Rapid Quench Technique Research Articles

Na+-Ca2+ exchange in cardiac sarcolemma: modulation of Ca2+ affinity by exercise.

The high activity of the cardiac Na+-Ca2+ exchanger has led to the suggestion that it plays an important role in the regulation of myocardial contractility. We have proposed that exercise training increases stroke volume as a consequence of an enhanced contractility caused by an adaptation in Ca2+ transport across the cardiac plasma membrane (sarcolemma). The present study examined the possibility that the Na+-Ca2+ exchanger in heart muscle is modified in response to training. Sprague-Dawley rats (female, n = 72) were randomly divided into exercise-trained (T) and sedentary control (C) groups. As a result of the 11-wk treadmill-training paradigm, group T had a 7.6% higher (P less than 0.005) heart-to-body weight ratio and a 36% increase (P less than 0.01) in gastrocnemius mitochondrial enzyme activity. Na+-Ca2+ exchange was studied in highly purified sarcolemmal vesicles using rapid-quenching techniques. The absolute initial rate of uptake was significantly higher in T vs. C at calcium concentrations [( Ca2+]) ranging from 10 to 80 microM. This increased uptake appears to be due solely to the fact that the apparent Km of the myocardial Na+-Ca2+ exchanger for Ca2+ was significantly lower in T vs. C (15.7 +/- 1.1 vs. 36.1 +/- 2.6 microM), since the maximum velocity was unchanged. The observed increase in the affinity of the exchanger for Ca2+ is not attributable to group differences in vesicular purity, cross-contamination, or passive Ca2+ efflux. This observation is consistent with observed alterations in sarcolemmal composition in response to exercise training. We propose that the modification of the Na+-Ca2+ exchanger may play an important role in the adaptation of the heart to exercise.

Vitrification and Crystallization Processes of High-<i>T</i><sub>c</sub> Superconducting Oxides in the System Bi-Ca-Sr-Cu-O

In a relatively wide region of the pseudoternary system BiO3/2-(CaO⋅SrO)1/2-CuO, CaO/SrO=1, glassy samples have been obtained by the rapid-quenching technique. The glass transition temperature in this system was found to be mainly dependent on the BiO3/2 content, with a maximum of 390°C at around 20mol% BiO3/2. The crystallization of the glasses of proper compositions such as BiCaSrCu2Ox and Bi2Ca2Sr2Cu3Ox produced superconductors consisting mainly of the low-Tc (80K) phase; this superconducting phase grew gradually with increasing heat-treatment temperature. The critical temperature Tc (zero) was 86K for Bi2Ca2Sr2Cu3Ox, whereas it can be expected to be increased by optimizing the heat-treatment conditions and/or using appropriate additives.

Pre-steady-state reduction kinetics of QH 2:cytochrome c oxidoreductase and the Q-pool: Evidence for a special quinone not in rapid equilibrium with the Q-pool

The pre-steady-state kinetics of the reduction of the prosthetic groups of QH 2: cytochrome c oxidoreductase in bovine heart submitochondrial particles were studied in relation to the kinetics of the Q-10 reduction, using duroquinol as substrate. The prosthetic groups, including semiquinone, were measured with EPR and low-temperature-diffuse reflectance spectroscopy, the samples being prepared with the rapid-freeze quench technique. For the determination of the redox state of ubiquinone in the pre-steady state the rapid chemical quench technique was used as an extension of the rapid-freeze quench technique, and Q-10 and QH 2-10 were measured with reversed-phase HPLC after extraction with petroleum ether. Ubiquinone was reduced biphasically, 8% of total Q-10 (equal to 1 mol Q-10/mol cytochrome c 1, being reduced within 5 ms, and the rest, the Q-pool, at a much lower rate. The initial rapid reduction of this special Q-10 was accompanied by rapid formation of Q ⨪ i and rapid reduction of a large part of the cytochrome b-562. Both semiquinone formation and reduction of b-562 showed transient kinetics due to a contribution of the reaction pathway via centre o when the iron-sulphur cluster and cytochrome c 1 were oxidised. The majority of the special quinol was located at centre i, probably bound, but also at centre o some bound quinol was formed. This was visible when antimycin was present, the antimycin-insensitive bound quinol being totally sensitive to myxothiazol. Myxothiazol alone accelerated the reduction of the Q-pool via centre i, but also the equilibration of cytochrome b-562 with the Q-pool. Antimycin drastically lowered the rate of reduction of the Q-pool and additionally seemed to block the rapid electron transfer from part of the Rieske iron-sulphur cluster to cytochrome c 1. It is concluded that, during the pre-steady-state, cytochrome b-562 is not in equilibrium with the Q-pool and that the rate of equilibration is probably determined by the rate of dissociation of the special bound quinol from centre i.

Binding of ADP and orthophosphate during the ATPase reaction of nitrogenase.

The pre-steady-state ATPase activity of nitrogenase from Azotobacter vinelandii was investigated. By using a rapid-quench technique, it has been demonstrated that with the oxidized nitrogenase complex the same burst reaction of MgATP hydrolysis occurs as observed with the reduced complex, namely 6-8 mol orthophosphate released/mol MoFe protein. It is concluded that the pre-steady-state ATPase activity is independent of electron transfer from Fe protein to MoFe protein. Results obtained from gel centrifugation experiments showed that during the steady state of reductant-independent ATP hydrolysis there is a slow dissociation of one molecule of MgADP from the nitrogenase proteins (koff less than or equal to 0.2 s-1); the second MgADP molecule dissociates much faster (koff greater than or equal to 0.6 s-1). Under the same conditions orthophosphate was found to be associated with the nitrogenase proteins. The rate of dissociation of orthophosphate from the nitrogenase complex, as estimated from the gel centrifugation experiments, is in the same order of magnitude as the steady-state turnover rate of the reductant-independent ATPase activity (0.6 mol Pi formed X s-1 X mol Av2(-1) at 22 degrees C). These data are consistent with dissociation of orthophosphate or MgADP being rate-limiting during nitrogenase-catalyzed reductant-independent ATP hydrolysis.

Age hardening of rapidly quenched Al-Zr-B alloys

Ternary alloys from the Al-Zr-B system were prepared as thin foils by a rapid quench technique. These foils were annealed isothermally as well as isochronally at various temperatures (150 to 550° C). The microstructures show that at high temperature, the grain growth is significantly retarded by the grain boundary pinning of boride precipitates. A strong age hardening is also a characteristic phenomenon in these alloys. It is found that microstructure and microhardness largely depend upon the zirconium/boron ratio of the alloy, indicating that the ratio determines the types of compounds occurring in this alloy system. It appears that in these alloys, high concentration of boron and the low ratio of zirconium/boron together yield stable precipitates at high temperatures.

Surface characteristics and electro-oxidation of formaldehyde and formate ion on amorphous Cu-Pd-Zr ternary alloys

A series of amorphous Cu-Pd-Zr alloys were fabricated by a rapid quench technique. Surface characterization before and after the treatment with aqueous HF was made mainly by surface area measurements and X-ray photoelectron spectroscopy. It was found that zirconium was leached out by the acid treatment and a Raney-type porous Cu-Pd alloy layer was effectively formed at the surface. Electrocatalytic properties for HCHO and HCOO − electro-oxidation were investigated in a 1.0 M NaOH solution at room temperature. Surface composition analyses showed that surface enrichment in copper or palladium was small even after the acid treatment. It was seen that the alloys rich in palladium were active both for HCHO and HCOO − electro-oxidation, while those rich in copper were active only for HCHO. The alloy with composition [ Cu]:[ Pd] = 1:3 had nearly the same activity for HCHO and HCOO − electro-oxidation.

Glass formation in the NiCrNb system

The development of metallic glasses which can be produced by rapid-quenching techniques has led to a new class of materials with extraordinary properties [1]. Depending upon the alloy composition, metallic glasses have been found to exhibit any of several attractive properties, such as good corrosion resistance, stability under irradiation, high strength and hardness and interesting magnetic and electrical properties. Many of the metallic glasses studied so far crystallize at relatively low temperatures and structural relaxation of the glass may occur as much as 200 K below the crystallization temperature leading to further deterioration in thermal stability. It is therefore desirable to identify glasses of higher crystallization temperature. Some effort has been made in this direction to develop metal-metalloid glasses [2], but little attention has been paid to glasses containing only metallic elements. Of the known metal-metal glass-forming alloys, the Ni60Nb40 (subscripts refer to at %) composition readily forms a glass of high crystallization temperature (Tx~923K) [3-6]. Since the addition of chromium ha's been found to be most effective in improving corrosion resistance in metalmetalloid glasses [1], we have investigated the effect of its addition on the glass-forming ability of Ni60Nb40 alloys. Alloy buttons of compositions Ni60CrxNb40 x and Ni60 yCryNb40 (x, y = 0, 5, 10, 13 and 15) were made by arc melting high-purity components in a pure argon atmosphere over a water-cooled copper crucible. The buttons were remelted at least six times to ensure homogeneity. Weight losses during melting were negligible and the nominal compositions were accepted. Rapidly quenched ribbons were prepared in s i tu by the melt-spinning technique. After induction melting in a quartz nozzle of around 1 mm orifice diameter, the molten metal was ejected onto a rotating polished copper wheel (~ 200mm diameter) using an ejection pressure of pure argon. Before melting, the nozzle containing the alloy pieces was repeatedly flushed with argon and an additional argon flow around the nozzle was maintained throughout the experiment by incorporating a concentric quartz tube of larger diameter to prevent oxidation of the melt pool. The ejection pressure and wheel velocity were adjusted to provide around 20 to 25 #m thick and 2 to 3 mm wide ribbons. X-ray-diffraction analyses of the asquenched ribbons were made using a Philips diffractometer with CoK~ radiation at a scanning rate of 1 ° (20)min -j . X-ray-diffractometer traces of the asquenched alloy ribbons are shown in Fig. 1. In the melt-spun Ni60CrxNb40 x alloy ribbons, X-ray diffraction revealed only diffuse intensity maxima characteristics of an amorphous

Crystallization and glass formation

The principal outstanding problems in the areas of glass formation, crystal nucleation and crystal growth are discussed. Each of these areas is seen to provide notable opportunity for improvements in our understanding. Also discussed are anticipated future directions in crystallization and glass formation. These include an expanded range of materials obtained and used as glasses, increasing utilization of rapid quench techniques, and wider use of sol-gel methods and forming glasses by viscous sintering.

Rate-limiting steps in the DNA polymerase I reaction pathway

The initial rates of incorporation of dTTP and thymidine 5'-O-(3-thiotriphosphate) (dTTP alpha S) into poly(dA) X oligo(dT) during template-directed synthesis by the large fragment of DNA polymerase I have been measured by using a rapid-quench technique. The rates were initially equal, indicating a nonrate-limiting chemical step. However, the rate of thionucleotide incorporation steadily diminished to 10% of its initial value as the number of consecutive dTMP alpha S residues in the primer strand increased. This anomalous behavior can be attributed to the helix instability inherent in phosphorothioate-containing duplexes. Positional isotope exchange experiments employing the labeled substrate [alpha-18O2]dATP have revealed negligible alpha, beta-bridging----beta-nonbridging isotope exchange in template-directed reactions of Escherichia coli DNA polymerase I (Pol I) both in the presence and in the absence of added inorganic pyrophosphate (PPi), suggesting rapid PPi release following the chemical step. These observations are consistent with a rate-limiting step that is tentatively assigned to a conformational change of the E X DNA X dNTP complex immediately preceding the chemical step. In addition, the substrate analogue (Sp)-dATP alpha S has been employed to examine the mechanism of the PPi exchange reaction catalyzed by Pol I. The net retention of configuration at the alpha-P is interpreted in terms of two consecutive inversion reactions, namely, 3'-hydroxyl attack, followed by PPi attack on the newly formed primer terminus. Kinetic analysis has revealed that while alpha-phosphorothioate substitution has no effect upon the initial rate of polymerization, it does attenuate the PPi exchange reaction by a factor of 15-18 fold.(ABSTRACT TRUNCATED AT 250 WORDS)

Kinetics of citrate-induced activation and polymerization of chick liver acetyl-CoA carboxylase.

The kinetic time course of citrate-induced activation and polymerization (into filaments) of the protomeric form of acetyl-CoA carboxylase were compared to assess the concertedness of the two processes. Rapid-quench techniques were employed to measure the kinetics of activation of the carboxylase-catalyzed reaction by citrate. When enzyme was preincubated with citrate prior to initiating the steady state turnover reaction with acetyl-CoA in the rapid-quench device, the observed rate of carboxylation of acetyl-CoA was apparently linear from the moment of mixing. However, when enzyme was mixed with citrate to initiate the reaction, a lag (t1/2 = 0.7 s) occurred in the approach to steady state carboxylation rate. This lag was independent of enzyme concentration over a 230-fold range and was marginally dependent upon citrate concentration. Over the same range of enzyme concentration, polymerization of carboxylase protomers, as determined by right angle light scattering, was enzyme concentration-dependent in a manner predicted by a single protomer activation step, followed by a rate-limiting dimerization of active protomer and subsequent polymerization. Polymerization is a second order process, with a second order rate constant of 597,000 M-1 s-1. There appear to be two steps that limit polymerization of the inactive carboxylase protomer: a rapid citrate-induced conformational change, which is independent of enzyme concentration and leads to an active protomeric form of the enzyme and the dimerization of the active protomer, which constitutes the first step of polymerization and is enzyme concentration-dependent. Dimerization is the rate-limiting step of acetyl-CoA carboxylase polymerization. On the basis these results, it is concluded that activation of catalysis and the polymerization of carboxylase protomers are not concerted. Furthermore, activation of carboxylation leading to the formation of an active protomer was faster than polymerization under all conditions, and therefore precedes polymerization. It was also shown that the activation constant (Kact) for citrate is altered in a predictable manner by the accumulation of the reaction product, malonyl-CoA, the Kact increasing with malonyl-CoA concentration. Depolymerization of fully polymerized acetyl-CoA carboxylase is caused by malonyl-CoA or ATP.Mg (and HCO3-). Both malonyl-CoA and ATP.Mg (and HCO3-) compete with citrate in the maintenance of a given state of the protomer-polymer equilibrium apparently by carboxylating the enzyme to form enzyme-biotin CO2- which destabilizes the polymeric form.

Glass Formation by Rapid Quenching in Lithium Silicates Containing Large Amounts of Li<sub>2</sub>O

The rapid-quenching technique extended the glass-forming region in the system Li2O-SiO2 toward the composition with high Li2O content. The ratio of the glass transition temperature Tg to the liquidus one T1 deviates largely from the so-called “two thirds rule” (Tg/T1=2/3) in the glasses containing large amounts of Li2O. The deviation was observed only in the glasses prepared by rapid quenching and it can be attributed to their high liquidus viscosity or small temperature variation of viscosity. The relation between the deviation from the “two thirds rule” and the glass-forming ability was discussed on the basis of the temperature and time variation curves of viscosity.

The mechanism of Klebsiella pneumoniae nitrogenase action. Pre-steady-state kinetics of an enzyme-bound intermediate in N2 reduction and of NH3 formation.

The reduction of N2 to 2NH3 by Klebsiella pneumoniae nitrogenase was studied by a rapid-quench technique. The pre-steady-state time course for N2H4, formed on quenching by the acid-induced hydrolysis of an enzyme-bound intermediate in N2 reduction, showed a 230 ms lag followed by a damped oscillatory approach to a constant concentration in the steady state. The pre-steady-state time course for NH3 formation exhibited a lag of 500 ms and a burst phase that was essentially complete at 1.5s, before a steady-state rate was achieved. These time courses have been simulated by using a previously described kinetic model for the mechanism of nitrogenase action [Lowe & Thorneley (1984) Biochem. J. 224, 877-886]. A hydrazido(2-) structure (=N-NH2) is favoured for the intermediate that yields N2H4 on quenching. The NH3-formation data indicate enzyme-bound metallo-nitrido (identical to N) or -imido (=NH) intermediates formed after N-N bond cleavage to produce the first molecule of NH3 and which subsequently give the second molecule of NH3 by hydrolysis on quenching. The simulations require stoichiometric reduction of one N2 molecule at each Mo and the displacement of one H2 when N2 binds to the MoFe protein. Inhibition by H2 of N2-reduction activity occurs before the formation of the proposed hydrazido(2-) species, and is explained by H2 displacement of N2 at the active site.

The mechanism of Klebsiella pneumoniae nitrogenase action. Pre-steady-state kinetics of H2 formation.

A comprehensive model for the mechanism of nitrogenase action is used to simulate pre-steady-state kinetic data for H2 evolution in the presence and in the absence of N2, obtained by using a rapid-quench technique with nitrogenase from Klebsiella pneumoniae. These simulations use independently determined rate constants that define the model in terms of the following partial reactions: component protein association and dissociation, electron transfer from Fe protein to MoFe protein coupled to the hydrolysis of MgATP, reduction of oxidized Fe protein by Na2S2O4, reversible N2 binding by H2 displacement and H2 evolution. Two rate-limiting dissociations of oxidized Fe protein from reduced MoFe protein precede H2 evolution, which occurs from the free MoFe protein. Thus Fe protein suppresses H2 evolution by binding to the MoFe protein. This is a necessary condition for efficient N2 binding to reduced MoFe protein.

The differential effects of short-time glutaraldehyde treatments on light-induced thylakoid membrane conformational changes, proton pumping and electron transport properties

A rapid quench technique utilizing the addition of excess buffer containing free amine groups (Tris, glycylglycine) to the reaction medium has enabled a detailed study of the time-course of glutaraldehyde inactivation on the spinach thylakoid membrane to be undertaken. The following light-induced parameters were inactivated in the sequence: slow transmittance changes (0–5 s) > coupling factor activity (5–20 s) > narrow angle 90° scattering changes (30–60 s). About 20% of PS II activity was lost by this treatment. No effect on activity, proton pumping and proton gradient formation was observed over the time-course studied. A consideration of these effects led to the proposal that the slow, light-induced transmittance changes reflect reversible thylakoid structural changes (unstacking, membrane flattening) in response to electron transport and the consequent proton pumping. The narrow angle 90° scattering changes were considered to reflect directly microconformational structural changes in response to the light-driven proton translocation as previously concluded from other workers.

Hard Magnetic MnAl and MnBi Ribbons Made from Molten State by Rapid Cooling

MnAl and MnBi ribbons were made from the molten alloys by the single-roller rapid-quenching technique, and their crystallographical, mechanical and magnetic properties were studied. Long, flexible MnAl ribbons were produced, and the ferromagnetic τ-phase was obtained by a simple annealing treatment. The best magnetic properties were Br=3.75 kG, IHc=1.83 kOe and (BH)max=2.1 MGOe for 56 at%MnAl ribbon. The MnBi ribbons were short and brittle, but they appear to be more stable chemically than the cast alloy. The best characteristics of the MnBi ribbons were Br=3 kG, IHc=1.5 kOe and (BH)max=1.6 MGOe.

Kinetics of activation of acetyl-CoA carboxylase by citrate. Relationship to the rate of polymerization of the enzyme.

The kinetics of citrate-induced activation and polymerization (into filaments) of the 450,000-dalton protomeric form of acetyl-CoA carboxylase were compared to assess the concertedness of the two processes. Rapid-quench techniques were employed to measure the time course of activation by citrate of the carboxylase-catalyzed reaction. When enzyme was preincubated with citrate prior to initiating the steady state turnover reaction with acetyl-CoA in the rapid-quench device, the observed rate of carboxylation of acetyl-CoA was apparently linear from the moment of mixing. However, when enzyme was mixed with citrate to initiate the reaction, a lag (t1/2 = 0.7 s) occurred in the approach to steady state carboxylation rate. This lag was independent of enzyme concentration over a 230-fold range and was marginally dependent upon citrate concentration. Over the same range of enzyme concentration, polymerization of carboxylase protomers, as determined by right angle light scattering, was enzyme concentration-dependent in a manner predicted by a single protomer activation step, followed by a rate-limiting dimerization of active protomer and subsequent polymerization. Based on these results, it is concluded that activation of catalysis and the polymerization of carboxylase protomers are not concerted. Furthermore, activation of carboxylation leading to the formation of an active protomer was faster than polymerization under all conditions, and therefore precedes polymerization. It was also shown that the activation constant (Kact) for citrate is altered in a predictable manner by the accumulation of the reaction product, malonyl-CoA, the Kact increasing with increasing malonyl-CoA concentration. Additional evidence is presented indicating that this change in Kact was not caused by autophosphorylation of the enzyme under these conditions and that phosphorylation does not affect the mechanism of activation elucidated above.

Initial products of photophosphorylation with AMP and [32P]Pi.

The role of AMP in photophosphorylation was studied using rapid mixing acid quench techniques. Fragmented spinach chloroplast membranes or subchloroplast particles were illuminated and rapidly mixed with [32P]orthophosphate and AMP at pH 7 for 10 ms to 60 s after which time perchloric acid was added to quench the reaction. ATP was found to be the primary and predominant nucleotide labeled. It was found that after illumination, an adenylate kinase-like activity carried out an AMP-dependent conversion of labeled ATP to labeled ADP which was inhibited by the presence of ADP. This reaction was characterized as being similar to chloroplast adenylate kinase in Mg2+ dependency and in sensitivity to phlorizin and tentoxin and distinct from chloroplast coupling factor 1. The small amounts of adenylate kinase activity present in fragmented well washed chloroplast membranes were found to be sufficient to carry out this rapid reaction. These results necessitated a reinterpretation of the earlier findings of Tiefert and Moudrianakis (Tiefert, M.A., and Moudrianakis, E.N. (1979) J. Biol. Chem. 254, 9500-9508) and no longer support the role of ADP as a phosphorylated intermediate in ATP synthesis.

MECHANISTIC STUDIES ON NITROGENASE FROM KLEBSIELLA PNEUMONIAE USING THE RAPID QUENCH TECHNIQUE

ABSTRACT A mechanism for the nitrogenase-catalysed reduction of dinitrogen (N2) to ammonia is presented. The mechanism is based on pre-steady state kinetic data for dihydrogen (H2), hydrazine (N2H4) and ammonia (NH3) formation obtained by a rapid quench technique and on a consideration of the chemistry of transition metal dinitrogen, dinitrogen hydride and hydride complexes. Stopped-flow spectrophotometry has demonstrated that electron transfer between the component proteins of nitrogenase is coupled to the hydrolysis of MgATP and that the dissociation of the oxidised Fe protein from reduced Mo-Fe protein is the rate limiting step in the catalytic cycle. A hydrazido(2–) species is suggested as an intermediate in N2 reduction and a nitride or nitrene species as an intermediate in both N2 and azide reduction.

New Materials Made by Rapid Quenching Technique

New Materials Made by Rapid Quenching Technique

Stabilization of β-Be at room temperature by a rapid quench technique

The retention of the ..beta..-Be phase at room temperature by the use of a rapid quench technique opens the way for a full investigation of its physical properties. Studied were thermal response, hardness, electrical resistivity, structural analysis, and superconductivity. Preliminary work indicates that the stabilization of the ..beta..-Be structure at room temperature is a very sensitive function of Ni content. This sensitivity is illustrated by the fact that the Be/sub 92/Ni/sub 8/ sample is mainly ..cap alpha..-phase while the Be/sub 91/ /sub 8/Ni/sub 8/ /sub 2/ is almost completely ..beta..-phase. Retention of the ..beta..-structure apparently requires the exact eutectoid composition and a high quench rate.