Indoleacetaldoxime hydro-lyase : II. Purification and properties

The purification and some properties of the enzyme indoleacetaldoxime hydrolyase (EC 4.2.1.29) from the fungus Gibberella fujikuroi, which dehydrates indoleacetaldoxime (IAOX) to indoleacetonitrile (IAN), are described. The enzyme activity in the fungus is present only under certain culture conditions. It is a soluble enzyme, has an optimum pH at 7, shows an energy of activation of —15,670 cal/mole, and has a Michaelis constant of 1.7 × 10−4 Image at 30 °. It appears to be specific for IAOX, and 1 mole of IAN is produced per mole of IAOX utilized. The enzyme is inhibited by a number of aldoximes of which phenylacetaldoxime (PAOX) is the most potent inhibitor. Inhibition by PAOX is competitive (Ki = 2.2 × 10−8 Image ). The enzyme is inhibited by SH reagents such as p-hydroxymercuribenzoate and N-ethylmaleimide, and by a number of SH compounds such as cysteine, β-mercaptoethanol, and 2,3-dimercaptopropanol (BAL). However, glutathione activates the enzyme. Metal chelating agents such as 8-OH-quinoline and diethyl dithiocarbamate inhibit the enzyme; the inhibition is partly reversed by ferric citrate. Ascorbic acid, and particularly dehydroascorbic acid (DHA), are good activators of the enzyme. Several other biological oxidants had either no action or had a slight effect. Potassium cyanide activates the enzyme at low concentration but inhibits at higher concentrations. Reduction of the enzyme with NaBH4 reduces activity, and the effect is partly reversed by pyridoxal phosphate and also by DHA. The above properties indicate that both an SH function and an oxidized function are required for activity.

Purification and properties of uridine hydrolase from mung-bean (Phaseolus radiatus) seedlings

The occurrence in plants of an enzyme system catalyzing the cleavage of uridine has been demonstrated. The enzyme from Phaseolus radiatus was purified about 132-fold with 24% recovery by a combination of procedures involving mild acid treatment, ammonium sulphate fractionation, negative adsorption on calcium phosphate gel and DEAE-cellulose chromatography. The enzyme cleaves uridine to uracil and ribose in the absence of phosphate indicating that the mechanism of cleavage was hydrolytic rather than phosphorolytic. The enzyme is specific to uridine and does not act on other purine and pyrimidine compounds. The enzyme shows maximum activity at pH 7.4 and has a temperature optimum of 45 °. It does not require metal ions for activity. Inhibition of the enzyme by p-chloromercuribenzoate as well as N-ethylmaleimide and the reversal of p-chloromercuribenzoate inhibition by sulfhydryl agents indicate the probable involvement of readily oxidizable sulfhydryl groups in enzyme activity.

Two-stream instability in plasma

The problem of two-stream instability in plasma is studied by specifying the importance of initial magnetic field associated with the motion of the charged particles and the boundary effects. In Part I the accurate initial steady state is studied when the streams of electrons and ions move with different uniform speeds in plasmas with plane and cylindrical geometry. In Part II, in order to show the effects of finiteness and inhomogeneity of the system, small transverse plasma oscillations are studied in the case of plane plasmas. The role of plasma-sheath oscillations at the boundaries is found to be very important in driving the instabilities associated with the electromagnetic modes. The numerical estimates of the growth rates of the instability are given for the specific case of the physical data in discharge tubes.

A Dynamic Migration Model for self-adaptive Genetic Algorithms

In this paper, we propose a self Adaptive Migration Model for Genetic Algorithms, where parameters of population size, the number of points of crossover and mutation rate for each population are fixed adaptively. Further, the migration of individuals between populations is decided dynamically. This paper gives a mathematical schema analysis of the method stating and showing that the algorithm exploits previously discovered knowledge for a more focused and concentrated search of heuristically high yielding regions while simultaneously performing a highly explorative search on the other regions of the search space. The effective performance of the algorithm is then shown using standard testbed functions, when compared with Island model GA(IGA) and Simple GA(SGA).

Studies on the metabolism of o-aminophenol purification and properties of isophenoxazine synthase from Bauhenia monandra

An enzyme which catalyzes the oxidative conversion of o-aminophenol to 2-amino-3-H-isophenoxazin-3-one has been purified 396-fold by using standard fractionation procedures. The enzyme is specific for o-aminophenol and has pH and temperature optima at 6.2 and 40 °, respectively. It is insensitive to metal chelating agents but is inhibited by several reducing substances. There is no cofactor or metal ion requirement for the reaction. A competitive type of inhibition was observed with structural analogs such as anthranilic acid and 3-hydroxyanthranilic acid. There are no free sulfhydryl groups in the enzyme, but preincubation of the enzyme with substrate or substrate analogs resulted in the liberation of titratable free sulfhydryl groups. The mechanism of biosynthesis of isophenoxazine ring is discussed.

Purification and properties of an inhibitor for nicotinamide-adenine dinucleotidase from Mycobacterium tuberculosis H37Rv

The excess of free inhibitor for the enzyme NADase present in the crude cell-free extracts of Mycobacterium tuberculosis H37Rv has been purified by chromatography on a DEAE-cellulose column and adsorption and elution from alumina Cγ-gel. Some of the properties of the purified inhibitor have been studied and attempts have been made to elucidate the nature of combination between the enzyme and the inhibitor. The purified inhibitor may be glycoprotein in nature, and considerable loss in the activity of the inhibitor preparations could be brought about by trypsin digestion. The inhibitor was specific for the enzymes from M. tuberculosis H37Rv or H37Ra and could be stored for at least 6 months in the frozen state below 0 ° without any significant loss in activity. The inhibition was noncompetitive with respect to the substrates, and the enzyme-inhibitor complex formed was undissociable.

Purification and properties of an inhibitor for nicotinamide-adenine dinucleotidase from Mycobacterium tuberculosis H37Rv

The excess of free inhibitor for the enzyme NADase present in the crude cell-free extracts of Mycobacterium tuberculosis H37Rv has been purified by chromatography on a DEAE-cellulose column and adsorption and elution from alumina Cγ-gel. Some of the properties of the purified inhibitor have been studied and attempts have been made to elucidate the nature of combination between the enzyme and the inhibitor. The purified inhibitor may be glycoprotein in nature, and considerable loss in the activity of the inhibitor preparations could be brought about by trypsin digestion. The inhibitor was specific for the enzymes from M. tuberculosis H37Rv or H37Ra and could be stored for at least 6 months in the frozen state below 0 ° without any significant loss in activity. The inhibition was noncompetitive with respect to the substrates, and the enzyme-inhibitor complex formed was undissociable.

Propagation of a spherical shock in an inhomogeneous self-gravitating or nongravitating system

The propagation of a shock wave of finite strength due to an explosion into inhomogeneous nongravitating and self-gravitating systems has been considered, using similarity principles, supposing that the density varies as an inverse power of distance from the centre of explosion. A large number of systems, characterised by different density exponents and different adiabatic coefficients of the gas have been considered for different shock strengths. The numerical integration from the shock inward has been continued to the surface of singularity where density tends to infinity and which acts like a piston in the self-gravitating case and to the surface where the velocity gradient tends to infinity in the nongravitating case. The effect of variation of shock strength, density exponent and adiabatic coefficient on the location of these singularities and on the distribution of flow parameters behind the shock has been studied. The initial energy of the system and the manner of release of the explosion energy influence strongly the flow behind the shock. The results have been graphically depicted.

Nucleotidases in plants : I. Partial purification and properties of the enzyme hydrolyzing flavine adenine dinucleotide from mung bean seedlings (Phaseolus radiatus)

The occurrence of an enzyme hydrolyzing flavine adenine dinucleotide (FAD) was demonstrated in a number of seed extracts. The enzyme from Phaseolus radiatus was purified 104-fold by fractionation with ammonium sulfate and ethanol and by negative adsorption on alumina Cγ gel. The enzyme cleaves the POP bond of FAD to yield flavine mononucleotide and adenosine monophosphate. When reduced glutathione is added to the enzyme, it cleaves FAD at the COP bond to yield riboflavine, adenosine, and pyrophosphate, Both the activities are optimal at a pH of 7.2 and at a temperature of 37 . The Km for both the activities is 1.65 × 10−5 M. The stoichiometry and the identity of the products of both the treated and untreated enzyme were established. The untreated enzyme was not inhibited by pCMB or arsenite, but the treated enzyme was sensitive to both these inhibitors. The inhibition by pCMB could be reversed by monothiols and the inhibition by arsenite by dithiols.

Biosynthesis of isoleucine and valine in mycobacterium tuberculosis H37Rv*1: II. Purification and properties of acetohydroxy acid isomerase

Acetohydroxy acid isomerase (AHA isomerase) was purified about 110-fold and separated from reductase and acetohydroxy acid isomeroreductase. The AHA isomerase was found to be homogeneous by agar and polyacrylamide gel electrophoreses at different pHs. The properties of AHA isomerase have been studied. The purified enzyme showed requirement for Image -ascorbic acid and sulfate ions for its activity. Synthetic ascorbic acid sulfate could replace Image -ascorbic acid and sulfate. α-Methyllactate and α-ketoisovalerate were found to inhibit AHA isomerase activity competitively whereas Image -valine and Image -isoleucine had no significant inhibitory effect. p-Hydroxymercuribenzoate inhibited AHA isomerase activity and the inhibition was reversed by β-mercaptoethanol.

Self-regularized pseudo time-marching schemes for structural system identification with static measurements

We explore the application of pseudo time marching schemes, involving either deterministic integration or stochastic filtering, to solve the inverse problem of parameter identification of large dimensional structural systems from partial and noisy measurements of strictly static response. Solutions of such non-linear inverse problems could provide useful local stiffness variations and do not have to confront modeling uncertainties in damping, an important, yet inadequately understood, aspect in dynamic system identification problems. The usual method of least-square solution is through a regularized Gauss-Newton method (GNM) whose results are known to be sensitively dependent on the regularization parameter and data noise intensity. Finite time,recursive integration of the pseudo-dynamical GNM (PD-GNM) update equation addresses the major numerical difficulty associated with the near-zero singular values of the linearized operator and gives results that are not sensitive to the time step of integration. Therefore, we also propose a pseudo-dynamic stochastic filtering approach for the same problem using a parsimonious representation of states and specifically solve the linearized filtering equations through a pseudo-dynamic ensemble Kalman filter (PD-EnKF). For multiple sets of measurements involving various load cases, we expedite the speed of thePD-EnKF by proposing an inner iteration within every time step. Results using the pseudo-dynamic strategy obtained through PD-EnKF and recursive integration are compared with those from the conventional GNM, which prove that the PD-EnKF is the best performer showing little sensitivity to process noise covariance and yielding reconstructions with less artifacts even when the ensemble size is small.

Self-regularized pseudo time-marching schemes for structural system identification with static measurements

We explore the application of pseudo time marching schemes, involving either deterministic integration or stochastic filtering, to solve the inverse problem of parameter identification of large dimensional structural systems from partial and noisy measurements of strictly static response. Solutions of such non-linear inverse problems could provide useful local stiffness variations and do not have to confront modeling uncertainties in damping, an important, yet inadequately understood, aspect in dynamic system identification problems. The usual method of least-square solution is through a regularized Gauss-Newton method (GNM) whose results are known to be sensitively dependent on the regularization parameter and data noise intensity. Finite time, recursive integration of the pseudo-dynamical GNM (PD-GNM) update equation addresses the major numerical difficulty associated with the near-zero singular values of the linearized operator and gives results that are not sensitive to the time step of integration. Therefore, we also propose a pseudo-dynamic stochastic filtering approach for the same problem using a parsimonious representation of states and specifically solve the linearized filtering equations through apseudo-dynamic ensemble Kalman filter (PD-EnKF). For multiple sets ofmeasurements involving various load cases, we expedite the speed of the PD-EnKF by proposing an inner iteration within every time step. Results using the pseudo-dynamic strategy obtained through PD-EnKF and recursive integration are compared with those from the conventional GNM, which prove that the PD-EnKF is the best performer showing little sensitivity to process noise covariance and yielding reconstructions with less artifacts even when the ensemble size is small. Copyright (C) 2009 John Wiley & Sons, Ltd.

A study of the purification and properties of tryptophan synthetase of Bengal gram (Cicer arietinum)

Active preparations of tryptophan synthetase were obtained from Bengal gram (Cicer arietinum) by the following procedure: (1) precipitation of inactive materials by manganous sulfate, (2) Adsorption of impurities on Alumina Cγ, (3) Adsorption of tryptophan synthetase on tricalcium phosphate gel, removal of inert protein from the gel by treatment with phosphate buffer (pH 7.2), and selective elution of the enzyme by 0.15 M phosphate buffer pH 7.2 containing 10% ammonium sulfate and 10−3 M serine. A 220-fold purification of the enzyme with 44% recovery of the activity was achieved. The pH optimum, effect of temperature, and substrate concentration and other properties of the purified enzyme have been studied in detail. Only the Image -isomer of serine takes part in the reaction. The Km values for indole, Image -serine, and Image -serine were calculated to be 0.66, 4.1, and 8.6 × 10−4 M, respectively. A kinetic study of the inhibition of tryptophan synthetase by indole-propionic acid has shown that it is of a competitive type. It has been demonstrated for the first time that 4-nitro-salicylaldehyde can replace pyridoxal phosphate as a coenzyme for the tryptophan synthetase reaction.

Biosynthesis of isoleucine and valine in mycobacterium tuberculosis H37Rv. II. Purification and properties of acetohydroxy acid isomeras

Acetohydroxy acid isomerase (AHA isomerase) was purified about 110-fold and separated from reductase and acetohydroxy acid isomeroreductase. The AHA isomerase was found to be homogeneous by agar and polyacrylamide gel electrophoreses at different pHs. The properties of AHA isomerase have been studied. The purified enzyme showed requirement for l-ascorbic acid and sulfate ions for its activity. Synthetic ascorbic acid sulfate could replace l-ascorbic acid and sulfate. α-Methyllactate and α-ketoisovalerate were found to inhibit AHA isomerase activity competitively whereas l-valine and l-isoleucine had no significant inhibitory effect. p-Hydroxymercuribenzoate inhibited AHA isomerase activity and the inhibition was reversed by β-mercaptoethanol.

Cloning,overexpression and purification of functionally active Saccharomyces cerevisiae Hop1 protein from scherichia coli

One of the major limitations to the application of high-resolution biophysical techniques such as X-crystallography and spectroscopic analyses to structure-function studies of Saccharomyces cerevisiae Hop1 protein has been the non-availability of sufficient quantities of functionally active pure protein. This has, indeed, been the case of many proteins, including yeast synaptonemal complex proteins. In this study, we have performed expression screening in Escherichia coli host strains, capable of high-level expression of soluble S. cerevisiae Hop1 protein. A new protocol has been developed for expression and purification of S. cerevisiae Hop1 protein, based on the presence of hexa-histidine tag and double-stranded DNA-Cellulose chromatography. Recombinant S. cerevisiae Hop1 protein was >98% pure and exhibited DNA-binding activity with high-affinity to the Holliday junction. The availability of the recombinant HOP1 expression vector and active Hop1 protein would facilitate structure-function investigations as well as the generation of appropriate truncated and site-directed mutant proteins, respectively. (C) 2010 Elsevier Inc. All rights reserved.