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.     

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.     

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.     

Mg and Ca binding to heparin in the presence of added univalent salt

Activities of Mg and Ca in solutions of heparin with added sodium or potassium chloride have been determined by means of a previously described dye spectrophotometric method. The polyion concentration, C_p (expressed as mol univalent anionic charges/I) is 0.001 in all cases, solution total ionic strengths are 0.005, 0.0075, 0.01, 0.02 and 0.04 mol/l. Divalent metal ion concentrations are varied between 0 and 1.0 C_p. The results for the metal ion activities are expressed in the form of binding isotherms, θ₂ vs. C₂/C_p (θ₂=C_2b/C_p; C_2b=bound divalent metal ion concentration) and Scatchard plots, K₂ vs. θ₂/(C₂-C_2b, at different ionic strengths. Results are correlated with a theoretical treatment of the mixed counterion system, called the ‘two variable theory’, recently developed by Manning. The ionic strength dependence of θ₂ and K₂ is found to agree with the prediction of the two variable theory. Mg interaction with heparin appears to be independent of the nature of the charged groups on the polyion and is in very good agreement with the two variable theory. Ca binding was found to be considerably higher than Mg binding and is in excess of theoretical predictions, suggesting a localized or specific interaction of Ca with heparin.     

Preparation,crystal structure and raman spectrum of tetrachloro phosphonium tetrachloro-iodate [PCl4] [ICl4]

The preparation of [PtCl₄] [ICl₄] is described. Its structure was determined by both X-ray analysis and Raman spectroscopy. This compound is orthorhombic, space group Imma with a = 12.315(1), b = 16.302(2), c = 12.215(1) Å V = 2452(1) ų and Z = 8. The structure was refined by least squares to R = 3.2% using 753 observed refiections. The PtCl₄⁺ cation is tetrahedral with T_d symmetry, whereas the two crystallographically independent ICl₄⁻ anions are square planar - one with an almost ideal D_4h symmetry and the other slightly distorted to a C_2v symmetry. The I-Cl distances are in the range of 2.461(4)-2.508(3) Å.     

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.     

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.     

Responses to respiratory stress in relation to blood pigment affinity in Goniopsis cruentata (Latreille) and (to a lesser extent) in Cardisoma guanhumi Latreille

The crabs Goniopsis cruentata (Latreille) and Cardisoma guanhumi Latreille are shown to be metabolic regulators at environmental temperatures.Haemocyanin P₅₀ in Goniopsis shows strong correlation with maximum standard respiratory rates (R_max) and critical oxygen tension (P_c) and also with lethal oxygen tensions (P_L), at different temperatures. The hypothesis that the P_c represents that P_o2 at which the blood pigment fails to become saturated in the gills, though qualitatively in accord with these correlations, is irreconcilable with metabolic regulation in these animals. An alternative hypothesis is tentatively suggested from which, like the previous hypothesis, the experimental relationship P_c ∝ (R_max) (P₅₀) can be derived. The new hypothesis leads to the conclusion that the P_c reflects the initiation of anaerobiosis. Altogether, the evidence is thought to suggest that the graphical correlations are causal and that pigment affinity plays a determining role in the responses to respiratory stresses, both thermal and anoxic.     

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.     

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 .     

Three coordination modes of the pentadentate ligand 2,6-diacetylpyridinedisemicarbazone

The pentadentate ligand 2,6-diacetylpyridinedisemicarbazone, DAPSC, reacts with Cr(NO₃)₃·9H₂O and forms two kinds of complexes. At pH=3, the ligand is singly-deprotonated and crystals of [Cr- (DAPSCH)(H₂O)₂](NO₃)₂·H₂O (Ia) are obtained. Evaporation of a solution at pH=0, yields crystals of [Cr(DAPSC)(H₂O)₂](NO₃)₃·2H₂O (II) in which the ligand is fully protonated. The reaction of DAPSC with UO₂(O₂CCH₃)₂ in methanol, followed by crystallization of the product from DMSO yields crystals of [UO₂(DAPSC2H)(H₂O)]·2DMSO (III) in which the ligand is fully deprotonated. Compound Ia is monoclinic, space group P2₁/n with a=11.746(1), b=14.752(2), c=11.866(1) Å,β=105.53(2)°, V= 1981(1) ų and Z=4. Compound II is monoclinic, space group, P2₁/n with a=38.000(3), b= 14.939(2), c=8.233(1) Å, β=96.12(2)°, V= 4647(1) Å and Z=8. Compound III is monoclinic, space group P2₁/n with a=18.048(2), b=15.207(2), c=8.842(1) Å,β=97.72(2)°, V=2405(1) ų and Z=4. The structures were refined using 2084, 4169 and 2516 reflections to R values of 4.4%, 7.8% and 4.8% respectively.     

A method to calculate binding equilibrium concentrations in the allosteric ternary complex model that supports ligand depletion

The allosteric ternary complex model is frequently used in pharmacology to represent the interaction of a receptor R with two ligands A and B. Certain well-known formulas are routinely used to calculate the fractions of the receptor bound at equilibrium with A only, B only, and both A and B. However, it is often omitted that these classical formulas presume that there is no ligand depletion, i.e. that the equilibrium concentrations [A] and [B] of the ligands are well approximated by their total concentrations [A]_T and [B]_T. We present a calculation method which is applicable without this or any restrictions. The equilibrium concentration [R] of the receptor is implicitly characterized by an equation which is solved with a very simple convergent numerical algorithm. The concentrations [A] and [B] are given by explicit formulas in terms of [R]. The required parameters are the equilibrium dissociation constants K_A and K_B, the cooperativity factor α, and the total concentrations [R]_T, [A]_T and [B]_T.     

Interaction of External K, Na, and Cardioactive Steroids with the Na-K Pump of the Human Red Blood Cell

The interaction of extracellular Na (Na_o), K (K_o), and strophanthidin with the Na-K pump of the human red blood cell has been investigated. Inhibition by submaximal concentrations of strophanthidin rapidly reaches a level which does not increase further over a relatively long period of time. Under these circumstances, it is possible to apply a steady-state kinetic analysis to the interaction of Na_o, K_o, and strophanthidin with the pump. In Na-free solutions, strophanthidin increases the apparent K_1/2 of the pump for K_o, but does not change the form of the relation between the reciprocal of the active K influx (ⁱM_K^P–1) and the reciprocal of [K_o] ([K_o]^–1); the relation both in the presence and absence of strophanthidin is adequately described by a straight line. In solutions containing Na, strophanthidin changes the form of the curve describing the relation between ⁱM_K^P–1 vs. [K_o]^–1; the curve becomes more parabolic in solutions containing strophanthidin. The rate of ouabain binding to K-free cells has also been measured; in the absence of K, the rate of binding is unaffected by Na_o. The data are considered in terms of a simple kinetic model. The findings can be explained if it is supposed that at low external K the form of the pump combined with one Na_o is more likely to combine with strophanthidin than is the uncombined form of the pump. The uncombined form of the pump is more likely to combine with K even at very low K_o than with strophanthidin.     

Thermodynamic and EPR characterization of mitochondrial succinate-cytochrome c reductase-phospholipid complexes

Succinate-cytochrome c reductase can be easily solubilized in a phospholipid mixture (1:1, lysolecithin:lecithin) in the absence of detergents. The resulting solution contains two b cytochromes with half-reduction potentials of 95 ± 10 mV (b₅₆₁), and 0 ± 10 mV (b₅₆₆) and cytochrome c₁ (E_{m 7.2} = +280±5 mV). The oxidation-reduction midpoint potentials obtained by optical potentiometric titrations are identical to those determined by the EPR titrations and are 40–60 mV higher than the corresponding midpoint potentials of these cytochromes in intact mitochondria. In contrast to detergent-suspended preparations, no CO-sensitive cytochrome b can be detected in the phospholipid-solubilized preparation or intact mitochondria. The half-reduction potential of cytochrome b₅₆₆ is pH-dependent above pH 7.0 (?60 mV/pH unit) while that of b₅₆₁ is essentially pH-independent from pH 6.7–8.5, in contrast to its pH dependence in intact mitochondria. EPR characterizations show the presence of three oxidized low-spin heme-iron signals with g values of 3.78, 3.41 and 3.37. The identification of these signals with cytochromes b₅₆₆ (b_T), b₅₆₁ (b_K) and c₁ respectively is made on the basis of redox midpoint potentials. No significant amounts of oxidized high-spin heme-iron are detectable. In addition, the preparation contains four distinct types of iron-sulfur centers: S₁ and S₂ (E_{m 7.4} = ?260 mV and 0 mV), and two iron-sulfur proteins which are associated with the cytochrome b-c₁ complex: Rieske's iron-sulfur protein (E_{m 7.4} = +280 mV) and Ohnishi's Center 5 (E_{m 7.4} = +35 mV).     

Mixed and uniform co-operativity of ligand binding to multisite proteins: the co-operativity types allowed by the adair equation and conditions for them

It is proved for the first time that the macroscopic co-operativity of binding to a protein with q binding sites may change signs over a single binding curve any number of times from 0 to (q-2), but no more than (q-2). n changes of sign of macroscopic co-operativity requires as a necessary condition at least n changes of sign of microscopic co-operativity, but this necessary condition is not a sufficient one. The necessary and sufficient condition that decides whether there are two changes of sign in a four-site protein is obtained. There are no changes when K₁K₃(K₂-K₄)+K₁K₂(K₃-K₂)+K₂K₃(K₄-K₃) is positive, and two changes when it is negative, presuming the above mentioned necessary conditions to be satisfied. The K's of this formula are the “intrinsic” per-site Adair constants. As a result, the conditions for all six co-operativity types possible with a four-site protein are now known.     

Current Separations in Myxicola Giant Axons

The effect of reducing the external sodium concentration, [Na]_o, on resting potential, action potential, membrane current, and transient current reversal potential in Myxicola giant axons was studied. Tris chloride was used as a substitute for NaCl. Preliminary experiments were carried out to insure that the effect of Tris substitution could be attributed entirely to the reduction in [Na]_o. Both choline and tetramethylammonium chloride were found to have additional effects on the membrane. The transient current is carried largely by Na, while the delayed current seems to be independent of [Na]_o. Transient current reversal potential behaves much like a pure Nernst equilibrium potential for sodium. Small deviations from this behavior are consistent with the possibility of some small nonsodium component in the transient current. An exact P_Na/P_K for the transient current channels could not be computed from these data, but is certainly well greater than unity and possibly quite large. The peak of the action potential varied with [Na]_o as expected for a sodium action potential with some substantial potassium permeability at the time of peak. Resting membrane potential is independent of [Na]_o. This finding is inconsistent with the view that the resting membrane potential is determined only by the distribution of K and Na, and P_Na/P_K. It is suggested that P_Na/P_K's obtained from resting membrane potential-potassium concentration data do not always have the physical meaning generally attributed to them.     

Electrocatalytic four-electron reduction of oxygen at the cytochrome c3-adsorbed electrode

The electrocatalytic activity of cytochrome c₃ for the reduction of molecular oxygen was characterized from the studies of the adsorption of cytochrome c₃ and the co-adsorption of cytochrome c₃ with cytochrome c on the mercury electrode by the a.c. polarographic technique. The adsorption of cytochrome c₃ on the mercury electrode is irreversible and is diffusion-controlled. The maximum amount of cytochrome c₃ adsorbed was 0.92 · 10^?11 mol · cm^?2 at ?0.90 V. The amount of cytochrome c₃ in the mixed adsorbed layer with cytochrome c was determined from the differential capacitance measurement. It was shown that the fractional coverage of cytochrome c₃ can be estimated from its bulk concentration and the diffusion coefficient (1.05 · 10^?6 cm² · s^?1). Cytochrome c₃ catalyzes the electrochemical reduction of molecular oxygen from the two-electron pathways via hydrogen peroxide to the four-electron pathway at the mercury electrode in neutral phosphate buffer solution. The catalytic activity varies with the bulk concentration of cytochrome c₃. The highest catalytic activity for the oxygen reduction (no hydrogen peroxide formation) is attained when one-half of the mercury electrode surface is covered by cytochrome c₃. The addition of cytochrome c or bovine serum albumin to the cytochrome c₃ solution inhibits the catalytic activity of cytochrome c₃. The reversible polarographic behavior of cytochrome c₃ through the mixed adsorbed layer of cytochrome c₃ and cytochrome c was also investigated.     

Iron complexes of chiral phenol-oxazoline ligands: Structural studies and oxidation catalysis

Iron complexes of two ligands, HphoxCOOH and HphoxiPr, have been synthesized and characterized by crystal structure analyses. The complexes (HNEt₃)₂[Fe(phoxCOO)₂](ClO₄) and [Fe(phoxiPr)₃] are reported. Reactions of the ligands rac-HphoxCOOH and rac-HphoxiPr with iron(II) or iron(III) perchlorate result in the formation of iron(III) complexes with pseudo-octahedral geometry around the metal center. The iron complex obtained from rac-HphoxCOOH crystallized in the centrosymmetric space group Cmca. The two ligands are bound in a tridentate manner generating a meridional coordination with both dianionic ligands on a metal center having the same chirality; due to the center of symmetry the complex with opposite chirality is also present. The complex (HNEt₃)₂[Fe(phoxCOO)₂](ClO₄) is the first accurate structural model of the iron complex of a siderophore analog commonly observed in mycobactins. The three didentate ligands in the complex [Fe(phoxiPr)₃] are bound with like atoms in a meridional manner to the metal center. The metal ion is surrounded by two ligands of the same chirality and one ligand of opposite chirality (ie. RRS or SSR); due to the presence of a center of symmetry both isomers are present in the crystal structure. The complex (HNEt₃)₂[Fe(phoxCOO)₂](ClO₄) shows promising activity in the oxidation of alkanes, such as toluene, ethylbenzene and cumene, while the complex [Fe(phoxiPr)₃] does not show any catalytic activity in alkane oxidations under the conditions tested. The complex (HNEt₃)₂[Fe(phoxCOO)₂](ClO₄) is reasonably efficient in the conversion of H₂O₂ to oxidation products.