Plots Of Initial Rates Research Articles

Homotropic cooperativity of cyclodextrin dimer as an artificial hydrolase

Three kinds of cyclodextrin (CD) dimer (one homo-dimer of β-CD and two types of hetero-dimer of α-CD and β-CD) were synthesized as artificial hydrolases. The initial rates of the cleavage reaction of p-nitrophenyl methoxyethoxyethoxyacetate ( 8) by each of the three dimers were measured under the condition of excess of substrate at 25 °C in a pH 7.4 phosphate buffer solution. In a plot of initial rate against concentration of the substrate, the reaction with the homo-dimer ( βCβH 5) gave a sigmoidal curve, whereas the reaction with either of the hetero-dimers gave a simple hyperbolic curve. Only βCβH 5 showed homotropic cooperativity, with a Hill constant of 1.8.

Highly Cooperative Tetrametallic Ruthenium-mu-Oxo-mu-Hydroxo Catalyst for the Alcohol Oxidation Reaction.

The tetrametallic ruthenium-oxo-hydroxo-hydride complex {[(PCy(3))(CO)RuH](4)(mu(4)-O)(mu(3)-OH)(mu(2)-OH)} (1) was synthesized in two steps from the monomeric complex (PCy(3))(CO)RuHCl (2). The tetrameric complex 1 was found to be a highly effective catalyst for the transfer dehydrogenation of alcohols. Complex 1 showed a different catalytic activity pattern towards primary and secondary benzyl alcohols, as indicated by the Hammett correlation for the oxidation reaction of p-X-C(6)H(4)CH(2)OH (rho = -0.45) and p-X-C(6)H(4)CH(OH)CH(3) (rho = +0.22) (X = OMe, CH(3), H, Cl, CF(3)). Both a sigmoidal curve from the plot of initial rate vs [PhCH(OH)CH(3)] (K(0.5) = 0.34 M; Hill coefficient, n = 4.2+/-0.1) and the phosphine inhibition kinetics revealed the highly cooperative nature of the complex for the oxidation of secondary alcohols.

Calcium oxalate crystallization kinetics studied by oxalate-induced turbidity in fresh human urine and artificial urine

We have studied the kinetics of oxalate-induced turbidity in fresh human urine and artificial urine. Assays are performed in 96-well plates, which allows many oxalate concentrations to be studied, repeatedly, in a short time. The metastable limit is defined in terms of the lowest oxalate concentration that gives a rate of change of attenuance significantly greater than the control. Interpretation of rates above this limit is based on ln/ln plots of initial rates against added oxalate concentration. This approach has a good theoretical basis, is well supported by our results and gives a turbidity rate index that is related to the product of the growth rate constant and a factor relating to the number and characteristics of the heteronuclei responsible for initiation of crystallization. This interpretation is posited upon the assumptions that second-order crystallization kinetics occur in unseeded urine when supersaturation exceeds the metastable limit and that aggregation during the initial phase of crystallization does not significantly contribute to changes in turbidity. Metastable limits of urine from healthy volunteers corresponded to a calcium oxalate supersaturation ratio of approx. 10. The turbidity rate index was higher in human urine than in artificial urine. The metastable limit, based on either oxalate concentration or supersaturation, for induction of calcium oxalate crystallization in normal human urine is higher than is likely to be found in normal subjects in vivo. The shape of the relationship between the metastable limit (based on oxalate concentration) and calcium concentration emphasizes the benefit of achieving a low urine calcium concentration. Comparison of the turbidity rate indices for human and artificial urine suggests that the role of nucleation promoters is more dominant than that of growth inhibitors.

Determination of Association Rate Constants by an Optical Biosensor Using Initial Rate Analysis

We show that initial rate analysis can be successfully applied to analyze experimental binding data generated by an optical biosensor. The initial rates of binding obtained from linear regression are concentration dependent, and plots of initial rate against ligate concentration yield a straight line that passes through the origin. The slope of this graph is the product of the association constant times the maximal binding capacity of the immobilized ligand. This latter parameter is easily obtained from a single binding curve at high ligate concentration, allowing rapid determination of the association rate constant. The association rate constant obtained in this manner is found to be in good agreement with that obtained by the more customary method of nonlinear regression analysis of the entire binding profile. Initial rate analysis is more simple than fitting the full association profile and needs less data collection time. It also requires fewer assumptions about the functional form of the association profile. This can be advantageous when fitting biosensor-derived data, which often show complex association kinetics. Furthermore, it avoids the potential complication of second-order kinetics which may be found at low ligate concentrations with high-affinity interactions.

Activation of nitrite reductase from Escherichia coli K12 by oxidized nicotinamide-adenine dinucleotide

Nitrite reductase from Escherichia coli K12 requires the presence of NAD+, one of the products of the reduction of NO2-by NADH, for full activity. The effect is observed with both crude extracts and purified enzyme. NAD+ also acts as a product inhibitor at high concentrations, and plots of initial rate against NAD+ concentration are bell-shaped. The maximum occurs at about 1 mM-NAD+, but increases with increasing NADH concentration. In the presence of 1 mM-NAD+ and saturating NO2-(2mM) the Michaelis constant for NADH is about 16 micron. The Michaelis constant for NO2-is about 5 micron and is largely independent of the NAD+ concentration. Similar but more pronounced effects of NAD+ are observed with hydroxylamine as electron acceptor instead of NO2-. The maximum rate of NADH oxidation by hydroxylamine is about 5.4 times greater than the maximum rate of NADH oxidation by NO2- when assayed with the same volume of the same preparation of purified enzyme. The Michaelis constant for hydroxylamine is 5.3 mM, however, about 1000 times higher than for NO2-. These results are consistent with a mechanism in which the same enzyme-hydroxylamine complex occurs as an intermediate in both reactions.

Isolation and characterization of 5-fluorotryptophan-resistant mutants with altered L-tryptophan transport.

Mutants of A9 mouse fibroblast, resistant to the killing effect of 0.4 mM 5-flurotryptophan (5-FT), have altered L-tryptophan transport properties. The resistant phenotype is stable for at least 90 generations of growth in MEM. A fluctuation test indicated that clones resistant to 0.4 mM 5-FT occurred spontaneously. An average mutation rate was estimated at 1.6 X 10(-6). Treatment with N-methyl-N'-nitro-N-nitrosoguanidine increased the frequency of these clones by at least 100-fold. These results indicate that the resistant clones arose as a result of a mutation. All the resistant mutant tested accumulate less 5-FT at near steady-state conditions than the wild type. Lineweaver-Burk plots of initial rates of tryptophan uptake yield a biphasic curve suggesting that tryptophan is transported by two transport systems. Kinetic constants determined by a computer program indicate that both proposed transport systems were modified in each of two 5-FT resistant mutants.

Kinetics of Insulin-Stimulated Accumulation of Glucose by Heart Cells from Newborn Rats

Cells isolated from the hearts of newborn (1-day-old) rat accumulate <sup>14</sup>C-glucose. Insulin stimulates glucose accumulation by the heart cells. Values for net accumulation of glucose by heart cells agree well with those values obtained earlier using whole hearts. Lineweaver-Burk plots of initial rates of glucose accumulation at different concentrations of glucose show that the effect of insulin is to increase the V<sub>max</sub>. It is concluded that the effect of insulin is to increase the rate of glucose accumulation rather than to alter the concentration of glucose required to half-saturate the system. Glucose accumulation is also stimulated by mild trypsin digestion of the cells. Increasing concentrations of concanavalin A inhibit glucose accumulation.

Wheat-germ aspartate transcarbamoylase. Kinetic behaviour suggesting an allosteric mechanism of regulation.

1. Some kinetic properties of aspartate transcarbamoylase (EC 2.1.3.2), that had been purified approx. 20-fold from wheat germ, were studied. 2. A plot of enzyme activity against pH showed a low maximum at pH8.4 and a second, higher, maximum at pH10.5. A plot of percentage inhibition by 0.2mm-UMP against pH was approximately parallel to the plot of activity against pH, except that between pH6.5 and 7.5 the enzyme was insensitive to 0.2mm-UMP. 3. Kinetics were studied in detail at pH10.0 and 25 degrees C. In the absence of UMP, initial-rate plots were hyperbolic when the concentration of either substrate was varied. UMP decreased both V(max.) and K(m) in plots of initial rate against l-aspartate concentration, but the plots remained hyperbolic. However, UMP converted plots of initial rate against carbamoyl phosphate concentration into a sigmoidal shape, without significantly affecting V(max.). Plots of initial rate against UMP concentration were also sigmoidal. 4. The theoretical model proposed by Monod et al. (1965) gave a partial explanation of these results. When quasi-equilibrium conditions were assumed analysis in terms of this model suggested a trimeric enzyme binding the allosteric ligands, carbamoyl phosphate and UMP, nearly exclusively to the R and T conformational states respectively, and existing predominantly in the R state when ligands were absent. However, the values of the Hill coefficients for the co-operativity of each allosteric ligand were somewhat less than those predicted by the theory. 5. Some of the implications of these results are discussed, and the enzyme is contrasted with the well-known aspartate transcarbamoylase of Escherichia coli.

Studies on the Allosteric Activation of Glycogen Phosphorylase b by Nucleotides: I. Activation of phosphorylase b by inosine monophosphate

Abstract The allosteric mechanism of phosphorylase b activation by inosine monophosphate has been investigated and compared with AMP activation of this enzyme. Enzyme activity was measured in the direction of glycogen synthesis. In contrast to AMP, IMP affected the Vmax but not the enzyme affinity toward glucose-1-P. The plots of initial rate (vi) against glucose-1-P concentration for IMP activation were sigmoidal, having Hill coefficients of 2, the homotropic cooperativity of glucose-1-P being independent of nucleotide concentration. The plots of vi against IMP concentration were sigmoidal at low glucose-1-P concentrations but hyperbolic at high substrate. In the presence of 0.3 m sodium fluoride the kinetics was normalized, resulting in an increased nucleotide affinity. Under these conditions the Km values of AMP and IMP activation were both independent of substrate concentration; only the Vmax changed with increasing glucose-1-P. The kinetic data suggest that the mechanism of nucleotide activation results in a two-stage allosteric transition of the enzyme, viz., a change in both catalytic efficiency and enzyme affinity toward glucose-1-P. This proposed mechanism receives support from the following physical criteria. AMP, IMP, and glucose-1-P show varying degrees of protection against cold inactivation and the removal of pyridoxal phosphate from the enzyme. While AMP causes partial dimerization of phosphorylase b, IMP may dimerize the enzyme only in the presence of glucose-1-P.

Kinetics of catalytic oxidation of methane: Application of initial rate technique for mechanism determination

AbstractA kinetic investigation on the oxidation of methane using a palladium catalyst was conducted. Conversions of methane were small and limited to about 10% to facilitate the use of initial rate approach. Plots of initial rate against total pressure and varying feed composition were used to interpret the data qualitatively. Regression coefficients from linearized rate equations and criteria of best fit were used to arrive at a plausible mechanism. The experimental results indicated that the reaction rate is controlled by surface reaction in which the adsorbed oxygen and adsorbed methane react to produce adsorbed carbon dioxide and adsorbed water.

Polymerization of acrylamide initiated by the persulfate–thiosulfate redox couple

AbstractConversion–time data were obtained for the polymerization of acrylamide initiated by the redox couple persulfate–thiosulfate by using a dilatometer. A plot of initial rate as a function of thiosulfate concentration shows a well‐defined maximum and three distinct regions of behavior. In each region the shape of the conversion–time curves demonstrates the differences in apparent order with respect to monomer arising from changes in initiator concentration during an individual run. A reaction mechanism is proposed to explain the results, and a limiting form of the rate expression is derived for each of the three regions. The ranges of concentration studied are: persulfate, 9.5 × 10−4−4.7 × 10−2M; thiosulfate, 2 × 10−5−2 × 10−2M; initial monomer, 0.05–1.0M; and temperature, 30–50°C. Within these ranges the initial rate shows a halforder dependence on persulfate and a first‐order dependence on initial monomer concentration.

The interpretation of non-hyperbolic rate curves for two-substrate enzymes. A possible mechanism for phosphofructokinase

1. A theoretical appraisal of the alternative pathway mechanism for a two-substrate enzyme shows that this mechanism is capable of giving rise to apparent substrate inhibition or substrate activation (Dalziel, 1958). It has now been shown that these phenomena may occur simultaneously in the following ways. With certain relationships between the kinetic parameters and the constant concentration of one substrate, A, the plot of initial rate, v, against the concentration of the other substrate, B, may show substrate ;activation' at low concentrations of B and substrate ;inhibition' at high concentrations of B. In other circumstances the plot of v against [B], with [A] constant, may be sigmoid (substrate activation), whereas the plot of v against [A], with [B] constant, may pass through a maximum (substrate inhibition). 2. Kinetic data for phosphofructokinase are of the latter type and it is suggested that the mechanism of this enzyme may involve a kinetically preferred pathway. It is emphasized that the phenomena of substrate inhibition and activation need not necessarily involve more than one binding site for each substrate on the enzyme molecule, nor more than one monomer per molecule.