Bi Reaction Research Articles

Expression, Purification, and Characterization of Choline Kinase, Product of the CKI Gene from Saccharomyces cerevisiae

In the yeast Saccharomyces cerevisiae, choline kinase (ATP:choline phosphotransferase, EC 2.7.1.32) is the product of the CKI gene. Choline kinase catalyzes the committed step in the synthesis of phosphatidylcholine by the CDP-choline pathway. The yeast enzyme was overexpressed 106-fold in Sf-9 insect cells and purified 71.2-fold to homogeneity from the cytosolic fraction by chromatography with concanavalin A, Affi-Gel Blue, and Mono Q. The N-terminal amino acid sequence of purified choline kinase matched perfectly with the deduced sequence of the CKI gene. The minimum subunit molecular mass (73 kDa) of purified choline kinase was in good agreement with the predicted size (66.3 kDa) of the CKI gene product. Native choline kinase existed in oligomeric structures of dimers, tetramers, and octomers. The amounts of the tetrameric and octomeric forms increased in the presence of the substrate ATP. Antibodies were raised against the purified enzyme and were used to identify choline kinase in insect cells and in S. cerevisiae. Maximum choline kinase activity was dependent on Mg2+ ions (10 mM) at pH 9.5 and at 30 degrees C. The equilibrium constant (0.2) for the reaction indicated that the reverse reaction was favored in vitro. The activation energy for the reaction was 6.26 kcal/mol, and the enzyme was labile above 30 degrees C. Choline kinase exhibited saturation kinetics with respect to choline and positive cooperative kinetics with respect to ATP (n = 1.4-2.3). Results of the kinetic experiments indicated that the enzyme catalyzes a sequential Bi Bi reaction. The Vmax for the reaction was 138.7 micromol/min/mg, and the Km values for choline and ATP were 0.27 mM and 90 microM, respectively. The turnover number per choline kinase subunit was 153 s-1. Ethanolamine was a poor substrate for the purified choline kinase, and it was also poor inhibitor of choline kinase activity. ADP inhibited choline kinase activity (IC50 = 0.32 mM) in a positive cooperative manner (n = 1.5), and the mechanism of inhibition with respect to ATP and choline was complex. The regulation of choline kinase activity by ATP and ADP may be physiologically relevant.

Neutron-induced peaks in Ge detectors from evaporation neutrons

We have studied the peak shapes at 596 and 691 keV resulting from fast neutron interactions inside germanium detectors. We have used neutrons from a 252Cf source, as well as from the 28Si(μ −, nv), and 209Bi(π −, xn) reactions to compare the peaks and to check for a dependence of peak shape on the incoming neutron energy. In our investigation, no difference between these three measurements has been observed. In a comparison of these peak shapes with other studies, we found similar results to ours except for those measurements using monoenergetic neutrons in which a significant variation with neutron energy has been observed.

Biochemical Studies of Saccharomyces cerevisiae Myristoyl-coenzyme A:Protein N-Myristoyltransferase Mutants

Saccharomyces cerevisiae myristoyl-CoA:protein N-myristoyltransferase (Nmt1p) is an essential 455-residue, monomeric enzyme that catalyzes the transfer of myristate from myristoyl-CoA to the NH2-terminal Gly residue of cellular proteins. Nmt1p has an ordered Bi Bi reaction mechanism with binding of myristoyl-CoA occurring before binding of peptide substrates. To define residues important for function, the polymerase chain reaction was used to generate random mutations in the NMT1 gene. A colony color sectoring assay was used to screen a library of 52,000 transformants for nmt1 alleles encoding enzymes with reduced activity. nmt1 alleles were identified that produced temperature-sensitive (ts) growth arrest due to substitutions affecting eight residues conserved in orthologous Nmts: Asn102, Ala202, Cys217, Ser328, Val395, Asn404, Leu420, and Asn426. Ala202 --> Thr, Cys217 --> Arg, Ser328 --> Pro, Asn404 --> Tyr, and Asn426 --> Ile produced the most severe ts phenotype. Their effects on the functional properties of the enzyme's myristoyl-CoA and peptide binding sites were defined by purifying each mutant from Escherichia coli and conducting in vitro kinetic analyses with acyl-CoA and peptide substrates and with two competitive inhibitors: S-(2-oxo)pentadecyl-CoA, a nonhydrolyzable myristoyl-CoA analog, and SC-58272, a peptidomimetic derived from the NH2-terminal sequence of an Nmt1p substrate (ADP-ribosylation factor-2, Arf2p). None of the substitutions affect the enzyme's acyl chain length selectivity. When compared with wild type Nmt1p, Cys217 --> Arg produces 3- and 6-fold increases in Ki for SC-58272 at 24 and 37 degrees C but no change in Ki for S-(2-oxo)pentadecyl-CoA, indicating that the substitution selectively affects Nmt1p's peptide binding site. Asn426 --> Ile selectively perturbs the myristoyl-CoA binding site, resulting in the most pronounced reduction in affinity for S-(2-oxo)pentadecyl-CoA (12- and 20-fold). Ala202 --> Thr, which confers the most severe ts phenotype, provides an example of a substitution that affects both sites, producing 3- and 6-fold increases in the Ki for S-(2-oxo)pentadecyl-CoA and 6- and 9-fold increases in the Ki for SC-58272 at 24 and 37 degrees C. An N-myristoylation-dependent change in the electrophoretic mobility of Arf1p was used to assay the effects of the mutants on cellular levels of protein N-myristoylation under a variety of growth conditions. The ts growth arrest produced by nmt1 alleles correlates with a reduction in myristoyl-Arf1p to </=50% of total cellular Arf1p.

Mass and charge distributions for the reaction 40Ca + 209Bi at 600 MeV.

The charge and mass of the projectile-like fragments produced in the 15-MeV per nucleon $^{40}\mathrm{Ca}$${+}^{209}$Bi reaction were determined for products detected near the grazing angle. Neutron number-charge (N-Z) distributions were generated as a function of the total kinetic energy loss and parametrized by their centroids, variances, and correlation coefficients. Although the initial system is very asymmetric, after the interaction, a drift of the charge and mass centroids toward further asymmetry is observed. The production of projectile-like fragments is consistent with a tendency of the projectile-like fragments to retain the projectile neutron-to-proton ratio 〈N〉/〈Z〉\ensuremath{\simeq}1. The correlation coefficient remains well below 1.0 for the entire range of total kinetic energy lost. Predictions of two nucleon exchange models, Randrup's and Tassan-Got's, are compared to the experimental results. The models are not able to reproduce the evolution of the experimental distributions, especially the fact that the variances reach a maximum and then decrease as function of the energy loss. This behavior supports the hypothesis that some form of projectile-like fragmentation or cluster emission is perturbing the product distribution from that expected from a damped mechanism.

Direct proton decay from the Gamow-Teller resonance in 208Bi.

Spin-isospin excitations in {sup 208}Bi have been investigated using the {sup 208}Pb ({sup 3}He,{ital t}){sup 208}Bi reaction at near {theta}{approx}0{degree} at {ital E}({sup 3}He)=450 MeV. The microscopic structure of the Gamow-Teller resonance (GTR), the isobaric analog state (IAS), and the spin-flip dipole ({Delta}{ital L}=1) resonance (SDR) in {sup 208}Bi has been studied by observing their direct proton decays to the low-lying neutron-hole states in {sup 207}Pb. Decay protons were measured at backward angles in coincidence with tritons detected at and near 0{degree}. The total branching ratio for proton decay from the GTR is determined to be only 4.9{plus_minus}1.3%. The total branching ratio for proton decay from the SDR amounts to 14.1{plus_minus}4.2%. The deduced total widths as well as the total and partial proton escape widths of the GRR and IAS are found to be in reasonable agreement with recent theoretical estimates obtained in the framework of the continuum Tamm-Dancoff approximation.

Fission of209Bi induced by6He ions

Secondary beams of4He and6He were produced through the transfer reactions with the 18.5 MeV/u11B and 35.5 MeV/u7Li primary beams. The fusion-fission cross sections have been measured for the4,6He +209Bi reactions at energies Ecm>1.5*Bfus. In the present experiment it was found that the fission excitation function for the neutron rich nuclei6He is significantly higher than for the4He nuclei.

Evidence for the possible synthesis of element 110 produced by the 59Co+209Bi reaction.

An experiment to synthesize element 110 by the Co59+209Bi reaction has been performed at the SuperHILAC at the Lawrence Berkeley Laboratory. One event with many of the expected characteristics of a successful synthesis of 110267 was observed. This event corresponds to a production cross section of about one picobarn.Received 22 November 1994DOI:https://doi.org/10.1103/PhysRevC.51.R2293©1995 American Physical Society

Entrance-channel dependence of the fission dynamics in 19F+209Bi vs 64Ni+165Ho.

Binary fragmentations of $^{19}\mathrm{F}$${+}^{209}$Bi (${\mathit{E}}_{\mathit{p}}$=491 MeV) and $^{64}\mathrm{Ni}$${+}^{165}$Ho (${\mathit{E}}_{\mathit{p}}$=778 MeV) leading to compound nuclei with ${\mathit{E}}^{\mathrm{*}}$\ensuremath{\approxeq}400 MeV and similar masses were investigated by measuring fission coincident neutrons and \ensuremath{\alpha} particles. The neutron field was decomposed into contributions from preequilibrium (PE), prescission, and the two fragment sources with a moving source analysis. It was performed for separate excitation energy classes deduced with the folding angle technique from the linear momentum transfer. The angular distribution of the PE neutrons from the $^{19}\mathrm{F}$${+}^{209}$Bi reaction indicates a pronounced out-of-plane anisotropy that would result in an overestimation of the PE multiplicity by 37% for a measurement only in plane. The PE multiplicities and temperatures are interpreted by a delay in formation for the more symmetric entrance channel $^{64}\mathrm{Ni}$${+}^{165}$Ho, which is typical for quasifission and in line with the enhancement of asymmetric mass splits. Prescission times derived from the neutron clock method extend from 30\ifmmode\times\else\texttimes\fi{}${10}^{\mathrm{\ensuremath{-}}22}$ s for $^{64}\mathrm{Ni}$${+}^{165}$Ho to 160\ifmmode\times\else\texttimes\fi{}${10}^{\mathrm{\ensuremath{-}}22}$ s for $^{19}\mathrm{F}$${+}^{209}$Bi. Here, a constant level density parameter of A/10 ${\mathrm{MeV}}^{\mathrm{\ensuremath{-}}1}$ was used which is an outcome of the simultaneous measurement of \ensuremath{\alpha} emission temperatures. The prescission times are qualitatively discussed in terms of the reaction dynamics.

Evidence for the synthesis of 267110 produced by the 59Co+ 209Bi reaction

An experiment to synthesize element 110 by the 59Co+ 209Bi reaction has been performed at the SuperHILAC at the Lawrence Berkeley Laboratory. One event with many of the expected characteristics of a successful synthesis of 267110 was observed. This event corresponds to a production cross section of about one picobarn.

Dynamical production of intermediate-mass fragments in peripheral 209Bi+ 136Xe collisions at [formula omitted

Intermediate-mass fragments (IMF) from the reaction 209Bi+ 136Xe at E lab A =28 MeV have been measured in coincidence with other charged reaction products, as well as with neutrons. The IMF emission patterns are seen to exhibit signatures of two mechanisms — statistical emission from the fully accelerated massive reaction partners and a fast dynamical emission from a single effective source. The latter mechanism, possibly involving a multiple neck rupture, becomes dominating for least dissipative collisions characterized by low associated light particle multiplicities.

The 4-fold fission in [formula omitted], 197Au and 159Tb reactions at 25 MeV/u

The paper presents the 4-fold fission or fragmentation of hot nuclei produced in 25 MeV/u 40Ar + 209Bi , 197Au and 159Tb reactions. The events with 4 massive fragments emitted with angles larger than 36 ° were detected by 8 PPACs with area of 25 × 20 cm 2. The TKE, distributions of mass and velocity for the four fragments have been obtained.

Cross sections of the (HI, αn) channel in the cold-fusion-type reactions 209Bi+ 40Ar and 208Pb+ 37Cl

By applying an off-line radiochemistry technique, cross sections for the production of the isotope 240 Cm(T 1 2 =27 d) in the cold-fusion-type reactions 209Bi+ 40Ar and 208Pb+ 37Cl at the bombarding energy E lab⩽230 MeV were determined to be 0.5±0.2 nb and 0.6±0.3 nb, respectively. The production of 240Cm was attributed to the 1n-deexcitation channel of the composite systems 249Md and 245Es. At the same time, the measured 240Cm production cross sections represent upper cross-section limits for the (HI, αn) channel of the reactions under study. These upper limits are about 100 times lower than the cross-section values reported recently by Nomura et al. for the ( 40 Ar, αx n) channels with x=1,2 of the 209Bi+ 40Ar reaction at E lab=208 MeV. In the context of the above measurements, presented and discussed is the up-to-date summary of the available evidence on cross sections of the (HI, αxn) channels in the cold-fusion-type reactions induced by projectiles ranging from 37Cl to 50Ti on targets of 203,205Tl, 208Pb, and 209Bi nuclei. Appreciable EC(β +)-delayed or/and spontaneous fission effects were detected in the 209Bi+ 40Ar, 208Pb+ 37Cl, and 206Pb+ 37Cl reactions. The obtained data point, in particular, to the EC(β +)-delayed fission occuring in the decay chains ▪ and ▪.

Isothermal titration calorimetric studies of Saccharomyces cerevisiae myristoyl-CoA:protein N-myristoyltransferase. Determinants of binding energy and catalytic discrimination among acyl-CoA and peptide ligands

Saccharomyces cerevisiae myristoyl-CoA:protein N-myristoyltransferase (Nmt1p) is an essential, monomeric enzyme that catalyzes the transfer of myristate from CoA to the amino-terminal Gly residue of cellular proteins. Product inhibition studies indicate that Nmt1p has an ordered Bi Bi reaction mechanism with myristoyl-CoA binding to the apo-enzyme to form a high affinity binary complex followed by binding of peptide with subsequent release of CoA and then the myristoylpeptide product. We have used isothermal titration calorimetry to quantify the effects of varying acyl chain length and removing the 3'-phosphate group of CoA on the energetics of interaction between Nmt1p and acyl-CoA ligands. Myristoyl-CoA binds to apo-Nmt1p with an affinity of 15 nM, corresponding to a binding free energy of -10.9 kcal/mol. This free energy is composed of a large favorable enthalpy of -24 kcal/mol and a large unfavorable entropic term. This large negative delta H degrees is consistent with a conformational change in the enzyme upon ligation, allowing synthesis of a functional peptide binding site. Binding of palmitoyl-CoA and lauroyl-CoA is driven by an exothermic enthalpy change which is much smaller than the corresponding parameter for myristoyl-CoA binding. The large differences in binding enthalpy and entropy (delta delta H degrees and T delta delta S degrees = 8-9 kcal/mol) demonstrate that the "off-length" acyl-CoAs bind to Nmt1p in a significantly different energetic fashion from myristoyl-CoA, even though the enzyme does not have a great deal of specificity among these ligands in terms of binding free energy (delta delta G degrees < or = 1 kcal/mol). The effect of removing the CoA 3'-phosphate group from myristoyl-CoA is similar to the effect of a two-carbon change in acyl chain length: i.e. an enthalpy dominated reduction in binding affinity. However, kinetic studies reveal that removing the 3'-phosphate from myristoyl-CoA has little effect on Nmt1p's catalytic efficiency, indicating that the 3'-phosphate group contributes binding free energy but little catalytic destabilization. The greater delta delta G degrees, with smaller delta delta H degrees and delta delta S degrees components, produced by removing the 3'-phosphate compared to increasing chain length suggests that it is not primarily the interactions of the 3'-phosphate which are disrupted when palmitoyl-CoA is substituted for myristoyl-CoA. No detectable interactions were noted between apo-Nmt1p and the substrate peptide, GAAPSKIV-NH2, providing additional support for the preferred ordered reaction mechanism.(ABSTRACT TRUNCATED AT 400 WORDS)

The substrate specificity of Saccharomyces cerevisiae myristoyl-CoA: protein N-myristoyltransferase. Polar probes of the enzyme's myristoyl-CoA recognition site.

Saccharomyces cerevisiae myristoyl-CoA:protein N-myristoyltransferase (Nmt1p) is a monomeric enzyme that is essential for vegetative growth. Nmt1p catalyzes the co-translational transfer of myristate from CoA to the amino-terminal Gly of cellular proteins in an ordered Bi Bi reaction mechanism that initially involves binding of myristoyl-CoA to the apoenzyme. Forty one fatty acid analogs were synthesized to define features in the acyl chain of myristoyl-CoA which are important determinants of its recognition by Nmt1p's acyl-CoA binding site as well as to help us deduce the structure of the binding site itself. These analogs included dicarboxylic acids, omega-nitrocarboxylic acids, analogs equivalent in length to C13:0-C15:0 which contain electronegative halogens at their omega-termini, hydroxytetradecanoic acids with hydrogen replaced by OH from C3 to C13, and azidophenyl-containing fatty acids with the linear azide unit attached either meta or para to phenyl and with variations in the length of their methylene chains. These compounds were converted to their CoA derivatives using Pseudomonas acyl-CoA synthetase and then surveyed as substrates for purified Nmt1p in an in vitro assay system that included an octapeptide derived from residues 1-8 of the human immunodeficiency virus Pr55gag polyprotein precursor. The results suggest that the myristoyl-CoA binding site contains a conical-shaped "receptor" that interacts with the omega-terminus of the bound acyl chain of acyl-CoAs. The acuteness of this cone determines the enzyme's capacity to accommodate steric bulk at the omega-terminus as well as Nmt1p's sensitivity to the distance between the eclipsed C5-C6 bond of a bound acyl chain and its omega-terminus. The activity profile of the various analog-CoAs also indicates that the enzyme's myristoyl-CoA binding site can accommodate fatty acid analogs with marked increases in polarity at their omega-terminus (compared to C14:0) as long as their chain length is equivalent to that of myristate.

High-spin spectroscopy of the reflection-asymmetric nucleus 221Ac

The high-spin state structure of the nucleus 221Ac was investigated by means of the 209Bi( 14C, 2n) reaction at 63 and 65 MeV. Measurements consisted of in-beam γγ coincidences using the Château de Cristal multidetector array and of e − γ coincidences. Two alternating parity bands could be established in 221Ac up to spin 35 2 h ̷ . Almost all of the states may be regrouped into parity doublets and the origin of these doublets is discussed.

On the determination of bismuth concentration in specimens by an alpha-activation technique

The α-activation technique for high-sensitivity Bi exploration in common rock minerals and alloys is developed. The technique based on bombardment of smooth surfaces of specimens being investigated with high fluxes of accelerated deuterium ions, Ed≥6 MeV / n total fluence ⊘ = 1013 - 1014 cm-2. The α-active polonium isotopes then are produced in nuclear reaction 209Bi(d,n)210Po. After deuterium beam exposure the induced activity of 210Po α-emitter is measured with α-sensitive plastic track detector— poly(allil)diglycolcarbone (CR-39) placed in close contact with the specimen surface for few hours or days. The control experiment with deuterium exposed Pb foils provides no α-particle track at all. The sensitivity obtained under these conditions corresponds to Bi concentrations at the level ≤10-6 g/g, the space resolution of Bi inclusions is ±20–25 μm. Our technique allows one to determine minor Bi concentrations in specimens which posess much higher content of Pb, Th and U.

MEASUREMENT AND ANALYSIS OF EXCITATION FUNCTIONS FOR ALPHA-INDUCED REACTIONS IN BISMUTH

Excitation functions for 209 Bi (α, 3n), 209 Bi (α, 4n) and 209 Bi (α, 5n) reactions have been measured in the energy range threshold to ≈50 MeV using stacked foil technique. Excitation functions have also been theoretically calculated and as expected, inclusion of pre-equilibrium contribution based on exciton model calculations along with compound nucleus calculations using Hauser–Feshbach formalism reproduces well the measured excitation functions. Interesting trend in the ratio of pre-equilibrium to equilibrium component with projectile energy has been observed.

Dynamics of the projectile-like fragment emission in the 40Ar+209Bi reaction at 26 MeV u-1

Angular distributions of fission fragments emitted in the 40Ar+209Bi reaction at 26 MeV u-1 were measured in coincidence with projectile-like fragments (PLF). Linear momentum transfer, angular momentum transfer ltr and spin alignment PZZ of the fissile nucleus were deduced as functions of the atomic number Z of PLF. Observed trend of ltr and PZZ suggests that a significant part of the PLF is emitted in dissipative process. Large missing momenta were observed. They are attributed to pre-equilibrium light-particle emission from the fissile nucleus.

A strong dynamic neutron source based on the 209Bi(γ, n) reaction

Abstract An intense dynamic n-source, I = 105 n/s having two n-groups En = 177 keV and 114 keV is reported. It is based on the Bi(γ, n) reaction where a chance overlap occurs between a γ-line of the Cu(n, γ) reaction and a nuclear level at 7637 keV in 209Bi. The thermal neutrons which induces the Cu(n, γ) reaction are obtained from a reactor. The spin, parity and total and radiative widths of the 7637 keV level in Bi were determined. The resonance cross sections for n-production are: σγn = 275 ± 40 mb and 93 ± 14 mb for the 177 keV and 114 keV groups respectively. The resulting dynamic n-source is the most intense which can be produced by this method.

Pre-equilibrium-equilibrium model calculations of nucleon radiative capture excitation functions

Excitation functions of activation cross sections for 130Te(p, gamma ), 142Ce(p, gamma ), 176Yb(p, gamma ) and 209Bi(p, gamma ) reactions and of integrated cross sections for 89Y(n, gamma ), 140Ce(n, gamma ) and 208Pb(n, gamma ) reactions were calculated using the pre-equilibrium-equilibrium exciton model. Results are compared to the available experimental data.