Rapid Quench Technique Research Articles

Insights into the HER-2 receptor tyrosine kinase mechanism and substrate specificity using a transient kinetic analysis.

The HER-2/erbB-2/c-neu proto-oncogene encodes for an EGF receptor-like protein which has been implicated in the pathogenesis of several human malignancies. Although much has been learned about the physiological significance of this receptor tyrosine kinase, its catalytic mechanism remains poorly understood. We have expressed, purified, and characterized two recombinant proteins corresponding to a full-length (HCD) and truncated (HKD) construct of the HER-2 intracellular tyrosine kinase domain and have identified an optimal substrate (GGMEDIYFEFMGGKKK; HER2Peptide) through screening of a degenerate peptide library. We have conducted a transient kinetic analysis of the HER-2 proteins (HCD and HKD) to illuminate mechanistic details of the HER-2 pathway. In particular, stopped-flow fluorescence studies with mant (N-methylanthraniloyl)-nucleotide derivatives provided direct measurements of the association and dissociation rate constants for these nucleotide interactions with the HER-2 recombinant proteins, thereby enabling the determination of nucleotide K(d) values. Moreover, the actual step of chemical catalysis was isolated using rapid chemical quench techniques and shown to occur approximately 3-fold faster than the steady-state rate which corresponds to product release. Evidence is also provided that suggests a conformational change that is partially rate-limiting at least in HCD. Furthermore, the role that the phosphorylation state of the protein may play on catalysis was examined. Studies carried out with pre-phosphorylated recombinant HER-2 proteins suggest that while autophosphorylation is not a prerequisite for enzymatic activity, this protein modification actually directly affects the catalytic mechanism by enhancing the rate of ADP release and that of the rate-limiting step. While a pre-steady-state kinetic analysis has been carried out on the catalytic subunit of cAMP-dependent serine/threonine kinase, to our knowledge, this study represents the first reported transient kinetic investigation of a receptor tyrosine kinase. This work serves as a basis for comparison of these two important protein kinase families and in this report we highlight these similarities and differences.

Phase relations in the Fe-rich part of the system Fe2O3(-Fe3O4)-CaO-SiO2 at 1240-1300°C and oxygen partial pressure of 5 × 10-3 atm: implications for iron ore sinter

Equilibrium phase relations in the Fe-rich portion of the system Fe2O3(-Fe3O4)-CaO-SiO2 (FCS) have been determined at 1240-1300°C and under an oxygen partial pressure of 5 × 10-3 atm, typical for iron ore sintering. Experiments were performed with the use of a rapid-quench technique and the chemical compositions of the melts and coexisting condensed phases were determined by combined optical microscopy and electron-probe microanalysis. At temperatures greater than ca 1250°C the major phase fields comprise Mt + Liq, C 2S + Liq, C2S + C2F + Liq and C2F + Liq (where Mt represents magnetite, C = CaO, F = Fe2O3, S = SiO2 and Liq denotes a quench liquid phase) plus an extensive, continuous, Liq-only phase field that extends from Fe-rich to SiO2-rich compositions. At less than about 1250°C the Liq-only melt region segregates into two distinct melt fields—one at high Fe contents and basicities greater than 1.8-2.0 and a second melt at low basicities (<1.8) and high SiO2 contents. At all temperatures examined the reduced oxygen conditions significantly enlarged the field of silicate melt present relative to that of Ca-ferrite melt. Magnetite (predominantly) or hematite may be the stable Fe-oxide phase; however, the occurrence of any particular Fe-oxide phase is strongly linked to the overall basicity (CaO/SiO2) of the charge.Experiments show that SFCss (silico-ferrite of calcia solid solution), which is the major ferrite bonding phase in low-Al sinter, cannot be produced as a single, unique, crystalline phase within the FCS system at 5 × 10-3 atm O2. At this oxygen partial pressure the bulk composition lies within the partially reduced Fe2O3(-Fe3O4)-CaO-SiO2 pseudo-ternary with the three-phase assemblage Mt + SFCss + Liqα stabilized at low temperatures (1240°C). Combined with data for the FCS ternary in air the results indicate that to maximize the formation of SFCss bonding phase a low-temperature (<1260°C), semi-reduced heating environment followed by a relatively slow, oxidizing, cooling stage is desirable.

Topoisomerase II drives DNA transport by hydrolyzing one ATP.

DNA topoisomerase II is a homodimeric molecular machine that couples ATP usage to the transport of one DNA segment through a transient break in another segment. In the presence of a nonhydrolyzable ATP analog, the enzyme is known to promote a single turnover of DNA transport. Current models for the enzyme's mechanism based on this result have hydrolysis of two ATPs as the last step, used only to reset the enzyme for another round of reaction. Using rapid-quench techniques, topoisomerase II recently was shown to hydrolyze its two bound ATPs in a strictly sequential manner. This result is incongruous with the models based on the nonhydrolyzable ATP analog data. Here we present evidence that hydrolysis of one ATP by topoisomerase II precedes, and accelerates, DNA transport. These results indicate that important features of this enzyme's mechanism previously have been overlooked because of the reliance on nonhydrolyzable analogs for studying a single reaction turnover. A model for the mechanism of topoisomerase II is presented to show how hydrolysis of one ATP could drive DNA transport.

An ATP-linked structural change in protein kinase A precedes phosphoryl transfer under physiological magnesium concentrations.

The kinetic mechanism for the catalytic subunit of protein kinase A was evaluated using physiological concentrations of free magnesium (0.5 mM) and a rapid quench flow technique. When the enzyme is pre-equilibrated with ATP, the peptide substrate, LRRASLG (Kemptide), is phosphorylated in a biphasic manner with a rapid, exponential "burst" phase (kb) followed by a slower, linear phase (kL) that corresponds to the steady-state kinetic rate. Both the amplitude and the substrate-rate dependence of the initial, burst phase indicate that the rate of phosphoryl transfer is fast (approximately 500 s-1) and does not limit turnover (45 s-1). Viscosity studies indicate that, while Kemptide is in rapid equilibrium, ATP does not exchange rapidly with the active site and kcat/KATP is limited by the rate constant for nucleotide encounter. When the pre-steady-state kinetic experiments are initiated with ATP, a lag phase is observed at low ATP concentrations consistent with rate-limiting association. At high ATP concentrations (>1 mM), a burst phase is observed but the rate and amplitude are low on the basis of the bimolecular rate constant for ATP association and the rate constant for phosphoryl transfer. The kinetic data indicate that the phosphoryl transfer step is fast at physiological magnesium concentrations, but an ATP-linked conformational change precedes this step, limiting the burst phase rate constant. Simulations of the pre-steady-state kinetic transients indicate that turnover (45 s-1) is limited both by net product release (70 s-1) and by this structural change (170 s-1). This structural change may also occur at high free magnesium concentrations, but it must be significantly faster than 170 s-1 and, consequently, not rate-limiting for turnover (kcat = 20 s-1 at 10 mM free Mg2+). We propose that this conformational event is an obligatory component of the kinetic pathway and includes a movement of the catalytic residues necessary for supporting phosphoryl group donation.

Kinetic Mechanism of the Enantioselective Conversion of Styrene Oxide by Epoxide Hydrolase from Agrobacterium radiobacter AD1

Epoxide hydrolase from Agrobacterium radiobacter AD1 catalyzes the enantioselective hydrolysis of styrene oxide with an E value of 16. The (R)-enantiomer of styrene oxide is first converted with a k(cat) of 3.8 s(-1), and the conversion of the (S)-enantiomer is inhibited. The latter is subsequently hydrolyzed with a k(cat) of 10.5 s(-1). The pre-steady-state kinetic parameters were determined for both enantiomers with stopped-flow fluorescence and rapid-quench techniques. For (R)-styrene oxide a four-step mechanism was needed to describe the data. It involved the formation of a Michaelis complex that is in rapid equilibrium with free enzyme and substrate, followed by rapid and reversible alkylation of the enzyme. A unimolecular isomerization of the alkylated enzyme precedes the hydrolysis of the covalent intermediate, which could be observed due to an enhancement of the intrinsic protein fluorescence during this step. The conversion of (S)-styrene oxide could be described by a three-step mechanism, which also involved reversible and rapid formation of an ester intermediate from a Michaelis complex and its subsequent slow hydrolysis as the rate-limiting step. The unimolecular isomerization step has not been observed for rat microsomal epoxide hydrolase, for which a kinetic mechanism was recently established [Tzeng, H.-F., Laughlin, L. T., Lin, S., and Armstrong, R. N. (1996) J. Am. Chem. Soc. 118, 9436-9437]. For both enantiomers of styrene oxide, the Km value was much lower than the substrate binding constant K(S) due to extensive accumulation of the covalent intermediate. The enantioselectivity was more pronounced in the alkylation rates than in the rate-limiting hydrolysis steps. The combined reaction schemes for (R)- and (S)-styrene oxide gave an accurate description of the epoxide hydrolase catalyzed kinetic resolution of racemic styrene oxide.

Solubility of Simple Pelitic and Granitic Mineral Assemblages in Chloride Rich Aqueous Solutions at 2 kbar, and 400°–650°C

Alkali chlorides complexes are the principal solutes of most hydrothermal fluids. An understanding of the solubility of alkali metal silicates will thus provide insight into the origins of hydrothermal fluids, and physical and chemical condi-tions of fluid-rock interaction during alkali metasomatism and ore formation. Several studies have shown the mobility of alkali metals during regional metamorphic processes (e.g. Dipple et al, 1990; Ague, 1991). Similarly, potassic alteration in porphyry copper deposits is abundant (e.g. Hemley, 1959). In order to obtain a better under-standing of fluid-rock interaction during such metamophic events, we have mea-sured the solubility of the assemblages albite(ab)+K-feldspar(kfs)+andalusite (and) +quartz (qtz) at 600 ~ and 650~ and ab+muscovite(mu)+and+qtz at 550~ at 2kbar over a total chloride range of 0.01 to 4 m C1 t~ These assemblages were selected to approximate high grade pelitic assemb-lages in contact aureoles. Additional studies with the simplified granitic mineral assemblage ab+mu+kfs+qtz were obtained over a temperature range of 400 ~ to 600~ at 2kbar total pressure and 1 m total chloride. The rapid-quench technique was used. Na, K, A1, Si, and pH were measured on quench. Starting

Participation of ADP dissociation in the rate-determining step in cAMP-dependent protein kinase.

Pre-steady-state kinetic analyses of the catalytic subunit of cAMP-dependent protein kinase showed that the rate constant for phosphoryl transfer is fast and either the release of one or both of the products or a conformational change controls turnover [Grant, B., & Adams, J. A. (1996) Biochemistry 35, 2022-2029]. To determine which step or steps control turnover in the wild-type enzyme, we used a catalytic trapping technique to measure directly the dissociation rate constant for ADP. The phosphorylation of two peptide substrates, LRRASLG and GRTGRRNSI, was monitored using a rapid quench flow technique under conditions where saturating concentrations of ADP were preequilibrated with the enzyme before excess ATP and one of the substrates were added to trap the free enzyme and to start the phosphorylation reaction. Under ADP preequilibration conditions, no 'burst' phase was observed, and although the rate of linear, steady-state turnover was unaffected, the net production of phosphopeptide lagged behind the non-preequilibrated control. This phenomenon occurs due to the slow release of the product, and kinetic modeling suggests that this effect can be explained if the dissociation rate constant for ADP is 24 s-1 and solely limits turnover (kcat = 23 s-1) for the phosphorylation of LRRASLG. Using GRTGRRNSI, the dissociation rate constant for ADP is 35 s-1 and limits turnover (kcat = 29 s-1) if the reaction is initiated by the addition of enzyme. Under preequilibration conditions with either ATP or GRTGRRNSI, turnover is approximately 50% lower, suggesting that ADP release may partially control this parameter. This preequilibration effect can be explained by slowly interconverting enzyme forms with specific peptide-induced turnover properties. These studies indicate that ADP release is an essential rate-limiting component for turnover but also suggests that other factors contribute subtly when the structure of the substrate is altered.

Is there a catalytic base in the active site of cAMP-dependent protein kinase?

The carboxyl group of an aspartic acid in the active site of the serine-specific protein kinase, cAMP-dependent protein kinase, is poised near the hydroxyl proton of a peptide substrate in the X-ray crystallographic structure (Madhusudan et al., 1994), suggesting that this residue may act as a general-base catalyst in the phosphoryl transfer reaction. Indeed, several proposals have been made in this regard. We measured the pre-steady-state kinetics in this enzyme using a rapid quench flow technique to understand the role of this putative base. The phosphorylation of the peptide substrate, GRTGRRNSI, by cAMP-dependent protein kinase exhibited "burst" kinetics consistent with a mechanism in which the peptide is phosphorylated rapidly (154 s(-1)) and the product(s) is (are) released slowly (16 s(-1)). The replacement of Mg2+ with Mn2+ leads to a 13-fold reduction in this observed "burst" rate constant, suggesting that this transient is limited either by the phosphoryl transfer step or by a metal ion-dependent conformational change step. The influence of deuterium oxide on the pre-steady-state kinetics was monitored in the presence of both divalent metal ions, and no solvent isotope effect was measured on either "burst" phase. A large solvent isotope effect is observed on k(cat) in the presence of either metal ion, and a proton inventory analysis in the presence of Mg2+ indicates that two or more protons are transferred in the product release step. Finally, no pH dependence is observed on the "burst" rate constant using either Mg2+ or Mn2+ over the pH range of 6-9. The combined data do not support a mechanism involving a general-base catalyst whose pK(a) is greater than 5 or less than 10 if the "burst" phase is cleanly limited by the phosphoryl transfer step. If the "burst" phase is limited by a metal ion-dependent conformational change step, the measurement of the phosphoryl transfer step is obscured, and the participation of a base catalyst is indeterminate.

Diiron-cluster intermediates in biological oxygen activation reactions

The activation of molecular oxygen is a fundamental process that is important in metabolic reactions in biological systems. In addition to the well-studied role of hemoproteins in participating in oxygen activation, there is increasing evidence that dinuclear iron centers in proteins also function in this role. Recent work on the interaction of the dinuclear iron centers found in ribonucleotide reductase and in methane monooxygenase with molecular oxygen is reviewed. Available spectroscopic data on reaction intermediates trapped by the rapid freeze quench kinetic technique allow insights into their structures and on the oxidation level of each Fe ion in the complex. These data suggest the initial intermediate formed on reaction of the diferrous center with oxygen is the formation of a diferric peroxo complex which undergoes further internal electron reorganization to form a diferryl-oxo complex termed intermediate Q. It is suggested that these intermediates are formed as initial steps in both methane monooxygenase and in ribonucleotide reductase activation of molecular oxygen. Further reaction steps of Q in each enzyme are distinguished by the source of the subsequent reducing equivalent. In the case of methane monooxygenase, the hydrocarbon substrate is the source of the required reducing equivalent to form a mixed valent ferryl/ferric complex which is proposed to donate a hydroxyl group to the hydrocarbon radical with resulting formation of a diferric ‘resting’ state and product. An external electron source supplies the single electron to the difrrryl center in ribonucleotide reductase to form a mixed valent complex that is capable of oxidizing a proximal tyrosyl residue (122) to its stable radical form with formation of the ‘resting’ diferric form of the enzyme. This ‘unified’ scheme for the activation of molecular oxygen by dinuclear iron centers in these two enzymes may have applicability to other enzymes containing these centers.

Biochemical kinetic characterization of the Acanthamoeba myosin-I ATPase.

Acanthamoeba myosin-IA and myosin-IB are single-headed molecular motors that may play an important role in membrane-based motility. To better define the types of motility that myosin-IA and myosin IB can support, we determined the rate constants for key steps on the myosin-I ATPase pathway using fluorescence stopped-flow, cold-chase, and rapid-quench techniques. We determined the rate constants for ATP binding, ATP hydrolysis, actomyosin-I dissociation, phosphate release, and ADP release. We also determined equilibrium constants for myosin-I binding to actin filaments, ADP binding to actomyosin-I, and ATP hydrolysis. These rate constants define an ATPase mechanism in which (a) ATP rapidly dissociates actomyosin-I, (b) the predominant steady-state intermediates are in a rapid equilibrium between actin-bound and free states, (c) phosphate release is rate limiting and regulated by heavy-chain phosphorylation, and (d) ADP release is fast. Thus, during steady-state ATP hydrolysis, myosin-I is weakly bound to the actin filament like skeletal muscle myosin-II and unlike the microtubule-based motor kinesin. Therefore, for myosin-I to support processive motility or cortical contraction, multiple myosin-I molecules must be specifically localized to a small region on a membrane or in the actin-rich cell cortex. This conclusion has important implications for the regulation of myosin-I via localization through the unique myosin-I tails. This is the first complete transient kinetic characterization of a member of the myosin superfamily, other than myosin-II, providing the opportunity to obtain insights about the evolution of all myosin isoforms.

Thiyl Radicals in Ribonucleotide Reductases

The ribonucleoside triphosphate reductase (RTPR) from Lactobacillus leichmannii catalyzes adenosylcobalamin (AdoCbl)-dependent nucleotide reduction, as well as exchange of the 5' hydrogens of AdoCbl with solvent. A protein-based thiyl radical is proposed as an intermediate in both of these processes. In the presence of RTPR containing specifically deuterated cysteine residues, the electron paramagnetic resonance (EPR) spectrum of an intermediate in the exchange reaction and the reduction reaction, trapped by rapid freeze quench techniques, exhibits narrowed hyperfine features relative to the corresponding unlabeled RTPR. The spectrum was interpreted to represent a thiyl radical coupled to cob(II)alamin. Another proposed intermediate, 5'-deoxyadenosine, was detected by rapid acid quench techniques. Similarities in mechanism between RTPR and the Escherichia coli ribonucleotide reductase suggest that both enzymes require a thiyl radical for catalysis.

Pre-steady-state kinetic analysis of cAMP-dependent protein kinase using rapid quench flow techniques.

The phosphorylation of a peptide substrate by the catalytic subunit of cAMP-dependent protein kinase was monitored over short time periods (2-1000 ms) using a rapid quench flow mixing device and a radioactive assay. The production of phosphokemptide [LRRAS(P)LG] as a function of time is characterized by a rapid "burst" phase (250 s-1) followed by a slower, linear phase (L/[E]t = 21 s-1) at 100 microM Kemptide. The amplitude of this "burst" phase varies linearly with the enzyme concentration and represents approximately 100% of the total enzyme concentration, indicating that the "burst" phase is not due to product inhibition. The observed rate constants for the "burst" and linear phases and the "burst" amplitude vary hyperbolically with the substrate concentration. From these dependencies, a maximum "burst" rate constant of 500 +/- 60 s-1 and a Km and Kd for Kemptide of 4.9 +/- 1.4 and 200 +/- 60 microM were determined. The kcat and Km data extracted from the linear portion of the rapid quench flow transients are indistinguishable from those obtained by standard steady-state kinetic analyses using low catalytic subunit concentrations and a spectrophotometric, coupled enzyme assay. Both rate constants for the "burst" and linear phases decreased in the presence of Mn2+. The data imply that the phosphorylation of Kemptide by the catalytic subunit occurs by a mechanism in which the substrate is loosely bound, is rapidly phosphorylated at the active site, and is released at a steady-state rate that is likely controlled by the dissociation rate constant for ADP. The combined pre-steady-state kinetic data establish a comprehensive, kinetic mechanism that predicts all the steady-state kinetic and viscosometric data. This study represents the first chemical observation and characterization of phosphoryl transfer at the active site of a protein kinase and will be useful for further structure-function studies on this and other protein kinases.

Evidence for nucleophilic catalysis in the aromatic substitution reaction catalyzed by (4-chlorobenzoyl)coenzyme A dehalogenase.

(4-Chlorobenzoyl)coenzyme A dehalogenase catalyzes the hydrolytic dehalogenation of (4-chlorobenzoyl)coenzyme A (4-CBA-CoA) to (4-hydroxybenzoyl)coenzyme A (4-HBA-CoA). Rapid-quench techniques were used in conjunction with [14C]-4-CBA-CoA to test for the formation of a covalent enzyme intermediate during catalysis. The rate of [14C]-4-CBA-CoA (37 microM) consumption in the presence of a 2-fold excess of dehalogenase (75 microM) was determined to proceed at k = 6.5 s-1, coincident with the formation of an enzyme intermediate containing covalently bound radiolabel. The radiolabeled enzyme reached a maximum level at 100 ms, corresponding to 27% of the starting [14C]-4-CBA-CoA, before declining. The kinetics of formation and consumption of the radiolabeled enzyme observed during turnover are consistent with its intermediacy in the overall reaction. A single turnover reaction carried out in 98% 18O-enriched water produced 4-HBA-CoA with 73-75% 16O and 27-25% 18O at the benzoyl ring C(4)-OH. In contrast, a multiple turnover reaction carried out in 93% H2(18)O produced 4-HBA-CoA labeled at the C(4)-OH with 89% 18O and 11% 16O. These results were interpreted as evidence for formation of an aryl enzyme intermediate during 4-CBA-CoA hydrolytic dechlorination in the dehalogenase active site.

Investigation of intrinsic point defects in rapidly quenched copper by nuclear quadrupole double resonance

Abstract This letter reports nuclear quadrupole double-resonance (NQDOR) measurements on Cu specimens that were rapidly quenched from the melt either by Duwez's gun technique or by melt spinning. Comparison with measurements of the recovery of the residual electrical resistivity and with previous NQDOR studies of electron-irradiated Cu leads to the conclusion that the defects annealing out in the so-called recovery stage III after low-temperature electron irradiation are self-interstitials. Among the vacancy-type imperfections introduced by the rapid-quenching techniques employed in the present work there is at least one defect (presumably the divacancy) that becomes mobile in the same temperature range as self-interstitials. This is in agreement with the evidence from other work that in Cu the enthalpies of migration of divacancies and of self-interstitials are almost the same.

Transformation kinetics of YBa2Cu3Ox in samples produced by the rapid-quenching technique

The samples were obtained by a rapid-quenching technique at 1430 degrees C in a Y-Ba-Cu-O system. The amorphous-to-crystalline transformation of YBa2Cu3Ox occurred in as-quenched samples during isothermal annealing in an oxygen atmosphere in the range 800-900 degrees C. The variation of electron resistivity of as-quenched samples was measured in situ during isothermal annealing. According to the changes of electron resistivity, it was suggested that the rate of the transformation process was mainly controlled by the rate of oxygen diffusion in the tetragonal YBa2Cu3Ox. The activation energy of the crystallization process obtained was Delta E=1.18 eV.

Calcium-activated myofibrillar ATPase: transient kinetics and the titration of its active sites

The transient kinetics of rabbit psoas Ca(2+)-activated myofibrillar Mg(2+)-ATPase were studied in a buffer of near physiological ionic strength at 4 degrees C by the rapid flow quench technique. The initial ATP binding steps were studied by the ATP chase and the cleavage and release of products steps were studied by the Pi burst method. The data obtained were interpreted by the simple scheme [formula; see text] represents the myosin heads with or without actin interaction. The constants obtained with myofibrils (where the molecules are highly organized) were compared with those with myosin subfragment 1 (S1) and cross-linked acto-S1 (where the molecules are dispersed in solution). Myofibrils appear to bind ATP as tightly as do S1 and cross-linked acto-S1. This suggests that with them k-2 less than kcat much less than k2, and it is proposed that the ATP chase method can be used to titrate the ATPase sites in myofibrils. The results of titration and single-turnover experiments revealed that myofibrils may contain partially active myosin heads. It is proposed that these heads bind ATP loosely without hydrolysis, as found with S1 [Tesi, C., N. Bachouchi, N., Barman, T., & Travers, F. (1989) Biochimie 71, 363-372]. There were large Pi bursts with the three preparations, showing that with all of them the release of products step (k4) is rate limiting.(ABSTRACT TRUNCATED AT 250 WORDS)

A new approach to preparing three-dimensional amorphous alloys

Considerable interest has been evident in preparing three-dimensional amorphous alloys for the past decade because the traditional rapid-quenching technique is applicable only to the production of very thin ribbon, strip and powder, of which the practical use is generally limited in the scope of electronics and power industry. Currently the preparation of three-dimensional amorphous alloys can be divided into two types of methods: solid-state synthesis and denucleation of liquid alloys. The former method, which has been increasingly developed in some countries such as the FRG and Japan, consists of solid-state reactions and powder consolidation. Amorphous Ni6sZr32 wire of 1 mm diameter has been produced by solid-state reactions from as-deformed composite under isothermal conditions (300 °C for 100 h) [1]. Power consolidation techniques including explosive compaction [2], warm extrusion [3] and high-temperature sintering [4] have been utilized to prepare bulk amorphous samples with rotational symmetry, such as rods, tubes or cones. Some key problems such as cracks and non-uniform properties of synthesized specimens by the above-mentioned technique are expected to be solved in spite of results attained recently. In 1984 an exciting achievement was made by Kui et al. [5], who succeeded in undercooling bulk Pd40Ni40P20 alloy to glassy spheroids of diameter 10 mm by fluxing. This confirms that the denucleation technique in which heterogeneous nuclei are removed leads to the formation of bulk metallic glasses at much lower cooling rates. However, the formation of bulk glass is possible only if the homogeneous nucleation frequency of the melt is sufficiently low and if the heterogenous nucleation centres are thoroughly eliminated. Obviously, most practical alloys, for which the,' lowest temperatures of crystal nucleation onset are above their glass transition temperatures (Tg) in the slow-cooling environment, can hardly meet these strict conditions. Therefore, rapid quenching is indispensable to trap the liquid alloys into three-dimensional metallic glasses. In this letter a new method of preparing three-dimensional amorphous alloys by a rapid-quenching denucleated melt (RQDM) is put forward. The RQDM experiment was composed of two steps: denucleating liquid alloys until a high undercooling is attained and rapid quenching the denucleated liquid alloys. The experiments were performed in a vacuum of < 2.7 x 10 .3 Pa on our self-made high-frequency electromagnetic levitation melter and hammer-and-anvil apparatus as shown in Fig. 1.

Investigation of the mechanism of phosphinothricin inactivation of Escherichia coli glutamine synthetase using rapid quench kinetic techniques

A number of slow tight-binding inhibitors are known for glutamine synthetase that resemble the geometry of the tetrahedral intermediate formed during the enzyme-catalyzed condensation of gamma-glutamyl phosphate and ammonia. One of these inhibitors, phosphinothricin [L-2-amino-4-(hydroxymethyl-phosphinyl)butanoic acid], has been investigated by rapid kinetic methods. Phosphinothricin not only exhibits the kinetic properties of a slow tight-binding inhibitor but also undergoes phosphorylation during the course of the ATP-dependent inactivation. The acid lability of phosphinothricin phosphate enabled investigation of the kinetics of glutamine synthetase inactivation using rapid quench kinetic techniques. The rate-limiting step in the inhibition reaction is the binding of inhibitor (0.004-0.014 microM-1 s-1) and/or a conformational change associated with binding, which is several orders of magnitude slower than the binding of ATP. The association rate of phosphinothricin depends on which metal ion is bound to the enzyme (Mn2+ or Mg2+). With Mn2+ bound to glutamine synthetase the rate of association and the phosphorylation rate are faster than when Mg2+ is bound. The data are interpreted with use of a model in which the binding of a substrate analogue with a tetrahedral moiety enhances the phosphorylation rate of the reaction intermediate; however, the initial binding interaction is retarded because the enzyme has to bind a molecule that has a "transition-state" geometry rather than a ground-state substrate structure. During the course of the inactivation, progressively slower rates for binding and phosphoryl transfer were observed, indicating communication between active sites.

Ethanol-induced inhibition of carbachol-stimulated uptake of calcium in PC12 pheochromocytoma cells

Using a rapid-quench technique the effects of ethanol on the uptake of 45Ca 2+ into PC12 pheochromocytoma cells were studied in suspension. At concentrations achieved during acute intoxication in man (25–100 mM), ethanol inhibited both the carbachol-stimulated and K +-induced uptake of calcium. Inhibition of carbachol-stimulated uptake of Ca 2+ occurred rapidly, within seconds at 27°C, whereas inhibition of K +-induced uptake of Ca 2+ developed more slowly. This disparity between the kinetics of these ethanol-induced inhibitions was unexpected, because the uptake of Ca 2+ evoked by either stimulus, is thought to occur predominantly through a common pathway, namely voltage-sensitive Ca 2+channels. This difference may reflect differential effects of ethanol on multiple carbachol-activated pathways for entry of Ca 2+. Alternatively, carbachol may facilitate the inhibitory action of ethanol on voltage-dependent channels. This apparent facilitation was manifested principally by a more rapid onset of inhibition, although the extent of inhibition by ethanol, in the presence of carbachol, was also increased. In preincubation experiments, ethanol did not enhance the apparent agonist-induced desensitization of carbachol-evoked uptake of Ca 2+. Nevertheless, an acute interaction between cholinergic agonists and ethanol, affecting homeostasis of Ca 2+ may play a role in the pathophysiology of alcohol intoxication.

Magnetic hardening of Pr-Co-B sintered magnets (abstract)

It is well known that the Pr2Co14B intermetallic compound has a high magnetocrystalline anisotropy (100 kOe at 294 K). This value is higher than that of the Pr2Fe14B compound (70 kOe at 294 K). Hard magnets, using the rapid-quenching technique, have been prepared for both the Co- and Fe-based materials. In the case of the conventional powder-metallurgy technique, only the Fe-based magnets have been successfully prepared. So far, there is no report of achieving any significant value of coercivity in the Pr-Co-B system. In this paper we report preliminary results on Pr-Co-B sintered magnets with room-temperature intrinsic coersivity, Hci, equal to 5.2 kOe and an energy product, (BH)max equal to 10.3 MGOe. At liquid-nitrogen temperature (77 K) Hci is greater than 18 kOe (which was the maximum available magnetic field) and (BH)max is equal to 12.5 MGOe. These values of (BH)max are twice those reported for the rapidly quenched magnets.1 The Curie temperature of the magnet is 988 K (corresponding to the presence of the Pr2Co14B phase). The microstructure of the magnets with high Hci is markedly different from that of the magnets with low Hci. The major difference is the lower concentration of voids and the well-defined shape of the grains. The grain size is between 5 and 30 μm. The major phases present in the magnet are Pr2Co14B, PrCo4B, and a minor amount of PrCo5. The metallurgical characteristics and phase-diagram features of the Pr-Co-B system seem to be substantially different from those of the Pr-Fe-B system, where the major phases present are the Pr2Fe14B, PrFe4B4, and the Pr-rich phase.