Transition kinetics of the conversion of blue to purple bacteriorhodopsin upon magnesium binding

Magnesium binding to cation-depleted blue bacteriorhodopsin (b-bR) was studied spectrophotometrically as well as by following stopped-flow kinetics. There exist three kinetically different steps in the binding process, yielding purple bacteriorhodopsin (p-bR). Since only the firtst step is dependent on the concentration of the reactants, the reaction scheme

can be proposed as the simplest model, with MgbR being the first intermediate and ΣI denoting a set of successive intermediates. According to this model k₁, k₋₁ and k₂ are calculated to be 2.8 × 10⁴ M⁻¹ · s⁻¹, 5.0 × 10 s⁻¹ and 1 × 10⁻² s⁻¹, respectively.     

Kinetics of the reversible association of ribosomal subunits: Stopped-flow studies of the rate law and of the effect of Mg2+

The kinetics of association and dissociation of Escherichia coli 30 S and 50 S ribosomal subunits appear to fit the simple scheme

over a wide range of Mg²⁺ and ribosome concentrations, for the preparations studied (which have a sharp [Mg²⁺]-dependence on the equilibrium degree of association, e.g. 10% to 90% for 1.5 mm to 3.5 mm). Both rate constants depend strongly upon magnesium ion concentration (k₂ goes from 0.04 × 10⁶ to 21 × 10⁶m⁻¹ s⁻¹ as [Mg²⁺] goes from 1.5 mm to 8 mm; k₁ goes from 150 to 2 s⁻¹ in the interval 1.0 to 3.0 mm), but k₁ may level off above 3.0 mm and k₂ increases slowly at high [Mg²⁺]. (The highest rate may not be far from the diffusion-controlled limit.) The primary effect of Mg²⁺, as calculated from the rather large changes in binding as a function of [Mg²⁺], is to decrease the contribution of electrostatic repulsion to the free energy of activation; specific, or class-specific, interactions of di- and multivalent cations with unknown ribosomal substructures may modulate this effect.     

Low Affinity and Slow Na+ Binding Precedes High Affinity Aspartate Binding in the Secondary-active Transporter GltPh

Glt_Ph from Pyrococcus horikoshii is a homotrimeric Na⁺-coupled aspartate transporter. It belongs to the widespread family of glutamate transporters, which also includes the mammalian excitatory amino acid transporters that take up the neurotransmitter glutamate. Each protomer in Glt_Ph consists of a trimerization domain involved in subunit interactions and a transport domain containing the substrate binding site. Here, we have studied the dynamics of Na⁺ and aspartate binding to Glt_Ph. Tryptophan fluorescence measurements on the fully active single tryptophan mutant F273W revealed that Na⁺ binds with low affinity to the apoprotein (K_d 120 mm), with a particularly low k_on value (5.1 m⁻¹s⁻¹). At least two sodium ions bind before aspartate. The binding of Na⁺ requires a very high activation energy (E_a 106.8 kJ mol⁻¹) and consequently has a large Q₁₀ value of 4.5, indicative of substantial conformational changes before or after the initial binding event. The apparent affinity for aspartate binding depended on the Na⁺ concentration present. Binding of aspartate was not observed in the absence of Na⁺, whereas in the presence of high Na⁺ concentrations (above the K_d for Na⁺) the dissociation constants for aspartate were in the nanomolar range, and the aspartate binding was fast (k_on of 1.4 × 10⁵ m⁻¹s⁻¹), with low E_a and Q₁₀ values (42.6 kJ mol⁻¹ and 1.8, respectively). We conclude that Na⁺ binding is most likely the rate-limiting step for substrate binding.     

MHC recognition by clones of Mls specific T-lymphocytes

To test whether M1s determinants, like other non-MHC or nominal antigens, are recognized by T-cells in association with H-2 determinants, the in vitro proliferative responses of T-cell lines and clones were studied. Lines and clones were prepared by soft agar cloning (B10.BR x BALB/c)F₁ (H-2^k/H-2^d, M1s^b/M1s^b) T-cells responding in a primary MLR to AKD2F1 (H-2^k/H-2^d, M1s^a/M1s^a) stimulator cells. All the T-cell clones obtained could respond equally well in a proliferative assay to the Mls^a determinant in association with the H-2 haplotype of either parent, i. e., DBA/2 (H-2^d, M1s^a), and AKR (H-2^k, M1s^a) both stimulated equally well. When the T-cell lines and clones were screened against stimulators from recombinant inbred (RI) strains, it became apparent that strains exhibiting the H-2^b, M1s^a genotype stimulated poorly or not at all. This shows that the T-cell response to M1s^a involves MHC recognition, and raises the possibility that the response to M1s^a can involve recognition of H-2 specificities shared between the H-2 ^k and H-2 ^d haplotypes.Abbreviations used in this paper MHC major histocompatibility complex - MLC mixed lymphocyte culture - IL-2 interleukin 2 - Con A concanavalin A - RI recombinant inbred Howard Hughes Medical Institute     

Synthesis and characterization of triaminecobalt(III) complexes and acid hydrolysis kinetics of fac-[Codpt(H2O)nX3−n]n+ (X = Cl,Br; n = 0,1,2)

The complexes CodptX₃ and [Codpt(H₂O)X₂]ClO₄ (X = Cl, Br; dpt = dipropylenetriamine = NH(CH₂CH₂CH₂NH₂)₂) have been prepared and characterized. Rate constants (s⁻¹) for aqueous solution at 25 °C and μ = 0.5 M (NaClO₄), for the acid-independent sequential ractions.

have been measured spectrophotometrically. For X = Cl: k₁ ⋍ 2 × 10⁻², k₂ = 1.7 × 10⁻⁴ and k₃ = 4.8 × 10⁻⁶, and for X = Br: k₁ ⋍ 2 × 10⁻², k₂ = 5.25 × 10⁻⁴ and k₃ = 2.5 × 10⁻⁵ The primary equation was found to be acid independent, while the secondary and tertiary aquations were acid-inhibited reactions. For the second step, the rate of the reaction was given by the rate equation

where C_t is the complex concentration in the aqua-and hydroxodihalo species, k′₂ is the rate constant for the acid-dependent pathway and K_a is the equilibrium constant between the hydroxo and aqua complex ions. The activation parameters were evaluated, for X = Cl: ΔH^≠₂ = 106.3 ± 0.4 kJ mol⁻¹ and ΔS^≠₂ = 40.2 ± 1.7 J K⁻¹ mol⁻, and for X = Br: ΔH^≠₂ = 91.6 ± 0.4 kJ mol⁻¹ and ΔS^≠₂ = 0.4 ± 1.7 J K⁻¹ mol⁻¹. The results are discussed and detailed comparisons of the reactivities of these complexes with other haloaminecobalt(III) species are presented.     

Mechanisms for acceleration of halide anation reactions of platinum(IV) complexes. REOA versus ligand assistance and platinum(II) catalysis Without central ion exchange

Chloride anation of trans-Pt(CN)₄ClOH₂⁻ has been studied with and without Pt(CN)₄²⁻ present at 25.0°C by use of stopped-flow and conventional spectrophotometry and a 1.00 M perchlorate medium. The rate law in the absence of Pt(CN)₄²⁻ is Rate=(p₁ + p₂ [H⁺] ) [Cl⁻]² [complex]/(1 + q [Cl₋]) with p₁=(3.0 ± 0.1) × 10⁻⁵ M⁻²s⁻¹, p₂=(3.6 ± 0.1) × 10⁻⁵ M⁻³ s⁻¹ and q=(0.62 ± 0.02) M⁻¹. It is compatible with a chloride assistance via an intermediate of the type Cl-Cl-Pt(CN)₄···OH₂²⁻, in which the reactivity of the aqua ligand is enhanced due to a partial reduction of the platinum. This mechanism of halide assistance is in principle the same as the modified reductive elimination oxidative addition (REOA) mechanism proposed by Poë, in which the intermediate is not split into free halogen, platinum(II) and water, and in which electron transfer not necessarily involves complete reduction to platinum(II). To avoid confusion with complete reductive eliminations, reactions without split of the intermediates are here termed halide-assisted reactions. The pH-dependence indicates acid catalysis via a protonated intermediate ClClPt(CN)₄···OH₃⁻.The Pt(CN)₄²⁻accelerated path has the rate law Rate=

[Cl-] [Pt(CN)₄²⁻] [complex] where k=(39.9±0.5) M⁻² s⁻¹ and K_a=(4.0±0.2)10⁻² M is the protolysis constant of trans-Pt(CN)₄ClOH²⁻.Reaction between PtCl₅OH₂⁻ and chloride is accelerated by Pt(CN)₄²⁻ and gives PtCl₆²⁻ as the reaction product. The rate law is Rate=k [Cl⁻] [Pt(CN)₄²⁻] [PtCl₅OH₂⁻] with k=(5.6 ± 0.2)10⁻³ M⁻² s⁻¹ at 35.0°C and for a 1.50 M perchlorate acid medium. The reaction takes place without central ion exchange. Alternative mechanisms with two consecutive central ion exchanges can be excluded. The role of Pt(CN)₄²⁻ in this reaction is very similar to that of the assisting halide in the halide assisted anations. [p ]Reaction between trans-Pt(CN)₄ClOH₂⁻ and PtCl₄²⁻ gives Pt(CN)₄²⁻ and PtCl₅OH₂⁻ as products and has the rate law Rate=k[PtCl₄²⁻] [trans-Pt(CN)₄ClOH₂⁻] with k=(3.32 ± 0.02) M⁻¹ s⁻¹ at 25 °C for a 1.00 M perchloric acid medium. The formation of an aqua complex as the primary reaction product and the rate independent of [Cl⁻] shows that formation of a bridged intermediate of the type Pt(II)Cl₄ClPt(IV)(CN)₄OH₂³⁻ is formed in the initial reaction step, not five-coordinated PtCl₅³⁻.     

Specificity and distribution of antigenic determinants on the polyoma virus capsid and nature of the reaction of immunoglobulin G antibody with the capsid surface.

Antisera to the LP and SP34 strains of polyoma virus have been prepared and their reactions with purified virions studied by double diffusion in agar and direct assay of antibody binding. One or more common antigenic determinants appear on the capsids of both strains. The form of this determinant varies slightly on each of the strains tested. The SP34 strain also carries its own strain-specific antigenic determinant. Both strains of virus were able to bind 150 anti-LP IgG³ molecules per virion and 50 anti-SP IgG molecules per virion. The slow rate of dissociation of bound IgG antibody (k_dissociation = 2 × 10⁻⁶ s⁻¹), and the rapid rate of antibody binding (k_dissociation = 2 × 10⁷m⁻¹ s⁻¹), suggest that IgG antibody is bound to the capsid surface through two antigen-antibody bonds. 50 anti-SP IgG molecules per capsid, divalently bound, completely inhibit the binding of 150 anti-LP IgG molecules, and vice versa. Consideration of the symmetry and molecular dimensions of the IgG molecule and the polyoma virus capsid leads to a model of the divalent interaction of IgG antibody with the common antigenic determinant(s). In this model, one species of antibody binds divalently to opposed subunits of a hexamer morphological unit. The other species of antibody binds divalently to the subunits on either side of the point of tangency of any two morphological units.     

Kinetics of the decomposition of hydrogen peroxide in the presence of ethylenediaminetetraacetatocerium(IV) complex

The rate of reaction of [Ce(EDTA)(OH)_nⁿ⁻] with H₂O₂ in 0.10 M KNO₃ solution was investigated at various temperatures. The presence of a peroxy intermediate is inferred from spectrophotometric measurements. The general rate equation,

is valid for pH 7-9 with n= 1 and 2 complexes involved. The rate constants k_l and k₂ were determined at 25 °C to be 0.054 and 0.171 M⁻¹ s⁻¹ respectively. The corresponding activation enthalpies, as calculated from Arrhenius plots, were δH₁^#= 51.3 ± 14.8 and δH₂^#= 41.8 ± 5.3 kJ m⁻¹ and the activation entropies were δS₁^#=-97 ± 47 and ΔS₂^#=−119±17 J K⁻¹ m⁻¹.     

Analysis of ligand-binding to the kringle 4 fragment from human plasminogen

The interaction of the isolated human plasminogen kringle 4 with the four -amino acid ligands -aminocaproic acid (ACA), N-acetyl-l-lysine (AcLys), trans-aminomethyl(cyclohexane)carboxylic acid (AMCHA) and p-benzylaminesulfonic acid (BASA) has been further characterized by ¹H-NMR spectroscopy at 300 and 600 MHz. Pronounced high-field shifts, reaching 3 ppm, are observed for AMCHA resonances upon binding to kringle 4, which underscores the relevance of ligand lipophilic interactions with aromatic side chains at the binding site. Ligand titration curves for the nine His and Trp singlets found in the kringle 4 aromatic spectrum reveal a striking uniformity in the kringle response to the various ligands. The average binding curves exhibit a clear Langmuir absorption isotherm saturation profile and the data were analyzed under the assumption of one (high affinity) binding site per kringle. Equilibrium association constants (K _a ) and first order dissociation rate constants (k _off) were derived from linearized expressions of the Langmuir isotherm and of the spectral line-shapes, respectively. The results for the four ligands, at 295 K, pH^* 7.2, indicate that: (a) AMCHA exhibits the strongest binding (K _a =159 mM ^-1) and ACA the weakest (K _a =21 mM ^–1) with AcLys and BASA falling in between; (b) ACA dissociates readily (k _off = 5.3 × 10³ s^–1) and AMCHA associates the fastest (k _off = 2.0 × 10⁸ M ^–1 s^–1) while the kinetics for BASA exchange is relatively slow (k _off = 0.8 × 10³ s^–1, k _on = 0.6 × 10⁸ M ^–1s^–1); (c) the ligand-binding kinetics is close to diffussion-controlled.Abbreviations ACA -aminocaproic acid - AcLys N-acetyl-l-lysine - AMCHA t-aminomethyl(cyclohexane)carboxylic acid - BASA p-benzylaminesulfonic acid - K4 kringle 4 - NOE nuclear Overhauser effect - ppm parts-per-million - pH^* glass electrode pH reading uncorrected for deuterium isotope effects - K _a ligand-kringle 4 equilibrium association constant - k _off ligand-kringle 4 dissociation rate constant - k _on ligand-kringle 4 association rate constant     

Enhancement of oxygen mass transfer in stirred bioreactors using oxygen-vectors. 1. Simulated fermentation broths

Oxygen mass transfer represents the most important parameter involved in the design and operation of mixing-sparging equipment for bioreactors. It can be described and analyzed by means of the mass transfer coefficient, k_La. The k_La values are affected by many factors such as geometrical and operational characteristics of the vessels, media composition, type, concentration and microorganism morphology, and biocatalysts properties. The efficiency of oxygen transfer could be enhanced by adding oxygen-vectors in broths, such as hydrocarbons or fluorocarbons, without increasing the energy consumption for mixing or aeration. The experimental results obtained for simulated broths indicated a considerable increase of k_La in the presence of n-dodecane, and the existence of a certain value of n-dodecane concentration that corresponds to a maximum mass transfer rate of oxygen. The magnitude of the positive effect of n-dodecane depends both on the broths characteristics and operational conditions of the bioreactor.Notation d stirrer diameter, mm - d oxygen electrode diameter, mm - D bioreactor diameter, mm - h distance from the inferior stirrer to the bioreactor bottom, mm - H bioreactor height, mm - k_La oxygen mass transfer coefficient, s^-1 - l impeller blade length, mm - I oxygen electrode immersed length, mm - P power consumption for mixing of non-aerated broths, W - P_a power consumption for mixing of aerated broths, W - (P_a/V) specific power input, W/m³ - s baffle width, mm - v_S superficial air velocity, m/s - V volume of medium, m³ - w impeller blade height, mm - volumetric fraction of oxygen-vector - _a apparent viscosity, Pa*s - density, kg/m³     

Permeabilities of theChara cell wall to saccharides,albumin and ficoll

A method was developed to enable the determination of the permeability coefficient of theChara cell wall to various solutes from a measurement of the water flow occurring in the solution-cell wall-water system. For this method, the cell wall tube, closed at one end with the natural septum, was connected to a pipette, which serves as a volumeter, by using a glass capillary and a needle. Permeability coefficientsk _s of the cell wall to glucose (M.W.=180.2), mannitol (M.W.=182.2), sucrose (M.W.=342.3), lactose (M.W.=342.3), raffinose (M.W.=504.5) and melezitose (M.W.=504.4) were 2.27, 2.36, 1.43, 1.38, 1.11 and 1.09×10⁻⁴ cm sec⁻¹, respectively. The reciprocal ofk _s is expressed as a linear function of molecular weight,M, by the equation 1/k _s =16M+1.5×10³ (cm⁻¹ sec) Albumin (M.W.=68,000) passed through the cell wall fairly well. Ficoll (M.W.=400,000±100,000) for practical purposes could not permeate the cell wall.     

A Conserved Active-site Threonine Is Important for Both Sugar and Flavin Oxidations of Pyranose 2-Oxidase

Pyranose 2-oxidase (P2O) catalyzes the oxidation by O² of d-glucose and several aldopyranoses to yield the 2-ketoaldoses and H²O². Based on crystal structures, in one rotamer conformation, the threonine hydroxyl of Thr¹⁶⁹ forms H-bonds to the flavin-N5/O4 locus, whereas, in a different rotamer, it may interact with either sugar or other parts of the P2O·sugar complex. Transient kinetics of wild-type (WT) and Thr¹⁶⁹ → S/N/G/A replacement variants show that d-Glc binds to T169S, T169N, and WT with the same K_d (45–47 mm), and the hydride transfer rate constants (k^red) are similar (15.3–9.7 s⁻¹ at 4 °C). k^red of T169G with d-glucose (0.7 s⁻¹, 4 °C) is significantly less than that of WT but not as severely affected as in T169A (k^red of 0.03 s⁻¹ at 25 °C). Transient kinetics of WT and mutants using d-galactose show that P2O binds d-galactose with a one-step binding process, different from binding of d-glucose. In T169S, T169N, and T169G, the overall turnover with d-Gal is faster than that of WT due to an increase of k^red. In the crystal structure of T169S, Ser¹⁶⁹ Oγ assumes a position identical to that of Oγ1 in Thr¹⁶⁹; in T169G, solvent molecules may be able to rescue H-bonding. Our data suggest that a competent reductive half-reaction requires a side chain at position 169 that is able to form an H-bond within the ES complex. During the oxidative half-reaction, all mutants failed to stabilize a C4a-hydroperoxyflavin intermediate, thus suggesting that the precise position and geometry of the Thr¹⁶⁹ side chain are required for intermediate stabilization.     

Pulse radiolytic study of α-tocopherol radical mechanisms in ethanolic solution

Pulse radiolytic studies of α-tocopherol (αTH) oxidation-reduction processes were carried out with low doses (5 Gy) of high-energy electrons in O₂₋, N₂₋, and air-saturated ethanolic solutions. Depending on the concentration of oxygen in solution, two different radicals, A· and B·, were observed. The first, A·, was obtained under N₂ and results from aTH reaction with solvated electron (k_aTH+csolv⁻ = 3.4 × 10⁸ mol⁻¹ liter s⁻¹) and with H₃C-ĊH-OH, (R·) (k_{aTH + R·} = 5 × 10⁵ mol⁻¹ liter s⁻¹). B·, observed under O₂, is produced by αTH reaction with RO₂ peroxyl radicals (k_aTH + RO₂^. = 9.5 × 10⁴ mol⁻¹ liter s⁻¹).     

Studies on the reactivity of malondialdehyde. I. Effects of acid concentration and solvent composition on deuterium exchange reactions

The rates of deuterium exchange reactions of malondialdehyde (MDA) and deuterated malondialdehyde (MDAd) have been studied as a function of acidity and the content of dimethyl sulfoxide (DMSO) in binary mixtures with D₂O . MDA incorporates deuterium from D₂O solutions in a first-order reaction with a rate constant (k_obs) that depends on the acid concentration. From this dependence, a catalytic constant, k_cat, can be derived (k_cat^MDA = 2.25 × 10⁵M^?s^?1). Similar kinetic behavior was found for MDAd in H₂O solutions, and in this case, k_cat^MDA = 1.56 × 10⁵M^?1s^?1. Results from reactions of MDA and MDAd in identical H₂OD₂O mixtures show that primary and secondary isotope effects are small (k_H/k_D = 1.13) and that solvent isotope effects cause most of the differences found between reactions in D₂O and H₂O. Reactions in binary DMSOd₆D₂O mixtures show a six-fold rate increase as the proportion of DMSOd₆ increases from 50% to 90%. These results also illustrate the relatively high reactivity of MDA at pH values well above its pK_a and the importance of medium composition on its reaction rate.     

Purification and Characterization of a Novel (<Emphasis Type="Italic">R</Emphasis>)-1-Phenylethanol Dehydrogenase from <Emphasis Type="Italic">Lysinibacillus</Emphasis> sp. NUST506

A novel (R)-1-phenylethanol dehydrogenase was successfully purified from Lysinibacillus sp. NUST506 by preparative polyacrylamide gel electrophoresis. The enzyme is a NAD+-dependent oxidoreductase. The molecular weight of the (R)-1-phenylethanol dehydrogenase measured by SDS-PAGE was about 28 kDa. Furthermore, the optimal reaction conditions for the oxidative reaction were 70°C and pH 9.5 and for the reductive reaction were 65°C and pH 6.5. Under the optimal conditions, the K_M and k_cat values with (R)-1-phenylethanol as a substrate were found to be 0.78 mM and 123 s^–1 and with acetophenone they were 0.56 mM and 125 s^–1, respectively. The (R)-1-phenylethanol dehydrogenase became more stable at pH 9.5 in comparison with pH 5.0 and high stability was noticed at 4 and 37°C. Properties of the enzyme place it as a promising candidate for industrial applications.     

Solubilization,characterization and photoaffinity labeling of the mitochondrial dihydropyridine receptor from bovine adrenal medulla

• 1.1. The mitochondrial dihydropyridine receptor was solubilized with Chaps at a detergent/ protein ratio of 2.5, during 45 min at 4°C.
• 2.2. From the rate constants of association (8.10 ± 0.25 × 10⁴ M⁻¹ min⁻¹) and dissociation (0.022 ± 0.001 min⁻¹ a K_d of 275 nM was calculated, while from saturation experiments a K_d of 270 ± 30 nM and a density of receptors of 106 ± 9 pmol/mg protein was obtained.
• 3.4. The solubilized receptors are heat-resistant, sensitive to the trypsin and to the reduction of disulfide bonds.
• 4.5. In native membranes, a polypeptide of 50 kDa was specifically photolabelled with [³H]Azidopine.

     

Kinetics of the formation and hydrolysis reactions of some thiomolybdate(VI) anions in aqueous solution

The kinetics of the formation of the thiomolybdate ions MoOS₃²⁻ and MoS₄²⁻ were determined spectroscopically from the addition of excess sulphide to MoO₂S₂²⁻ in pH buffered media (6–8) at 30 °C. The reverse (hydrolysis) reactions of MoO₂S₂²⁻ and MoOS₃²⁻ were measured under the same conditions. The reaction rates measured are shown below:

Values of the rate-constants (s⁻¹) obtained at pH 7.0 were k₁₀ 2.4 × 10⁻³, k₂₁ 1.5 × 10⁻⁵, k₃₀ 2.1 × 10⁻⁵, k₂₃ 6.0 × 10⁻⁴, and k₃₄ 1.9 × 10⁻⁵; where the results are comparable they are in good agreement with those obtained by earlier workers, although different conditions were used. However, in this work it was found that certain reactions had to be mathematically treated as two consecutively occurring reactions. There is also a difference in interpretation of the mechanism of the hydrolysis reactions of the tri- and tetrathio ions. In general the lability towards further S replacement of O atoms, and the reverse reaction, decreased with increased S substitution. All reaction rates increased with increasing H⁺ ion concentration, mostly this was a linear relationship over the limited pH range examined. The effect of the H⁺ ion is interpreted in terms of protonation of the oxythiomolybdate ions at an O atom leading to increased lability.