Initial Reduction Rate Research Articles

Electron Transfer from α-Reduced Nicotinamide Adenine Dinucleotide to Flavoprotein, Cytochromes, and Mixed Function Oxidases of Rat Liver Microsomes

Abstract Cytochrome b5 of rat liver microsomes is reduced by α-NADH; the extent of reduction is equal to that obtained with β-NADH. With both nucleotides, the rates of reduction of cytochrome b5 are very fast; alternatively, rates of reduction of cytochrome c and dichloroindophenol have been measured. The rates observed with α-NADH are about 10% of the rates observed with β-NADH. Conditions have been established for the measurement of first order kinetic parameters in these reductions: Km of α- and β-NADH are 13.0 and 3.3 µm for cytochrome c reductase activity and 5.1 and 6.7 µm, respectively, for dichloroindophenol reduction. Three lines of evidence suggest that α-NADH and β-NADH may reduce cytochrome c and dichloroindophenol via the same microsomal flavoprotein. (a) There is competition between α- and β-NADH. (b) The relative rates of cytochrome c reduction are diminished equally for α- and β-NADH when the enzyme is inhibited or denatured. (c) The relative rates of reduction of both cytochrome c and dichloroindophenol are increased equally for α- and β-NADH when microsomal cytochrome b5 reductase is enriched. Both α- and β-NADH are electron donors to cytochrome P-450 in microsomes. The initial rates of reduction of cytochrome P-450 by α- and β-NADH are equal (K = 0.126 min-1) and considerably slower than reduction by NADPH. In a second, slower phase of cytochrome P-450 reduction, the rate is somewhat less rapid with α-NADH (K = 0.030 min-1) than with β-NADH (K = 0.042 min-1). Compared to β-NADH and NADPH, α-NADH is a more efficacious donor of electrons for microsomal mixed function oxidation of a methyl sterol intermediate of cholesterol biosynthesis. Furthermore, the substrate-independent rate of oxidation of α-NADH is very slow (10% of β-NADH). To date, only diaphorase-like activities have been reported for oxidation of α-NADH. Although the present report shows clearly that α-NADH may function as an electron donor to microsomal mixed function oxidases, the physiological significance of these findings remains obscure because enzymic reduction of α-NAD+ has not yet been reported.

Study of promoting effect of amines in reduction of cyclohexanone with 2-propanol, catalyzed by tris(triphenylphosphine)ruthenium (II) bichloride complex

1. Aqueous solutions of triethylamine and piperidine promote the homogeneous reduction of cyclohexanone by 2-propanol in the presence of the RuCl2(PPh3)3 complex. 2. At low concentrations of the indicated amines the initial reduction rate of the ketone correlates satisfactorily with the change in the relative pH* value of the solution. 3. Pyridine and n-amylamine do not promote the process and completely destroy the promoting effect of triethylamine and piperidine. Tribenzylamine and diphenylamine lower five- and 20-fold, respectively.

Evaluation of Opsonic and Leukocyte Function With a Spectrophotometric Test in Patients With Infection and With Phagocytic Disorders

Abstract Paraffin oil droplets containing Oil Red O and coated with Escherichia coli lipopolysaccharide were ingested rapidly by human peripheral blood phagocytes only if they were pretreated with normal serum. This property formed the basis of a screening test in which lipopolysaccharide-coated particles were opsonized with patients’ serums and then were added to autologous leukocytes suspended in two portions, one of which contained nitroblue tetrazolium. After these incubations the cells were washed and extracted with dioxane. Oil Red O and nitroblue tetrazolium formazan in the dioxane extracts were spectrophotometrically assayed, thereby providing simultaneous determinations of the initial rates of ingestion and nitroblue tetrazolium reduction. The test differentiated opsonically-deficient serums and chronic granulomatous disease phagocytes from normals. Serums from individuals with bacterial infections had supernormal opsonic activity, and leukocytes from these patients had increased rates of nitroblue tetrazolium reduction in response to ingestion.

Photolysis of Oxalato-Titanium (IV) Complex in the Presences of an Excess of Oxalic Acids

By irradiation with a low pressure mercury lamp, photolys is of oxalato-titanium(N) complex was studied in aqueous solution at constant pH with excess amount of oxalic acid to prevent any secondary reactions, such as hydrolysis of the primary product.In the presence of excess amount of oxalic acid, the composition of oxalato-titanium(N) and ()complexes were investigated spectrophotometrically and were assumed t be TiO(C20i)2 2 and Ti(C20k)2Irradiation of a solution of oxalato-titanium(N) complex in excess amount of oxalic acid, in which the molar ratio of Ti(N) and oxalic acid is 1: 10, led to photoreduction and hydrolysis of the complex. The photoreduction reaction forms an oxalato-titanium()complex with evolution of carbon dioxide. The hydrolysis reaction gives a precipitate of titanium(N) hydroxide with evolution of carbon dioxide and carbon monoxide.Furthermore, a photosensitized reaction of oxalic acid by the oxalato-titanium()complex oceured during the irradiation. Consequently, oxalic acid decomposed to carbon dioxide and earbon monoxide.The initial reduction rate and the final reduction ratio decreased linearly with increase of pH value of the sample solution. Thus, it was found that H ion contributed to the photoreduction of oxalato-titanium(N) complex.

Kinetics of the reaction of cyclohexene with the surface of a manganese dioxide

The reaction of cyclohexene with the surface oxygens of MnO 2 produces benzene and water. The kinetics of this irreversible chemisorption has been studied in a flow system. The influence of cyclohexene pressure on the rate of benzene formation, suggests that the surface reaction is rate determining. The number of reactive oxygens has been shown to be increasing with the temperature, reaching a value close to half of the total surface oxygen. The temperature dependency of the number of reactive oxygens, follows the same pattern as the initial rate of reduction (8 kcal/mole). The rate-determining step could thus be the availability of the sites, as was already assumed by Taylor and Thon for the kinetics of chemisorption.

Factors governing the kinetics and steady state of the mitochondrial nicotinamide nucleotide transhydrogenase system.

The mitochondrial nicotinamide nucleotide transhydrogenase catalyzes a rapid reduction of NAD+ by NADPH as compared to the reduction of NADP+ by NADH. By assuming pseudo first-order kinetics it is shown that the rate constant of the reaction is strongly dependent on the actual [NAD+] [NADPH] / [NADP+] [NADH] ratio, giving a half-maximal rate constant at [NAD+] [NADPH]/[NADH] [NADP+] of approx. 0.003. The altered rate constant can possibly be explained on the basis of a reversible conformational change of the enzyme between an active and an inactive state, controlled by the prevailing concentrations of oxidized and reduced nicotinamide nucleotides. Possible similarities with bacterial transhydrogenases are considered. In the presence of an energy source the overall transhydrogenase reaction involves a strong shift of the equilibrium towards the conversion of NADH and NADP+ into NAD+ and NADPH. Evidence is presented that the final extent of the overall energy-linked transhydrogenase reaction is strikingly dependent on the ratio of the initial rates of the energy-linked reduction of NADP+ by NADH and the nonenergy-linked reduction of NAD+ by NADPH. Thus, the final extent of the transhydrogenase reaction in the presence of an energy source can by explained in terms of a steady state rather than an equilibrium. The possibility of an energy-linked conformational change of transhydrogenase, involving a conversion of the enzyme from an inactive to an active state, is considered.

A Comparative study on the effects of phenobarbital and 3,4-benzpyrene on the hydroxylating enzyme system of rat-liver microsomes

The effects of phenobarbital and 3,4-benzpyrene on the hydroxylating enzyme system of rat-liver microsomes have been compared. Repeated injections of phenobarbital into rats caused a marked increase in the concentration of cytochrome P-450, and in the activities of NADPH-cytochrome P-450 reductase and NADPH-cytochrome b 5 reductase, as well as an enhancement of the rate of aminopyrine demethylation, whereas 3,4-benzpyrene hydroxylation was only moderately stimulated. The latter activity, however, was enhanced several-fold by the injection of a single dose of 3,4-benzpyrene. Associated herewith there was a formation of cytochrome P-448 but there was no obvious increase in the rate of reduction of the hemoprotein by NADPH. When added to the assay system in vitro, aminopyrine and to a minor extent 3,4-benzpyrene stimulated the initial rate of NADPH-cytochrome P-450 reduction catalyzed by liver microsomes from controls. Using liver microsomes from 3,4-benzpy-rene-treated rats, 3,4-benzpyrene was more potent than aminopyrine in stimulating the rate of NADPH-cytochrome P-448 reduction. The concentration of the polycyclic hydrocarbon needed to obtain maximum stimulation of the rate of reduction of the cytochrome by NADPH was considerably lower with liver microsomes from 3,4-benz-pyrene-treated rats than from controls. Furthermore, aminopyrine, which markedly inhibited 3,4-benzpyrene hydroxylation catalyzed by control liver microsomes, had considerably less effect on this activity when microsomes from 3,4-benzpyrene-treated rats were used as the source of enzyme. The results support the hypothesis that in contrast to phenobarbital, 3,4-benzpyrene yields a modified cytochrome (P-448) in the liver microsomes with an increased affinity for 3,4-benzpyrene as well as a decreased affinity for aminopyrine. Possible mechanisms for cytochrome P-448 formation are discussed.

The catalytic oxidation of 1-butene over bismuth molybdate: V. The kinetics of the oxidation: B. Experiments with continuous flow and recirculation; reduction and reoxidation of BiMo

The kinetics of the oxidation of 1-butene over the Bi 2O 3 · MoO 3 (koechlinite) catalyst were investigated in continuous flow experiments and in experiments with recirculation of the feed. The reaction was strongly inhibited by the product butadiene especially at lower temperatures and is zero-order in the oxygen pressure. The order of the butene partial pressure could not be determined because of the predominance of the inhibition by the product. The initial rate of reduction of the catalyst by 1-butene is approximately equal to that of the catalytic reaction: it is also strongly inhibited by butadiene and the rate appears to be determined by the combined influences of inhibition and progressive oxygen depletion. Contrary to reduction by hydrogen, that follows the usual parabolic law, it does not progress further than Mo 5+ and Bi 3+. Reoxidation of catalyst once reduced is fast and not inhibited by butadiene. Therefore, the diene inhibition has to be viewed as a competition between butene and butadiene for the same oxidized site. It is concluded that the absence of diene inhibition in pulse experiments is caused by the diene adsorption being slow in comparison to the residence time in pulse technics. By combining the data the conclusion can be drawn that the curved log k vs. 1 T relation observed is caused by the interference of the diene inhibition. The diene inhibited reaction has an activation energy of 36 kcal mole −1, the noninhibited reaction, of 11 kcal mole −1.

Kinetics of the reduction of tetrathionate by intact bacterial cells.

The kinetics of the reduction of tetrathionate by washed nongrowing intact cells ofCitrobacter were studied. The rate of the reduction in most cases is not constant. The course of reduction usually resembles the first order kinetics within a limited, varying initial time period. This finding makes it possible to evaluate quantitatively the curves obtained experimentally of the relationship between concentration of the product and time, applyingequations valid for this reaction order, to determine the rate constant of the first order reaction and thus to determine the initial rate of the reduction of tetrathionate as the measure of the tetrathionate-reductaso activity of intact cells. Deviations from the first order kinetics, observed during later phases of the reduction are caused by excessive acidification of the medium. The effect of various experimental conditions on the initial rate of reduction of tetrathionate by the cells was also studied. The results presented served as a basis of a general method for the quantitative evaluation of the tetrathionate-reductase activity of intact bacterial cells.

The effect of steroid hydroxylase inhibitors on the rate of reduction of adrenal mitochondrial cytochrome P-450

When added to adrenocortical mitochondria, metyrapone, amphenone and aminoglutethimide induce type II difference spectra, and block both the initial rate of malate-linked cytochrome P-450 reduction and also the DOC-induced increase in this rate. Removing the -NH 2 group from aminoglutethimide shifts the spectrum to shorter wavelengths and reduces its effectiveness in blocking P-450 reduction. These compounds appear to block steroid hydroxylation by binding to oxidized P-450 and only very slowly allowing the P-450 to be reduced.

Equilibrium kinetic study of the mechanism of mitochondrial and supernatant malate dehydrogenases of bovine heart

The kinetics at equilibrium have been examined for mitochondrial and supernatant malate dehydrogenases ( l-malate:NAD + oxidoreductase, EC 1.1.1.37) from bovine heart at catalytic concentration by means of isotopic exchange of reactants to compare their catalytic mechanisms. In accord with previous studies of lactate, alcohol and pig heart mitochondrial malate dehydrogenases the rate of substrate interchange exceeded the NAD + ⇌ NADH rate, in some cases by more than 100-fold, except at lowest substrate concentration. At pH 8.0 a marked depression in the NAD + ⇌ NADH rate to near zero without inhibition in the oxaloacetate ⇌ malate rate was compatible with a compulsory binding order with substrate binding to E (enzyme)-coenzyme complexes, but not to free enzyme. At pH 9.0 only a moderate depression in the NAD + ⇌ NADH rate was found, indicating a partially compulsory pathway in which coenzyme dissociation occurs from both E- coenzyme and E- coenzyme-substrate complexes, but is more rapid from the binary complex. Kinetics with variable concentration of non-reactive pairs of substrate and coenzyme suggested the formation of the abortive complex E- NADH malate, but not E- NAD +-oxaloacetate . The initial rate of reduction of oxaloacetate was in all cases greater than the equilibrium rates. Minimum estimates were obtained for some dissociation constants. Despite considerable structural differences, the similar kinetic behavior at equilibrium of the mitochondrial and supernatant enzymes suggests a uniform catalytic mechanism which includes a compulsory binding order at pH 8.0 with coenzyme binding prior to substrate, a partially compulsory pathway at pH 9.0, non-rate-limiting chemical transformation, and formation of the abortive complex, E- NADH-malate . This mechanism is also in accord with that previously found with pig heart mitochondrial malate dehydrogenase.

Kinetics of enzyme-substrate reactions in single living cells

SUMMARY Changes in NAD (or NADP) reduction due to microelectrophoretic additions of metabolites are followed in localized areas of single living cells (EL2 cells) by means of a modified Chance-Legallais microfluorimeter or an optimized photon counting method with a time resolution from 1 to 1 100 second. The glycolytic activity is dependent upon mitochondrial-extramitochondrial competition for activators and the removal of reducing equivalents. At 23° the glycolytic rate is decreased by several times in presence of lactate, with recovery at 37°. While the LDH pathway is predominant, the contributions of the mitochondrial oxidations and a-glycerophosphate shuttle are also significant. In rapid microfluorimetric recordings the initial rate of NAD reduction can be made to vary with substrate concentration or changes in metabolites and cell type.

The role of ubiquinone in the respiratory chain of Acetobacter xylinum

1. Whole cells of Acetobacter xylinum were found to contain a quinone of the ubiquinone (coenzyme Q) group. The quinone was isolated from the cells and crystallized. It was identified by its physical, chemical and spectroscopic properties as a ubiquinone with 10 isoprene units (ubiquinone-10). No naphthaquinone was detected in the cells. 2. Cell-free extracts prepared by means of a French pressure cell were separated into three fractions by differential centrifugation. The ubiquinone was located predominantly in the particulate fraction sedimenting at 33000g, which also contained most of the NADH oxidase and malate oxidase activities. The concentration of ubiquinone-10 in extracts was similar to that of the flavoproteins and about three times the concentration of the individual cytochromes. 3. Aerobic incubations of crude extracts with either NADH or malate resulted in reduction of the endogenous ubiquinone-10 to steady-state concentrations of 55 and 40% of the total quinone respectively. In the presence of cyanide more than 95% of the endogenous ubiquinone-10 was reduced by either NADH or malate. 4. The initial rate of reduction of endogenous ubiquinone-10 by malate and the rate of ubiquinol oxidation, in A. xylinum extracts, were found to be compatible with the overall rate of malate oxidation with oxygen. 5. The effects of various respiratory inhibitors on the oxidation-reduction reactions of the endogenous quinone indicate that its position on the respiratory chain is between the malate flavoprotein dehydrogenase and the cytochrome chain.

Study on the Precipitation of Copper Powder from Aqueous Cupric Sulphate Solution by Hydrogen Pressure Reduction

This paper is concerned with the precipitation of copper powder from aqueous cupric sulphate solution by hydrogen pressure reduction. Experiments have been done in 3l stainless autoclave, equipped with the sampling device and the magnetic induction agitation system. The results obtained are as follows: (1) It is easy to precipitate metallic copper from acidic copper sulphate solution, but the addition of certain organic compounds to the reduction charge is indispensable to prevent the plating and plastering of copper on the wall of the autoclave. A kind of acryl compounds have been used in this research. The addition of this dispersion agent effects the reduction rate and the physical characteristics of the precipitated powder. (2) Although the reaction proceeds without catalyst, the addition of fine copper powder as seed increases the rate a little.(3) The addition of ammonium sulphate to the reduction charge effects a marked buffering action to depress the increase of the acidity from the reaction. The reduction rate increases with the amount of ammonium sulphate added and the initial reduction rate is in proportion to it, (4) The initial reduction rate is proportional to the hydrogen partial pressure and the initial copper concentration.(5) The following reducing conditions appear to be optimum: Cu 50g/l, dispersion agent 0.1g/l. (NH4)2SO4/Cu=1 (molar ratio), 150°C, PH2 20-30kg/cmcm2.(6) From the results as stated above, the initial reduction rate can be represented by an equation of the form:(Received June 4, 1965)

Action spectrum, kinetics and quantum requirement of phosphopyridine nucleotide reduction and cytochrome oxidation in the blue-green alga Anacystis nidulans

By means of fluorescence and absorption-difference spectrophotometry the kinetics were measured of phosphopyridine nucleotide reduction and cytochrome oxidation in intact cells of the blue-green alga Anacytis nidulans upon illumination with light of various wavelengths. The action spectrum of phosphopyridine nucleotide reduction was found to be proportional to that of cytochrome oxidation; both action spectra showed activity of chlorophyll a and of phycocyanin, but a relatively higher activity of chlorophyll a than the action spectrum of photosynthesis. 3-(3,4-Dichlorophenyl)- i,i-dimethylurea, known as a very potent inhibitor of photosynthesis, did only slightly affect the initial rate of phosphopyridine nucleotide reduction upon illumination. The efficiency of cytochrome oxidation was appreciably increased by 3-(3,4-dichlorophenyl)- i,i-dimethylurea in light of 620 mμ, mainly absorbed by phycocyanin, but not or only slightly in light of 680 mμ, mainly absorbed by chlorophyll a. The number of quanta, required for the reduction of a phosphopyridine nucleotide molecule, was found to be about 5 at 620 mμ; the number needed for the oxidation of a molecule of cytochrome was 7–10 at 680 mμ. The results, together with other evidence, support the hypothesis that in algal photosynthesis two different photochemical oxidation-reduction reactions occur, mediated by two different pigment systems. The two systems operate in series: the first one reduces phosphopyridine nucleotide and oxidizes the cytochrome, the other one reduces the cytochrome and produces O 2 by means of the oxidation of water. Possibly in both light reactions one quantum is needed for the transfer of one hydrogen equivalent.

Enzymatic reduction and oxidation of glutathione by illuminated chloroplasts

1. 1. The photochemical reduction of oxidized glutathione has been demonstrated in the presence of washed spinach chloroplast fragments, glutathione reductase preparations, and a catalytic concentration of triphosphopyridine nucleotide. 2. 2. Reduction of as much as 90 % of the oxidized glutathione added was obtained. Initial rates of reduction were observed which approached 50 % of the rate of reduction of quinone measured under similar conditions. 3. 3. Oxygen production never exceeded 80 % of the equivalent amount of reduced glutathione formed. Possible explanations for this discrepancy are discussed. 4. 4. A light-catalyzed oxidation of reduced glutathione by molecular oxygen was observed in the presence of chloroplasts and certain of the reductase preparations.