Solvent isotope effects on α-glucosidase

The solvent kinetic isotope effects (SKIE) on the yeast α-glucosidase-catalyzed hydrolysis of p-nitrophenyl and methyl-d-glucopyranoside were measured at 25 °C. With p-nitrophenyl-d-glucopyranoside (pNPG), the dependence of k_cat/K_m on pH (pD) revealed an unusually large (for glycohydrolases) solvent isotope effect on the pL-independent second-order rate constant, ^DOD(k_cat/K_m), of 1.9 (±0.3). The two pK_as characterizing the pH profile were increased in D₂O. The shift in pK_a2 of 0.6 units is typical of acids of comparable acidity (pK_a=6.5), but the increase in pK_a1 (=5.7) of 0.1 unit in going from H₂O to D₂O is unusually small. The initial velocities show substrate inhibition (K_is/K_m～200) with a small solvent isotope effect on the inhibition constant [^DODK_is=1.1 (±0.2)]. The solvent equilibrium isotope effects on the K_is for the competitive inhibitors d-glucose and α-methyl d-glucoside are somewhat higher [^DODK_i=1.5 (±0.1)]. Methyl glucoside is much less reactive than pNPG, with k_cat 230 times lower and k_cat/K_m 5×10⁴ times lower. The solvent isotope effect on k_cat for this substrate [=1.11 (±0. 02)] is lower than that for pNPG [=1.67 (±0.07)], consistent with more extensive proton transfer in the transition state for the deglucosylation step than for the glucosylation step.     

Evolution of enzyme catalytic power. Characteristics of optimal catalysis evaluated for the simplest plausible kinetic model

1. Evolutionary changes in the structure of an enzyme that provide an increase in its K_m value are considered. Provided that K_m increases as a result of increases in the forward rate constants of the catalysis relative to the reverse rate constants, the enzyme catalyses the conversion of a fixed concentration of its substrate more rapidly when its structure provides that K_m>[S] than when K_m<[S]. 2. Catalytic efficiency of enzymes is discussed in terms of the simplest plausible model, the Haldane [(1930) Enzymes, Longmans, London] reversible three-step model: [Formula: see text] The rate equation for the forward reaction of this model (formation of P) may be written in the simple form: [Formula: see text] K_eq. is the equilibrium constant (=[P]_eq./[S]_eq.), and k_cat.=V/[E]_T, where [E]_T is the total enzyme concentration. 3. To assess the effectiveness of an enzyme, it is necessary only to determine the extent to which the constraints of a particular kinetic mechanism permit v₂ (v when K_m»[S]) to approach v_d (the diffusion-limited rate). 4. The value of the optimal rate of catalysis (v_opt., the maximal value of v₂) is dictated by the equilibrium constant for the reaction, K_eq.; v₂=v_d/a, where [Formula: see text] when k₊₁ is assumed equal to k₋₃, and v_opt.=v_d/a_min.. When K_eq.≥1, it is necessary that k₊₂»k₋₁ for a to take its minimum value, a_min.; when K_eq.«1, it is necessary only that k₊₂»K_eq.·k₋₁, i.e. a can equal a_min. even if k₊₂<k₋₁. When K_eq.»1, v_opt.=v_d; when K_eq.=1, v_opt.=v_d/2, and when K_eq.«1, v_opt.=K_eq.·v_d. 5. The analysis, together with predicted effects of evolutionary pressure, suggests that in practice the rates of the fastest enzyme-catalysed freely reversible reactions might be expected to be lower than the value of k₊₁[E]_T[S] by about an order of magnitude, particularly if K_eq.<1. 6. The existing literature suggests that, in general, appropriate values of K_m have evolved for the provision of high rates of catalysis but that many values of k_cat. are not large enough to provide optimal rates of catalysis unless the value of k₊₁ in vivo is lower than its value in free solution.     

The cytochromes of mitochondria from Tetrahymena pyriformis strain ST

1. Mitochondria of the obligately aerobic ciliate protozoon, Tetrahymena pyriformis strain ST, are unusual in that they possess a cytochrome oxidase system that does not react with reduced mammalian cytochrome c; the presence of cytochromes a₆₀₃+a₃ is masked in the α-band region of spectra by the broad absorption band of cytochrome a₆₂₀. 2. Other haemoproteins present include cytochromes b₅₆₀, b₅₅₆, c₅₅₃ and c₅₄₉. 3. The reaction of reduced cytochrome a₃ with CO is reversed by flash photolysis, and in the presence of O₂ the subsequent oxidation of this cytochrome is followed by that of cytochrome a₆₀₃. 4. Cytochromes a₆₂₀ and b₅₆₀ also react with CO and with KCN; the latter cytochrome corresponds with that designated cytochrome o by other workers. 5. The contribution of cytochrome a₆₀₃ to difference spectra is revealed by making use of the fact that it does not react with KCN. 6. Cytochrome a₆₂₀ is unstable, and its α-absorption band is lost from spectra of mitochondria which have been aged or treated with ultrasound, detergents or organic solvents. 7. Possible pathways of electron transport via the several different terminal oxidases in Tetrahymena mitochondria are proposed.     

Calculation of the energy confinement time and power threshold for L-H transitions in a tokamak with allowance for the parameters of the transport barrier

The ASTRA-ETL code is used to simulate L-H transition scenarios and calculate the energy confinement time and the threshold power of the L-H transition as functions of the averaged electron density 〈n〉, the averaged magnetic field B, the neutral density n _n , and the neutral temperature T _n , as well as the values of T _Se , T _Si , and n _S at the separatrix. It is shown that the linear dependence of the threshold power of the L-H transition on the averaged electron density, Q _L-H∝〈n〉, is associated with an increase in the viscosity of a poloidally rotating plasma due to charge exchange and is governed exclusively by an increase in the neutral density n _n . When the averaged electron density 〈n〉 is low, the threshold power rises because T _Si and T _Se increase. The accuracy of predictions for the power threshold of the L-H transition can be improved if the scaling of Q _L-H versus 〈n〉 and B is derived by processing experimental data from discharges with close parameter values at the separatrix. The hysteresis effect during an L-H-L transition triggered by varying the input power is modeled. The global energy confinement time τ_E is shown to increase linearly with 〈n〉 in the range 〈n〉<3.6×10¹⁹ m^?3 and to saturate at higher electron densities; this behavior is found to be characteristic of the Ohmic, L-, and H-modes. The saturation is associated with the fact that losses via the ion channel (when the transport coefficients are density-independent) dominate over losses via the electron channel. The dependence of τ _E on the input power is determined from the calculated database and is found to be τ _E =0.12Q _L-H ^?0.46 at a fixed averaged electron density 〈n〉. In the simulations of the L-H transition, the energy confinement time τ _E increases by a factor of 2 only if the thermal diffusivity inside the transport barrier is lower than that in the central plasma by a factor of more than 6.     

New Mutational Variants of Neurospora Nadp-Specific Glutamate Dehydrogenase

The am locus of Neurospora codes for NADP-dependent glutamate dehydrogenase (GDH). Four new am mutants that produced mutationally altered GDH have been characterized. Mutant am₁₁₉ is a CRM-negative, complementing mutant that maps between am₂ and am₁. The other three mutants are CRM formers that produce varieties of GDH that can be activated by glutamate or succinate. The GDH of am₁₃₀ and am₁₃₁ is similar in terms of activation properties to that of am₃. The GDH of am₁₂₂ requires very high concentrations of dicarboxylate for activity. The mutation in am₁₃₀ maps between am₁₄ and am₂ and resulted in a replacement at residue 75 of the GDH (pro → ser). The mutation in am₁₂₂ maps near am₁₁ and apparently resulted in the replacement of the tryptophan residue at position 389 with an unknown amino acid. The mutation in am₁₃₁ maps between am₂ and am₁.     

Isolation,characterization and transposition of an (IS2)2 intermediate

We have isolated and characterized a dimer derivative of the extensively studiedEscherichia coli insertion sequence IS2. The dimer structure — called (IS2)₂ — consists of two IS2 elements arranged as a direct repeat, separated by 1 bp. The junction between the (IS2)₂ dimer and target sequences is located at various positions in independent isolates; however, one position was preferred. The transposition of (IS2)₂ into a target plasmid resulted in cointegrate-type structures. The transposition frequency of the (IS2)₂ dimer itself was significantly higher than that of the isogenic monomer IS2 insertion. The poor stability and high activity of (IS2)₂ indicates that this is an active transposition intermediate. The mode of transposition of (IS2)₂ is analogous to the joined dimer model described in the case of (IS21)₂ and (IS30)₂.     

Reversible linkage isomerization of pentaamminecobalt(III) complexes of urea and its N-methyl derivatives

The (NH₃)₅CoOC(NH₂)₂³⁺ ion is consumed in water according to the rate law k(obs.) = k₁ + k₂[OH⁻], where k₁ = 4.0 × 10⁻⁵ s⁻¹ and k₂ = 14.2 M⁻¹ s⁻¹ (0–0.1 M [OH⁻];μ = 1.1 M, NaClO₄, 25 °C). A hitherto unrecognized intramolecular O- to N- linkage isomerization reaction has been detected. In strongly acid solution only aquation to (NH₃)₅CoOH₂³⁺ is observed, but in 0.1–1.0 M [OH⁻], 7% of the directly formed products is the urea-N complex (NH₃)₅CoNHCONH₂²⁺ which has been isolated. In the neutral pH region a much greater proportion (25%) of the products is the urea-N species. These results are interpreted in terms of an urea-O to urea-N linkage isomerization reaction competing with hydrolysis for both spontaneous (k₁) and base-catalyzed (k₂) pathways; the rearrangement is not observed in strongly acidic solution (pH ⩽ 1) because the protonated N-bonded isomer (pK′_a ≈ 3) is unstable with respect to the O-bonded form. The appearance of the isomerization pathway as the pH is raised in the 0–6 region is commensurate with a rate increase which cannot be attributed to a contribution from the base catalysis term k₂[OH⁻]. It is argued that this observation establishes, for the spontaneous pathway, that hydrolysis and linkage isomerization are separate reaction pathways — there is no common intermediate. The product distribution and rate data lead to the complete rate law, k(obs.) = k₁ + k₂[OH⁻] = (k_s + k_ON) + (k_OH + k′_ON) [OH⁻] for the reactions of the O-bonded isomers, where k_s, k_OH are the specific rates for hydrolysis, and k_ON, k′_ON are the specific rates for O- to N-linkage isomerization, by spontaneous and base-catalyzed pathways respectively; k_ON = 1.3 × 10⁻⁵ s⁻¹ and k′_ON = 1.1 M⁻¹ s⁻¹ (μ = 1.0 M, NaClO₄, 25 °C). The O- to N- linkage isomerization has been observed also for complexes of N-methylurea, N,N-dimethylurea and N-phenylurea, but not for the N,N′-dimethylurea species. There is an approximately statistical relationship among the data for −NH₂ capture (versus H₂O), while −NHR and −NR₂ do not compete with water as nucleophiles for Co(III) in either the spontaneous or base-catalyzed hydrolysis processes. For each urea-O complex, O- to N-isomerization is a more significant parallel reaction in the spontaneous as opposed to the base-catalyzed pathway. This is interpreted as being indicative of more associative character in the spontaneous route to products, a conclusion supported by other evidence. Some activation parameter data have been recorded and the effect of the N-substitution on the rates of solvolysis (H₂O, Me₂SO) is discussed. The urea-N complexes have been isolated as their deprotonated forms, [(NH₃)₅CoNHCONRR′](ClO₄)₂·xH₂O (R,R′ = H, CH₃). They are kinetically inert in neutral to basic solution but in acid they protonate (H₂O, pK′_a 2–3; μ = 1.0 M, 25 °C) and then isomerize rapidly back to their O-bonded forms. Some solvolysis accompanies this N- to O-rearrangement in H₂O and Me₂SO. Specific rates and activation parameters are reported. The kinetic data follow a rate law of the form k_NO(obs.) = (k + k_NO)[H⁺]/(K′_a + [H⁺]) and the active species in the reaction is the protonated form; k, k_NO are the specific rates for hydrolysis and isomerization, respectively. Proton NMR data establish that the site of protonation (in Me₂SO) is the cobalt-bound nitrogen atom. For the unsubstituted urea species (NH₃)₅CoNH₂CONH₂³⁺, diastereotopic exo-NH₂ protons arising from restricted rotation about the CN bond are observed. The relevance to the mechanism of the linkage isomerization process is considered. ¹³C and ¹H NMR and electronic absorption spectral data are presented, and distinctions between linkage isomers and the solution structures (electronic and conformational) are discussed. The urea-N/urea-O complex equilibrium is governed by the relation K′_NO(obs.) = K′_NO[H⁺]/[H⁺](K′_a), where K′_NO is the equilibrium constant = [(NH₃₅Co(urea-O)³⁺]/[(NH₃)₅Co(urea-N)³⁺]. Values for K′_NO(=k_NO/k_ON = 260 and pK′_a ≈ 3 for the NH₂CONH₂ system are consistent with the stability of the N-isomer in feebly acidic to basic solution (e.g. pH 6, K′_NO(obs.) = 2.6 × 10⁻²) and instability in acid solution (e.g. pH 1, K′_NO(obs.) = 240). The equilibrium data for this and other urea complexes of (NH₃)₅Co(III) are contrasted with the result for the analogous Rh(III)NH₂CONH₂ system K′_NO ≈ 1).     

Effect of atoms evaporated from the anode on the structure of the vacuum arc space charge sheath

The structure of the anode space charge sheath of a vacuum arc is studied with allowance for the dependence of the negative anode fall on the ratio of the directed electron velocity v ₀ to the electron thermal velocity v _T for different values of the flux density of atoms evaporated from the anode. Poisson’s equation for the sheath potential is solved taking into account the electron space charge, fast cathode ions, and slow ions produced due to the ionization of atoms evaporated from the anode. The kinetic equation for atoms and slow anode ions is solved with allowance for ionization in the collision integral. Analytic solutions for the velocity distribution functions of atoms and slow ions and the density of slow ions are obtained. It is shown that the flux of slow ions substantially affects the spatial distribution of the electric field E(z) in the sheath. As the flux density increases, the nonmonotonic dependence E(z) transforms into a monotonic one and the sheath narrows. For a given flux of evaporated atoms Πa, the increase in the ratio of the directed electron velocity to the electron thermal velocity leads again to a nonmonotonic dependence E(z). As z increases, the electric field first increases, passes through the maximum, decreases, passes through the minimum E _min, and then again increases toward the anode. There is a limiting value of the ratio (v ₀/v _T )* at which E _min(z) vanishes. At v ₀/v _T > (v ₀/V _T )*, the condition for the existence of a steady-state sheath is violated and the profiles of the field and potential in the sheath become oscillating. The dependence of (v ₀/v _T )* on the flux density of evaporated atoms Π _a is obtained. It is shown that the domain of existence of steady-state solutions in the sheath broadens with increasing Π _a .     

Ozone alteration of transport of cations and the Na+/K+-ATPase in human erythrocytes.

We have purified glutaminase 65-fold from cow brain; the final specific activity is 24 μmol/min/mg. The enzyme is stable between pH 7.5 and 9.0 and has maximal activity at pH 8.8. It requires P_i for activity. The dependence of activity on P_i concentration is sigmoidal; 50 mmP_i gives half-maximal velocity at pH 8.8. At 0.2 mP_i, pH 8.8, the dependence of activity on glutamine concentration is hyperbolic; the observed K_Gln was 30 mm. Increasing P_i concentrations increase the apparent V_m and decrease the apparent K_Gln. NH₄⁺ does not inhibit at concentrations up to 0.1 m. Glutamic acid inhibits competitively with respect to glutamine; at 0.2 mP_i pH 8.8, K_Gln was 30 mm and K_Glu was 19 mm. The results are consistent with a model in which NH₄⁺ is released irreversibly from the enzyme-substrate complex and is the first product released. The activity of glutaminase appears to be independent of the nature of the buffer with which it is equilibrated before being assayed.     

ESEEM and Mössbauer studies of the ferriheme model compound bis(3-aminopyrazole)tetraphenylporphyrinatoiron(III) chloride, [TPPFe(NH2PzH)2]Cl

A model heme complex, bis(3-aminopyrazole)tetraphenylporphinatoiron(III) chloride, [TPPFe (NH₂PzH)₂]Cl, for which the EPR g-values lead to a rhombicity V/Δ=1.2 if g _zz is the largest g-value, have been investigated by electron spin echo envelope modulation (ESEEM) and Mössbauer spectroscopies. The ESEEM studies focus on the proton sum frequency peaks at near twice the proton Larmor frequency. Analysis of the distant proton peak (mainly due to the pyrrole-H) at exactly twice the proton Larmor frequency shows conclusively that g _zz is aligned along the normal to the porphyrin plane, and thus the electron configuration is (d _xy )²(d _xz ,d _yz )³, with g _zz >g _yy >g _xx . This system is thus another violation to Taylor's "proper axis system" rule. The near proton (the α-H and N-H of the axial ligands) peaks provide distance information for those protons from the metal. Magnetic Mössbauer studies of the same complex confirm the (d _xy )²(d _xz ,d _yz )³ ground state and indicate that, as is the case for cytochrome P450_cam, A _xx is the largest magnitude A-value, and is negative in sign. Other low-spin iron(III) porphyrinates also have A _xx of negative sign, but usually the magnitude is only about half that of A _zz , which is always positive in sign.     

The structural dependence of amino acid hydrophobicity parameters

Amino acid hydrophobicity parameters, G_hp log P (partition coefficient) values, free energies of solution, G_sol and hydration numbers, are well correlated by equations derived from the relationship O_X = Aα_X + Lσ_IX + Sν_X + I_iX + H₁n_HX + H₂n_nX + b₀ where O is the quantity correlated; X denotes the amino acid side chain; α is a polarizability parameter; σ_I, a localized electrical effect parameter; ν, a steric parameter; i, an indicator variable which accounts for an ionic X ; n_H and n_n the number of OH or NH bonds and of full nonbonding orbitals in X, respectively, and b₀ is the intercept. The equation is based on the assumption that Δ_hp log P and ΔG_sol are all functions of the difference in intermolecular forces between the amino acid and some medium, and the amino acid and water. The parameters were chosen to model the intermolecular forces of interest.Generally the most important factor is α_x. This is followed by ν, i, and n_H. Least important is σ_I. ΔG_sol depends on α, n_H and n_n. Hydration numbers depend on i, n_H and n_n. The hydrophobicity of amino acid side chains is the result of a preference for a nonpolar medium as a increases and for a polar medium as i, n_H and σ_I increase. It is quantitatively accounted for by the model, and no special “hydrophobic bond” need be involved. The results show that log P values for amino acids are composite quantities whose composition is variable.     

Simulation of internal transport barriers by means of the canonical profile transport model

Models with critical gradients are widely used to describe energy balance in L-mode discharges. The so-called first critical gradient can be found from the canonical temperature profile. Here, it is suggested that discharge regimes with transport barriers can be described based on the idea of the second critical gradient. If, in a certain plasma region, the pressure gradient exceeds the second critical gradient, then the plasma bifurcates into a new state and a transport barrier forms in this region. This idea was implemented in a modified canonical profile transport model that makes it possible to describe the energy and particle balance in tokamak plasmas with arbitrary cross sections and aspect ratios. The magnitude of the second critical gradient was chosen by comparing the results calculated for several tokamak discharges with the experimental data. It is found that the second critical gradient is related to the magnetic shear s. The criterion of the transport barrier formation has the form (a ²/r)d/drln(p/p _c ) > z ₀ (r), where r is the radial coordinate, a is the plasma minor radius, p is the plasma pressure, p _c is the canonical pressure profile, and the dimensionless function z _O(r) = C _O + C ₁ s (with C _0i ～1, C _0e ～3, and C _1i,e ～2) describes the difference between the first and second critical gradients. Simulations show that this criterion is close to that obtained experimentally in JET. The model constructed here is used to simulate internal transport barriers in the JET, TFTR, DIII-D, and MAST tokamaks. The possible dependence of the second critical gradient on the plasma parameters is discussed.     

The roles of Rhodobacter sphaeroides copper chaperones PCuAC and Sco (PrrC) in the assembly of the copper centers of the aa3-type and the cbb3-type cytochrome c oxidases

The α proteobacter Rhodobacter sphaeroides accumulates two cytochrome c oxidases (CcO) in its cytoplasmic membrane during aerobic growth: a mitochondrial-like aa₃-type CcO containing a di-copper Cu_A center and mono-copper Cu_B, plus a cbb₃-type CcO that contains Cu_B but lacks Cu_A. Three copper chaperones are located in the periplasm of R. sphaeroides, PCu_AC, PrrC (Sco) and Cox11. Cox11 is required to assemble Cu_B of the aa₃-type but not the cbb₃-type CcO. PrrC is homologous to mitochondrial Sco1; Sco proteins are implicated in Cu_A assembly in mitochondria and bacteria, and with Cu_B assembly of the cbb₃-type CcO. PCu_AC is present in many bacteria, but not mitochondria. PCu_AC of Thermus thermophilus metallates a Cu_A center in vitro, but its in vivo function has not been explored. Here, the extent of copper center assembly in the aa₃- and cbb₃-type CcOs of R. sphaeroides has been examined in strains lacking PCu_AC, PrrC, or both. The absence of either chaperone strongly lowers the accumulation of both CcOs in the cells grown in low concentrations of Cu^2 +. The absence of PrrC has a greater effect than the absence of PCu_AC and PCu_AC appears to function upstream of PrrC. Analysis of purified aa₃-type CcO shows that PrrC has a greater effect on the assembly of its Cu_A than does PCu_AC, and both chaperones have a lesser but significant effect on the assembly of its Cu_B even though Cox11 is present. Scenarios for the cellular roles of PCu_AC and PrrC are considered. The results are most consistent with a role for PrrC in the capture and delivery of copper to Cu_A of the aa₃-type CcO and to Cu_B of the cbb₃-type CcO, while the predominant role of PCu_AC may be to capture and deliver copper to PrrC and Cox11. This article is part of a Special Issue entitled: Biogenesis/Assembly of Respiratory Enzyme Complexes.     

Optical measurements of intracellular oxygen concentration of rat heart in vitro

The optical characteristics of hemoglobin-free perfused rat heart have been examined in detail. Ethyl hydrogen peroxide is found to convert myoglobin into “ferryl compound” in the perfused heart, as is also seen in vitro. After pretreatment with ethyl hydrogen peroxide, a typical mitochondrial absorption spectrum, similar to that of isolated rat heart mitochondria, is obtained in perfused heart. The overall absorption spectrum of the heart obtained by the aerobic to anaerobic transition is a superposition of the mitochondrial spectrum on that of myoglobin. By comparing these spectra, it is found that measurement of cytochrome a + a₃ at 605–620 nm is possible in spite of the absorbance change due to the oxygenation-deoxygenation of myoglobin, whereas the wavelength pairs for cytochrome c at 550-540 nm, cytochrome b at 562–575 nm and cytochrome a + a₃ at 445–450 nm can not be used in the heart because of interference from the absorption change of myoglobin. The partial pressure of O₂ (P₅₀) which is required for half maximal deoxygenation (or oxygenation) of myoglobin in perfused heart is found to be 2.4 mm Hg at room temperature and the Hill constant, n, is 1.1; these values are similar to those of myoglobin purified from rat heart. The steady-state O₂ titration has been performed by using absorbancy changes of myoglobin and cytochrome a + a₃ as intracellular O₂ indicators. In the perfused heart, the percentage change of oxygenation-deoxygenation of myoglobin parallels the oxidation-reduction of cytochrome a + a₃, while the mixture of purified myoglobin and isolated mitochondria shows a deviation, reflecting the difference of O₂ affinities between myoglobin and cytochrome a + a₃. The results indicate that there may be an O₂ gradient between cytosolic and mitochondrial compartments in the hemoglobin-free perfused heart. The absorption changes of myoglobin and of cytochrome a + a₃ can be measured in a single contraction-relaxation cycle. A triple beam method was introduced to eliminate the effect of light scattering changes in these measurements. The results demonstrated that myoglobin is more oxygenated during the systolic and diastolic periods and deoxygenated in the resting period, whereas cytochrome a + a₃ is more reduced in systole and diastole and oxidized in the resting state. Changing the perfusion conditions greatly alters the time course of the events which occur during the contraction-relaxation cycle of the perfused heart.     

Generalized Bohm’s criterion and negative anode voltage fall in electric discharges

The value of the voltage fall across the anode sheath is found as a function of the current density. Analytic solutions are obtained in a wide range of the ratio of the directed velocity of plasma electrons v ₀ to their thermal velocity v _T . It is shown that the voltage fall in a one-dimensional collisionless anode sheath is always negative. At the small values of v ₀/v _T , the obtained expression asymptotically transforms into the Langmuir formula. Generalized Bohm’s criterion for an electric discharge with allowance for the space charge density ρ(0), electric field E(0), ion velocity v _i (0), and ratio v ₀/v _T at the plasma-sheath interface is formulated. It is shown that the minimum value of the ion velocity v _i ^* (0) corresponds to the vanishing of the electric field at one point inside the sheath. The dependence of v _i ^* (0) on ρ(0), E(0), and v ₀/v _T determines the boundary of the existence domain of stationary solutions in the sheath. Using this criterion, the maximum possible degree of contraction of the electron current at the anode is determined for a short high-current vacuum arc discharge.     

Isocitrate lyase from Neurospora crassa: pH dependence of catalysis and interaction with substrates and inhibitors.

The maximal velocity, V, for isocitrate cleavage by isocitrate lyase from Neurospora crassa is dependent on two dissociable groups with pK_a values of 6.1 and 8.6. A dissociable group with a pK_a of 8.5 on the enzyme-substrate complex affects the pK_m for isocitrate. The pK_i for homoisocitrate is affected in a like manner. The pH dependence of the pK_i's for succinate, a product of isocitrate cleavage, and the succinate analog maleate is similar to the pH dependence of the pK_m of isocitrate below pH 7.3, but is markedly different above this pH. Both the K_m for isocitrate and the K_i for succinate were dependent upon Mg²⁺ concentration. The pK_i for oxalate, an analog of glyoxylate which is also a product of isocitrate cleavage, is dependent on a group with a pK_a of 6.8 on the enzyme-inhibitor complex. The pH dependence of the pK_i for phosphoenolpyruvate, which binds to the succinate site, suggests that it is dependent on two dissociable groups, one on phosphoenolpyruvate and one, by analogy to the pK_m for isocitrate, on the enzyme-glyoxylate-inhibitor complex.