence of 5'-methylthioadenosine phosphorylase as an auxiliary enzyme to prevent the accumulation of the inhibitory product, 5'-methylthioadenosine, and thus to obtain linearity of the reaction with time. Initial-velocity studies gave intersecting or converging linear double-reciprocal plots. No substrate inhibition by decarboxylated S-adenosylmethionine was observed at concentrations up to 0.4 mM. Apparent Michaelis constants were 60 microM for spermidine and 0.1 microM for decarboxylated S-adenosylmethionine. Spermine was a competitive product inhibitor with respect to decarboxylated S-adenosylmethionine, but a mixed one with respect to the other substrate, spermidine. 5'-Methylthioadenosine showed a mixed inhibition with both substrates, predominantly competitive with respect to decarboxylated S-adenosylmethionine and predominantly uncompetitive with respect to spermidine. The observed kinetic and inhibition patterns are consistent with a compulsory-order mechanism, where both substrates add to the enzyme before products can be released.

Steady state kinetics of chloroperoxidase-catalyzed N-demethylation reactions.

The kinetic mechanism of the chloroperoxidase-catalyzed N-demethylation of N,N-dimethylaniline supported by ethyl hydroperoxide was determined from initial rate and inhibition studies. When the concentration of N,N-dimethylaniline was varied in the presence of several different fixed concentrations of ethyl hydroperoxide, a double reciprocal plot of the data gave a series of parallel lines. Parallel lines were also observed when the data were plotted as a function of the concentration of ethyl hydroperoxide. A linear double reciprocal plot was obtained when the concentrations of both substrates were varied in a constant ratio. Competitive substrate inhibition was observed for N,N-dimethylaniline with a KI of 4.78 mM. Competitive substrate inhibition was also observed for ethyl hydroperoxide but the secondary replot of the slopes of the double reciprocal lines versus the concentrations of ethyl hydroperoxide intercepted the origin, indicating that no abortive binary complex between ethyl hydroperoxide and the enzyme was formed. These results suggested that the inhibition of the demethylation reaction by ethyl hydroperoxide was due to the reaction of ethyl hydroperoxide with chloroperoxidase compound I to evolve oxygen. N,N-Dimethylaniline inhibited the chloroperoxidase-catalyzed evolution of oxygen from ethyl hydroperoxide with a KI (0.111 mM) essentially identical with its KM for demethylation (0.122 mM). The inhibition of the demethylation reaction by 2,5-dimethylfuran was competitive with respect to N,N-dimethylaniline and uncompetitive with respect to ethyl hydroperoxide, consistent with dead-end inhibition in a ping-pong system. The results of the initial rate and inhibition studies are consistent with a Ping Pong Bi Bi mechanism as the minimal kinetic model for chloroperoxidase-catalyzed N-demethylation reactions. In this model, ethyl hydroperoxide reacts with chloroperoxidase to form the oxidized enzyme intermediate compound I with the concomitant release of ethanol. N,N-Dimethylaniline then binds to compound I and is oxidized, resulting in the formation of N-methylaniline and formaldehyde and the regeneration of the native peroxidase.