Properties and reaction mechanism of C4 leaf pyruvate,Pi dikinase

The properties and reaction mechanism of maize leaf pyruvate,Pi dikinase are described. Km values were determined for the forward reaction substrates, pyruvate, ATP, and Pi, at pH 7.4 and 8.0 and for reverse reaction substrates at pH 7.4. Enzyme activity was almost totally dependent on added monovalent cations in both directions. NH+4 was most effective, with Ka values of about 0.38 mM for the forward reaction and 2 mM for the reverse reaction. K+ also completely activated the enzyme in the forward direction (Ka = 8 mM) but only partially activated in the reverse direction. Na+ had little effect on either reaction. The pH optimum for the forward reaction was about 8.2; the reverse reaction optimum was about 6.9. Maximum activity for the reverse direction was about twice the maximum forward direction rate. From data on the requirements for the ATP-AMP exchange reaction, on the mechanism of inhibition of the forward reaction by PEP, AMP, and PPi, and from the kinetics of the interaction of varying certain substrate pairs, it was concluded that the maize leaf pyruvate,Pi dikinase reaction proceeded by the two-step Bi Bi Uni Uni mechanism. This differs from the mechanism of catalysis by the bacterial enzyme.     

Estimation of the contribution of the bioluminescent reaction rate and quantum yield to the enhancement of firefly bioluminescence in the presence of cationic liposomes.

Cationic liposomes containing phosphatidylcholine, cholesterol and distearyldimethylammonium chloride (DSDAC) enhanced maximum light emission (BL intensity) and total light emission from the firefly bioluminescence (BL) reaction. The increase in BL intensity was interpreted on the basis of the increase in both BL reaction rate and BL quantum yield (PhiBL) of the BL reaction. The increase in BL reaction rate was due to the increase in the localized concentration of BL reactants on the surface of cationic liposomes by electrostatic interaction. On the other hand, the increase in PhiBL was due to the change of light-emitting species in the presence of cationic liposomes. Each contribution of BL reaction rate and PhiBL to the enhancement of the BL intensity was estimated by measuring the BL reaction rate and PhiBL in the presence of cationic liposomes containing various amounts of DSDAC. The contribution of the BL reaction rate to the increase in the BL intensity was found to be two-fold greater than that of PhiBL.     

Kinetic modeling of omega-transamination for enzymatic kinetic resolution of alpha-methylbenzylamine

A kinetic model for omega-transaminase from Bacillus thuringiensis JS64 was developed by using the King-Altman method to simulate the kinetic resolution of alpha-methylbenzylamine (alpha-MBA). Starting from a ping-pong bi-bi mechanism, a complete kinetic model including substrate inhibition only in the reverse reaction (i.e., transamination between acetophenone and L-alanine) was developed. The asymmetric synthesis of (S)-alpha-MBA proved to be difficult due to a much lower maximum reverse reaction rate than the maximum forward reaction rate, thermodynamically exergonic forward reaction (i.e., transamination between (S)-alpha-MBA and pyruvate), and the severe product and substrate inhibition of the reverse reaction. Experimental values for kinetic parameters show that the product inhibition constant of (S)-alpha-MBA is the most important parameter on determining the resolution reaction rate, suggesting that the resolution reaction rate will be very low unless (S)-alpha-MBA strongly inhibits the reverse reaction. Using the kinetic model, the kinetic resolution of alpha-MBA in aqueous buffer was simulated, and the simulation results showed a high degree of consistency with experimental data over a range of reaction conditions. Various simulation results suggest that the crucial bottleneck in the kinetic resolution of alpha-MBA lies mainly in the accumulation of acetophenone in reaction media as the reaction proceeds, whereas L-alanine exerts a little inhibitory effect on the reaction. The model predicts that removing acetophenone produced during the reaction can enhance the reaction rate dramatically. Indeed, the biphasic reaction system is capable of extracting acetophenone from the aqueous phase, showing a much higher reaction rate compared to a monophasic reaction system. The kinetic model was also useful in predicting the properties of other, better enzymes as well as the optimal concentrations of amino acceptor and enzyme in the resolution reaction.     

Ultrasonic monitoring of glycerol settling during transesterification of soybean oil

A low-intensity ultrasonic measurement system was used to monitor the products of transesterification of soybean oil in methanol to FAME (biodiesel). The byproducts of the transesterification reaction are methyl esters, glycerol and other products. During the transesterification reaction, the glycerol, having a higher density than the methyl ester, settles at the bottom of the reaction vessel. The aim of this study was to measure the glycerol deposition rate during transesterification and to assess the reaction rate and end time. Soybean oil was converted into biodiesel at four temperature levels. The amount of catalyst (KOH) used in the transesterification reactions was determined by titration. The ultrasonic waveforms captured during the reaction were recorded and analyzed automatically. The ultrasonic system monitored the effects of reaction temperatures on the glycerol settling rate and the reaction end times. The ultrasonic measurement of glycerol settling would be a useful non-destructive method for evaluating the effects of parameters such as catalyst amount, mixing time and temperature on transesterification reactions.     

Kinetic study on the reaction mechanism of pantothenase: existence of an acyl-enzyme intermediate and role of general acid catalysis.

A kinetic study was performed on the reaction mechanism of pantothenase (EC 3.5.1.22) catalyzed hydrolysis of the pantothenic acid. A nonlinear progress curve is derived if the reaction occurs at low buffer concentrations. The nonlinearity is due to partial reversibility of the reaction; an acylenzyme (pantoyl-enzyme) is formed during the reaction, and beta-alanine, the other end product, is able to react with the acyl-enzyme and return back to pantothenate. The dependence of the beta-alanine return reaction on buffer concentration and on pH suggests a general acid catalysis during the reaction. A reaction mechanism is suggested, in which the -NH3+ form of beta-alanine participates in the return reaction, and the deacylation of the acyl-enzyme is acid catalyzed.     

Extra-weak chemiluminescence of drugs. XII. Effect of the molar ratio of amino acid to sugar on extra-weak chemiluminescence in the Maillard reaction

The extra-weak chemiluminescence in the Maillard reaction caused by the reaction between L -lysine and D -arabinose was measured, and a linear relationship was found between the chemiluminescence and the amount of L -lysine added. After a 1-hour reaction equimolar amounts of D -arabinose and L -lysine were consumed regardless of the initial concentration of D -arabinose. The chemiluminescence of the Maillard reaction originates from Maillard reaction products formed by the equimolar reaction between sugar and amino acid and depends on the concentration of amino acid.     

Catecholamine sensitivity of the hypophyseal-adrenal system in the early ontogeny of rats]

The dynamics of the hypophyseal-suprarenal system reaction to catecholamines of different type of effect was studied in the Wistar rats during the first 16 days of life. The reaction to epinephrine was noted in 4 days old and to novodrin in 8 days old rats. Since the reaction to epinephrine acting on alpha- and beta-receptors appears ahead of that to novodrin acting selectively on beta-receptors, a suggestion was put forward to the effect that alpha-receptors matured more rapidly that beta-receptors. The increase of the system reaction to the stimulation of recepors sensitive to catecholamines was noted on the 12-14th days of life and appears to be related to the formation of central adrenergic mechanisms.     

Immunoelectron microscopic demonstration of estrogen receptors in osteogenic cells of Japanese quail

The localization of estrogen receptors (ERs) in osteogenic cells was immunoelectron microscopically examined in the femurs of female and estrogen-treated male Japanese quail. An electron dense reaction product showing ER localization was observed in the nuclei of osteoblasts and immature osteocytes in the medullary bone of the female quail. However, reaction product was not seen in the osteoclasts. On the endosteal bone surface of male quail, nuclear reaction product was detected in bone lining cells. After 24 h of estrogen treatment, reaction product was observed in the nuclei of preosteoblasts on the endosteal bone surface. After 48 h, the medullary bone partly appeared along the endosteal surface. Nuclear reaction product was seen in osteoblasts on the medullary bone surface.     

Immunoelectron microscopic demonstration of estrogen receptors in osteogenic cells of Japanese quail

Summary The localization of estrogen receptors (ERs) in osteogenic cells was immunoelectron microscopically examined in the femurs of female and estrogen-treated male Japanese quail. An electron dense reaction product showing ER localization was observed in the nuclei of osteoblasts and immature osteocytes in the medullary bone of the female quail. However, reaction product was not seen in the osteoclasts. On the endosteal bone surface of male quail, nuclear reaction product was detected in bone lining cells. After 24 h of estrogen treatment, reaction product was observed in the nuclei of preosteoblasts on the endosteal bone surface. After 48 h, the medullary bone partly appeared along the endosteal surface. Nuclear reaction product was seen in osteoblasts on the medullary bone surface.     

Engineering aspects of immobilized lipases on esterification: A special emphasis of crowding,confinement and diffusion effects

Cross‐linked enzyme crystal (CLEC) and sol‐gel entrapped pseudomonas sp. lipase were investigated for the esterification of lauric acid with ethanol by considering the effects of reaction conditions on reaction rate. The activation energy for the reaction was estimated to be 1097.58 J/mol and 181.75 J/mol for sol‐gel and CLEC entrapped lipase respectively. CLEC lipase exhibited a marginal internal diffusion effect on reaction rate over sol‐gel lipases and found to be interesting. The overall reaction mechanism was found to conform to the Ping Pong Bi Bi mechanism. The higher efficiency of sol‐gel lipases over CLEC lipases in esterification reaction is mainly due to the combined effects of crowding, confinement and diffusional limitations.     

The ultrastructural localization of adenosinetriphosphatase and alkaline phosphatase activity in eosinophil leukocytes

Summary The previously undescribed localization of reaction products of adenosinetriphosphatase and of alkaline phosphatase in eosinophil leukocytes was demonstrated by cytochemical studies of the rat intestine. Alkaline phosphatase reaction product was found only in minimal amounts on the plasma membrane but was distinct on the nuclear membranes and outer compartment of mitochondria but not on the cristae. The Golgi membranes and the endoplasmic reticulum reacted but less intensely. The specific granules showed no alkaline phosphatase activity.The adenosinetriphosphatase reaction, on the other hand, was found on the plasma membrane, vesicular or tubular profiles of the endoplasmic reticulum and on the matrix of the specific granules. The crystalloid of the granules did not show any reaction.Recipient of a postdoctoral fellowship from the muscular distrophy association of Canada.     

The action of chloride peroxidase on 4-chloroaniline. N-oxidation and ring halogenation.

Chloride peroxidase catalyses both the ring halogenation and N-oxidation reactions of 4-chloroaniline by H2O2 and either KCl or KBr. In the absence of any halide salt only the N-oxidation reaction was observed, with the resulting conversion of 4-chloroaniline into 4-chloronitrosobenzene. The N-oxidation reaction proceeded even more rapidly in the presence of Cl- or Br-, in spite of the fact that ring halogenation was also a rapid reaction. The enhancement of N-oxidation was highly dependent on the pH of the media and displayed an optimum in the region of pH 3.5-4.0. No rate enhancement was observed above pH 5.5. KF partially inhibited the rate of N-oxidation in a pH-dependent manner. On the basis of calculated catalytic-centre activity the N-oxidation reaction was the major reaction at pH 3.5 or higher, whereas the ring-halogenation reaction became the major reaction below pH 3.5. In the presence of high concentrations of 4-chloroaniline relative to H2O2 the reaction intermediate, 4-chlorophenylhydroxylamine, was detected for the first time in a chloride peroxidase-catalysed reaction with this arylamine substrate. These findings were interpreted on the basis of current knowledge concerning the mechanism of action of chloride peroxidase.     

Tissue expression of proliferative antigens (PCNA and Ki-67) in oral lichen ruber related to clinical status

The aim of this study was to determine the expression intensity of PCNA and Ki-67 tissue antigens related to pathologically modified oral mucosa in OLR lesions, and to determine the reaction intensity of these antigens in individual clinical forms, i.e. lichen ruber planus (LRP) and lichen ruber erosivus (LRE) comparing the reaction intensity with the inflammation grade and the degree of hyperkeratosis in lesions of 30 patients. Control group included patients (n = 15) with oral leukoplakia simplex. Tissue antigens were observed by immunohistochemical analysis using APAAP and LSAB methods. The reaction on tested tissue antigens was focal positive and of mosaic type. The reaction of the PCNA antigen was intensely high in OLR lesions regardless on the clinical form of the lesion. The reaction intensity positively correlated with the inflammation grade and the degree of hyperkeratosis in lesions. The reaction on Ki-67 tissue antigen ranged from low to moderately high intensity. Intensely high reaction was observed in lesions of lichen ruber erosivus. The reaction positively correlated with the inflammation grade and the degree of hyperkeratosis in lesions. Observed modified reaction of analyzed tissue antigens related with individual clinical forms of OLR might be the indicator of transformed nature of these lesions.     

Effect of additives on gas-phase catalysis with immobilised Thermoanaerobacter species alcohol dehydrogenase (ADH T)

This paper presents a strategy for preparing an efficient immobilised alcohol dehydrogenase preparation for a gas-phase reaction. The effects of additives such as buffers and sucrose on the immobilisation efficiency (residual activity and protein loading) and on the gas-phase reaction efficiency (initial reaction rate and half-life) of Thermoanaerobacter sp. alcohol dehydrogenase were studied. The reduction of acetophenone to 1-phenylethanol under in situ cofactor regeneration using isopropanol as co-substrate was used as a model reaction at fixed reaction conditions (temperature and thermodynamic activities). A strongly enhanced thermostability of the enzyme in the gas-phase reaction was achieved when the enzyme was immobilised with 50 mM phosphate buffer (pH 7) containing sucrose five times the protein amount (on weight/weight basis). This resulted in a remarkable productivity of 200 g L⁻¹ day⁻¹ even at non-optimised reaction conditions. The interaction of additives with the enzyme and water affects the immobilisation and gas-phase efficiencies of the enzyme. However, it was not possible to predict the effect of additives on the gas-phase reaction efficiency even after knowing their effect on the immobilisation efficiency.     

Effect of mitochondria on the redox reaction between oxyhemoglobin and ferricytochrome c

The effect of mitochondria on the redox reaction between oxymyoglobin (oxy-Mb) and ferricytochrome c was studied. The parameters of this reaction in the absence of mitochondria have been investigated earlier. It is shown that the course of oxidation of oxymyoglobin by cytochrome c in the presence of mitochondria differs from that without mitochondria: no reduced cytochrome c is observed; in addition, the order of this redox reaction and its dependence on pH and ionic strength change. The factors influencing the state of mitochondrial membrane and uncouples enhance markedly the reaction rate. The conclusion was drawn that mitochondria directly participate in the oxymyoglobin-cytochrome c redox reaction. The possibility of this reaction in vivo under extreme conditions and during pathological processes is discussed.     

Evaluation of antifungal volatile compounds on the basis of the elongation rate of a single hypha

A novel method is proposed for the evaluation of the activity of an antifungal agent administered as a gas. This system is composed of a batch-flow type reaction vessel, a gas flow system, and a microscopic observation system. The agar plate was prepared on the ceiling of the reaction vessel, and the mycelium of a fungus (Aspergillus niger or Rhizoctonia solani) was inoculated onto it. After preincubation at 25 degrees C for 24 h, the reaction vessel was connected to the gas flow system. An appropriate hypha was selected, and its elongation rate was measured. Then a sample holder containing an antifungal compound was inserted into the reaction vessel from the side hole to saturate the atmosphere inside with its vapor. The retardation or inhibition of the hypha elongation was observed on a television monitor and recorded on a video tape recorder. The antifungal compound was then removed, and the reaction vessel was flushed with air. If the hypha lived, it began to elongate again. By this method, antifungal activity of seven odor compounds could be evaluated quantitatively within several hours.     

Evaluation of antifungal volatile compounds on the basis of the elongation rate of a single hypha.

A novel method is proposed for the evaluation of the activity of an antifungal agent administered as a gas. This system is composed of a batch-flow type reaction vessel, a gas flow system, and a microscopic observation system. The agar plate was prepared on the ceiling of the reaction vessel, and the mycelium of a fungus (Aspergillus niger or Rhizoctonia solani) was inoculated onto it. After preincubation at 25 degrees C for 24 h, the reaction vessel was connected to the gas flow system. An appropriate hypha was selected, and its elongation rate was measured. Then a sample holder containing an antifungal compound was inserted into the reaction vessel from the side hole to saturate the atmosphere inside with its vapor. The retardation or inhibition of the hypha elongation was observed on a television monitor and recorded on a video tape recorder. The antifungal compound was then removed, and the reaction vessel was flushed with air. If the hypha lived, it began to elongate again. By this method, antifungal activity of seven odor compounds could be evaluated quantitatively within several hours.     

Stimulation of the Streptococcus pneumoniae RecA protein-promoted three-strand exchange reaction by the competence-specific SsbB protein

The effect of the transformational competence-specific Streptococcus pneumoniae single-stranded DNA binding protein, SpSsbB, on the ATP-dependent three-strand exchange activity of the SpRecA protein was investigated. Although SpRecA exhibited only a trace level of strand exchange activity in the absence of SpSsbB, an extensive strand exchange reaction was observed when SpSsbB was added to the reaction solution after SpRecA. A more limited strand exchange reaction was observed, however, when SpSsbB was added to the reaction solution before SpRecA. This dependence on the order of addition, together with additional DNA-dependent ATP hydrolysis experiments, indicated that the mechanism of stimulation may involve the postsynaptic binding of SpSsbB to the displaced linear single-stranded DNA reaction product. When dATP was provided in place of ATP as the nucleotide cofactor (to suppress a potentially inhibitory effect of SpSsbB on the interaction of SpRecA with the circular ssDNA reaction substrate), the stimulatory effect of SpSsbB on the strand exchange reaction was apparent regardless of the order in which it was added to the reaction solution. These findings suggest that SpSsbB may be able to facilitate SpRecA-promoted DNA recombination reactions during natural transformation in S. pneumoniae.     

Characterization of eosin 5-isothiocyanate binding site in band 3 protein of the human erythrocyte

The characteristics of the anion transport system in human erythrocyte, which can be modified by eosin 5-isothiocyanate (EITC), were studied using the pH titration method and by measuring the sulfate efflux. Based on the pH dependence of EITC binding to the erythrocyte ghosts, it was found that the reaction rate was maximal at about pH 6.4, and that the pH profile of EITC binding was similar to that of divalent anion transport. The interaction between EITC and ghosts was interpreted by a two-step reaction, a fast ionic-binding reaction and a slow covalent-binding reaction. The induced CD spectrum of the EITC-ghost system was also dependent on pH. The intensity of the CD band at 530 nm was decreased in acidic pH region, and the inflection point was observed at about pH 6.3, indicating a participation of the histidine residue in the interaction of EITC with band 3. In order to characterize the EITC-binding site, the kinetics of sulfate efflux in intact and EITC-modified cells were examined at various pH values. The inhibitory effect of EITC was dependent on pH. From the experimental results, the followings are suggested. The rate of ionic interaction in the early stage is much slower than that in a general ionic reaction. A conformational change may participate in the reaction. The conformation of the EITC-binding site depends on pH, relating to the dissociation of the histidine residues. The EITC molecules act also as a competitive inhibitor to the sulfate efflux after binding covalently to band 3 protein.     

Short synthesis of a benzyl ether-protected building block for the synthesis of carbocyclic galactopyranose mimics

A versatile intermediate for the synthesis of galactose-mimicking carbasugars was synthesised from tetrabenzyl galactose in five steps and 30% overall yield. The reaction sequence uses an l-proline-mediated aldol reaction as key step. The reaction sequence was run on several grams of material.