Slow Steady State Research Articles

Selective atomic sieving across metal/oxide interface for super-oxidation resistance

Surface passivation, a desirable natural consequence during initial oxidation of alloys, is the foundation for functioning of corrosion and oxidation resistant alloys ranging from industrial stainless steel to kitchen utensils. This initial oxidation has been long perceived to vary with crystal facet, however, the underlying mechanism remains elusive. Here, using in situ environmental transmission electron microscopy, we gain atomic details on crystal facet dependent initial oxidation behavior in a model Ni-5Cr alloy. We find the (001) surface shows higher initial oxidation resistance as compared to the (111) surface. We reveal the crystal facet dependent oxidation is related to an interfacial atomic sieving effect, wherein the oxide/metal interface selectively promotes diffusion of certain atomic species. Density functional theory calculations rationalize the oxygen diffusion across Ni(111)/NiO(111) interface, as contrasted with Ni(001)/NiO(111), is enhanced. We unveil that crystal facet with initial fast oxidation rate could conversely switch to a slow steady state oxidation.

Pre-Steady-State and Steady-State Kinetic Analysis of Butyrylcholinesterase-Catalyzed Hydrolysis of Mirabegron, an Arylacylamide Drug.

The β-adrenergic drug Mirabegron, a drug initially used for the treatment of an overactive bladder, has new potential indications and is hydrolyzed by butyrylcholinesterase (BChE). This compound is one of the only arylacylamide substrates to be catabolized by BChE. A steady-state kinetic analysis at 25 °C and pH 7.0 showed that the enzyme behavior is Michaelian with this substrate and displays a long pre-steady-state phase characterized by a burst. The induction time, τ, increased with substrate concentration (τ ≈ 18 min at maximum velocity). The kinetic behavior was interpreted in terms of hysteretic behavior, resulting from a slow equilibrium between two enzyme active forms, E and E'. The pre-steady-state phase with the highest activity corresponds to action of the E form, and the steady state corresponds to action of the E' form. The catalytic parameters were determined as kcat = 7.3 min-1 and Km = 23.5 μM for the initial (burst) form E, and kcat = 1.6 min-1 and Km = 3.9 μM for the final form E'. Thus, the higher affinity of E' for Mirabegron triggers the slow enzyme state equilibrium toward a slow steady state. Despite the complexity of the reaction mechanism of Mirabegron with BChE, slow BChE-catalyzed degradation of Mirabegron in blood should have no impact on the pharmacological activities of this drug.

Biochemical characterization of Caulobacter crescentus xylose dehydrogenase

d-Xylose sugar is a common component of hemicellulose, the second largest fraction of biomass. Many groups have developed biological conversions of d-xylose to value-added products by recombinant expression of the xylose dehydrogenase enzyme from Caulobacter crescentus. This enzyme uses NAD+ as a cofactor to oxidize d-xylose to d-xylono-1,4-lactone. A detailed understanding of the mechanism of this enzyme could be useful in engineering more efficient versions. Therefore, we have conducted kinetic studies including both the forward and reverse physiological reactions of this enzyme. We demonstrate that the enzyme's substrate binding mode follows a sequential steady state ordered mechanism with NAD+ or NADH binding first. Furthermore, the kcat of the reaction in the direction of NAD+ reduction is 10-fold higher than that of the reverse reaction. From rapid reaction studies, we demonstrate the binding of NAD+ and NADH to the free enzyme and that hydride transfer occurs in a fast step followed by a much slower steady state. We calculate that the dissociations of the sugar products from the enzyme complexes are the major rate limiting steps in both directions.

Regulation of Actin Dynamics by Cross-Linking Molecules

The ability of cells to control the internal dynamics of actin filaments plays a crucial role in many cellular processes, including cell motility and cell division. We demonstrate that spontaneous fragmentation is the crucial process in the comparably slow steady state dynamics of in vitro actin filaments. Yet, in vivo, actin turn-over is enhanced by many different regulator proteins. Simultanously, cells need mechanisms to locally stabilize actin filaments against induced depolymerisation in order to maintain certain actin structures for longer times. Using biochemical and fluorescence microscopy techniques we demonstrate that employment of cross-linking and bundling proteins not only gives cells a powerful tool to govern the architecture of cytoskeletal systems, but also allows for a drastic stabilization against internal kinetics by suppressing disassembly of cytoskeletal filaments. Even the actin depolymerizing factors are not sufficient to disintegrate highly cross-linked actin networks unless molecular motors are used simultaneously.View Large Image | View Hi-Res Image | Download PowerPoint Slide

Elucidating the function of the unknown Escherichia coli GTPase YjeQ through genetic interactions

The protein YjeQ is a small GTPase that associates with the 30S ribosomal subunit. The slow steady state GTPase activity of YjeQ of 10 h−1 is stimulated 160 fold by this interaction, implicating the ribosome as the site of YjeQ function in the cell. Although dispensable in both Escherichia coli and Bacillus subtilis, strains lacking this protein are severely comprised for growth. Additionally, we have shown that this protein is a virulence determinant in a Staphylococcus aureus mouse infection model, implicating it as a potential target for the development of new antimicrobial agents. In this study we are looking at the genetic interactions of yjeQ to more precisely determine its role in the cell. Two methods were used to identify genetic interactions with yjeQ. The first was a screen of overexpression strains for correction of the yjeQ deletion slow growth phenotype. Using this method all essential E. coli genes were screened as well as 40 non-essential translation related genes. A number of suppressors were identified, with one third functioning in translation or ribosome biogenesis. The suppressors were further screened for mechanism of suppression, and infB, era, and glnS were found to partially correct a ribosome defect associated with yjeQ deletion. As a second method to identify genetic interactions with yjeQ, double deletion strains were created with yjeQ and the non-essential translation related genes. These double deletions were characterized for growth and implicate a number of genes as functionally related to yjeQ. Using this data we are creating a network of genes that interact with yjeQ and if they function up- or downstream in the cell. This work was funded by CIHR.

A kinetic analysis of the catalase activity of myeloperoxidase.

The predominant physiological activity of myeloperoxidase is to convert hydrogen peroxide and chloride to hypochlorous acid. However, this neutrophil enzyme also degrades hydrogen peroxide to oxygen and water. We have undertaken a kinetic analysis of this reaction to clarify its mechanism. When myeloperoxidase was added to hydrogen peroxide in the absence of reducing substrates, there was an initial burst phase of hydrogen peroxide consumption followed by a slow steady state loss. The kinetics of hydrogen peroxide loss were precisely mirrored by the kinetics of oxygen production. Two mols of hydrogen peroxide gave rise to 1 mol of oxygen. With 100 microM hydrogen peroxide and 6 mM chloride, half of the hydrogen peroxide was converted to hypochlorous acid and the remainder to oxygen. Superoxide and tyrosine enhanced the steady-state loss of hydrogen peroxide in the absence of chloride. We propose that hydrogen peroxide reacts with the ferric enzyme to form compound I, which in turn reacts with another molecule of hydrogen peroxide to regenerate the native enzyme and liberate oxygen. The rate constant for the two-electron reduction of compound I by hydrogen peroxide was determined to be 2 x 10(6) M(-1) s(-1). The burst phase occurs because hydrogen peroxide and endogenous donors are able to slowly reduce compound I to compound II, which accumulates and retards the loss of hydrogen peroxide. Superoxide and tyrosine drive the catalase activity because they reduce compound II back to the native enzyme. The two-electron oxidation of hydrogen peroxide by compound I should be considered when interpreting mechanistic studies of myeloperoxidase and may influence the physiological activity of the enzyme.

Age of crustal melting, emplacement and exhumation history of the Shivling leucogranite, Garhwal Himalaya

We report a U–Pb monazite age of 23.0±0.2 Ma for the Shivling leucogranite, a tourmaline+muscovite±biotite leucogranite at the top of the High Himalayan slab in the Garhwal Himalaya, north India. The Shivling–Bhagirathi leucogranite is a viscous near-minimum melt, emplaced as a foliation parallel laccolith via a dyke network not far from its source region. Prograde heating occurred soon after the India–Asia collision at c. 50 Ma up to melting at 23 Ma and high temperatures (>550 °C) were maintained for at least 15 Ma after garnet growth. The leucogranite was emplaced at mid-crustal depths along the footwall of the Jhala fault, a large-scale low-angle normal fault, part of the South Tibetan Detachment system, above kyanite and sillimanite grade gneisses. The geometry of the leucogranite laccolith shows biaxial extension and boudinage both perpendicular (north-northeast–south-southwest) and parallel to the strike (west-northwest–east-southeast) of the mountain range. Unroofing occurred by underthrusting beneath the High Himalayan slab along the Main Central Thrust zone, progressively ‘jacking up’ the leucogranites, removal of material above by low-angle normal faulting, and erosion. Very rapid cooling at rates of 200–350 °C/Ma between 23–21 Ma immediately followed crystallization, as tectonic unroofing and erosion removed 24–28 km of overburden during this time. K–Ar muscovite ages are 22±1.0 Ma and fission track ages of zircons from >5000 m on the North Ridge of Shivling are 14.2±2.1 and 8.8±1.2 Ma and apatites are 3.5±0.79 and 2.61±0.23 Ma. Slow steady state cooling at rates of 20–30 °C/Ma from 20–1 Ma shows that maximum erosion rates and unroofing of the leucogranite occurred during the early Miocene. This timing coincides with initiation of low-angle, north-dipping normal faulting along the South Tibetan Detachment system.

Anomalous Kinetics of the Trapping Reaction in One Dimension Under Steady State Conditions

ABSTRACTWe study the A + B→B trapping reaction under steady state conditions for the case in which both A particles and traps(B) are mobile. Using Monte Carlo simulations, we follow the kinetic rate law in one dimension. Anomalies arise due to self-organization of the A particles, which results in a slower steady state reaction rate than is predicted classically. We find a partial order of reaction with respect to trap density of X = 2, and an overall order for the reaction of Z = 3.2. These results are in agreement with other works which predict an exponential rather than an algebraic decay law with respect to the A particle density.

Transient state kinetic effects of calcium ion on sarcoplasmic reticulum adenosine triphosphatase.

A rapid mixing technique was used to investigate the effects of Ca2+ ion on the kinetics of ATP hydrolysis by sarcoplasmic reticulum vesicles. "Basic" ATPase measured in the absence of Ca2+ showed an initial burst of inorganic phosphate production. Similarities in the transient state kinetic properties of basic and "extra" or Ca2+-dependent ATPase suggest that the two activities represent a single enzyme species. At low concentrations of Ca2+ (less than 10(-6) M) the time course of the partial reactions of extra ATPase appeared to fit a simple scheme in which the acid-stable, phosphorylated enzyme (E approximately P) breaks down directly to inorganic phosphate and free enzyme. A similar mechanism seemed to apply to moderate levels of ATP and high external concentrations of Ca2+ known to inhibit transport activity. In the intermediate range of Ca2+ concentrations inorganic phosphate production was resolved into two phases consisting of a fast initial rate (burst) and slow steady state. Acid-stable phosphorylated protein showed a transient decay which coincided with the appearance of the burst. This behavior is consistent with a scheme in which E approximately P breaks down to an acid-labile or noncovalent intermediate state (E-P). A slow secondary increase in phosphorylation followed the transient decay in E approximately P. This late phase of protein labeling was eliminated following pretreatment with Triton X-100, sodium oxalate, or diethyl ether which decrease or prevent the formation of a transport gradient. An analysis of the dependence of the steady state level of phosphorylation and rate of inorganic phosphate production on Ca2+ concentration indicated that the phosphorylation mechanism involves interaction of two Ca2+ ions with the enzymatic carrier. The pathway by which E approximately P breaks down, i.e. whether it goes to E + Pi or E-P, may depend on the extent to which these sites are occupied by Ca2+. The transport of Ca2+ is discussed in terms of a flip-flop mechanism in which E approximately P and E-P represent high and low affinity Ca2+ binding states occurring in separate halves of an enzyme dimer.

Kinetics of Substrate Hydrolysis by Molecular Variants of Escherichia coli Alkaline Phosphatase

Abstract Two isozymes of Escherichia coli alkaline phosphatase have been compared in the stopped flow spectrophotometer with respect to the kinetics of hydrolysis of two phosphate esters. Hydrolysis of 2,4-dinitrophenyl phosphate at pH 5.5 is biphasic; release of chromophoric alcohol occurs in a rapid burst followed by a slower steady state. The two isozymes show similar burst rates and amplitudes but their steady state rates differ by a factor of 2. Hydrolysis of p-nitrophenyl phosphate at pH 8.0 shows no significant burst and the homogeneous steady state rate is essentially the same for both isozymes. These observations and the ionic strength dependence of the rates of these reaction steps support the hypothesis, proposed previously in the literature, that the molecular process which determines the steady state rate at pH 8.0 is responsible for the presteady state burst at pH 5.5. The steady state process at pH 5.5 must be a subsequent step in the catalytic mechanism. A molecular hybrid containing one subunit of wild type alkaline phosphatase and one subunit from an inactive mutationally altered alkaline phosphatase shows one-half the normal burst amplitude at pH 5.5. All transient and steady state rates observed for the hybrid equal those seen for wild type enzyme, once corrected for the halved number of active sites inferred from the burst amplitude. This finding casts doubt on catalytic mechanisms involving alternately functioning subunits that several workers have proposed for E. coli alkaline phosphatase.

The flow of a viscous liquid in a converging tube

The problem of the viscous flow of an incompressible Newtonian liquid in a converging tapered tube has been solved in spherical polar coordinates. The method of the solution involves the Stokes' stream function and a technique introduced by Stokes in the study of a sphere oscillating in a fluid. The theory for the flow in a rigid tube includes: (1) the pulsatile flow with both radial and angular velocity components; (2) the steady state flow with both radial and angular velocity components and (3) the very slow steady state flow with only a radial velocity component present. For a tapered elastic tube, the velocity of the propagated pulse wave is determined. The solution given is in terms of the elastic constants of the system and the coordinates for this type of geometry. The pulse velocity is then related to the velocity in an elastic cylindrical tube with the necessary correction terms to account for the tapered tube.

The Specific Sorption of Cobalt and Zinc by Layer Silicates

AbstractThe sorption of Co and Zn by layer silicates was studied in dilute mineral suspensions containing, in most cases, about 10-6 M Co or Zn. Electrostatic adsorption was eliminated by the presence of 0.1 N CaCl2. Co and Zn were determined by a radioisotopie technique.Detailed studies on montmorillonite and to a lesser extent vermiculite, muscovite and biotite revealed the presence of at least two forms of specifically sorbed Co, one of which was exchangeable by certain ions such as Cu, Ni, Zn, Fe, Mn or more Co and the other, occurring in much smaller amounts, was not exchangeable by these cations. The latter form is considered to result from lattice penetration; the former is associated with surface groups. These forms may be separated quantitatively by several desorption procedures including successive extractions with dilute acetic acid.The total amount and relative proportion of these forms of sorbed Co and Zn depend on the pH of the system, time of reaction, mineral species used, and amount of Co or Zn added. Equilibrium is not readily attained but tends to approach a slow steady state after several days. Isotherms indicate a variation in bonding energy with surface coverage. The sorption of Co and Zn from dilute solutions by any mineral is related to its stability.These studies, considered in conjunction with published data, suggest that a common mechanism may be involved in the specific sorption of many heavy metal cations by many minerals.