Studies on NADPH-cytochrome c reductase I: Isolation and several properties of the crystalline enzyme from ale yeast.

NADPH-cytochrome c reductase has been isolated from a top-fermenting ale yeast, Saccharomyces cerevisiae (Narragansett strain), after ca. a 240-fold purification over the initial extract of an acetone powder, with a final specific activity (at pH 7.6, 30 °C) of ca. 150 μmol cytochrome c reduced min^?1mg^?1 protein. The preparation appears to be homogeneous by the criteria of: sedimentation velocity; electrophoresis on cellulose acetate in buffers above neutrality; and by polyacrylamide gel electrophoresis. Although the reductase appeared to partially separate into species “A” and “B” on DEAE-cellulose at pH 8.8, the two species have proven to be indistinguishable electrophoretically (above pH 8) and by sedimentation. By sedimentation equilibrium at 20 °C, a molecular weight of ca. 6.8 (± 0.4) × 10⁴ was obtained with use of a $\text{V}\text{?}{}_{\mathrm{20\; °}}\text{= 0.741}$ calculated from its amino acid composition. After disruption in 4 m guanidinium chloride- 10 mm dithioerythritol- 1 mm EDTA, pH 6.4 at 20 °C, an $\text{M}\text{?}{}_{\mathrm{<mtext>r</mtext>}}$ of 3.4 (± 0.1) × 10⁴ resulted, which points to a subunit structure of two polypeptide chains per mole. Confirmatory evidence of the two-subunit structure with similar, if not identical, polypeptide chains was obtained by polyacrylamide gel electrophoresis in dodecyl-sulfate, after disruption in 4 m urea and 2% sodium dodecyl sulfate, and yielded a subunit molecular weight of ca. 4 × 10⁴. Sulfhydryl group titration with 4,4′-dithiodipyridine under acidic conditions revealed one sulfhydryl group per monomer, which apparently is necessary for the catalytic reduction of cytochrome c. NADPH, as well as FAD, protects this-SH group from reaction with 5,5′-dithiobis (2-nitrobenzoate). The visible absorption spectrum of the oxidized enzyme (as prepared) has absorption maxima at 383 and 455 nm, typical of a flavoprotein. Flavin analysis (after dissociation by thermal denaturation of the “A” protein) conducted fluorometrically, revealed the presence of 2.0 mol of FAD per 70,000 g, in confirmation of the deduced subunit structure. The identity of the FAD dissociated from either “A” or “B” protein was confirmed by recombination with apo-d-amino acid oxidase and by thin-layer chromatography. A kinetic approach was used to estimate the dissociation constant for either FAD or FMN (which also yields a catalytically active enzyme) to the apoprotein reductase at 30 °C and pH 7.6 (0.05 m phosphate) and yielded values of 4.7 × 10^?8m for FAD and 4.4 × 10^?8m for FMN.     

Resonance Raman spectroscopy of cytochrome c oxidase and electron transport particles with excitation near the Soret band

We report the resonance Raman spectra of cytochrome $\text{c}$ oxidase, both solubilized and in electron transport particles using laser excitation near the Soret band. As in the spectra of other hemoproteins, such as cytochrome $\text{c}$, the shape and intensity of a number of bands change when the oxidation state is varied. However, one of the hemes of solubilized cytochrome $\text{c}$ oxidase shows redox behavior which is anomalous. Spectra of electron transport particles are dominated by cytochrome $\text{c}$ oxidase. There are, however, definite differences between spectra of solubilized cytochrome $\text{c}$ oxidase and electron transport particles in the oxidized states.     

Electron transfer from pyridinyl radicals, hydrated electrons, CO2.- and O2.- to bacterial cytochrome P450.

Several rate constants for one-electron reduction of cytochrome P450 are more rapid in the absence than in the presence of the specific substrate. The respective values for methyl viologen, nicotinamide adenine dinucleotide and the 1-methyl-4-(and -3-)carbamidopyridinium radicals are 2.6, 3.4, 6 and 35 × 10⁷ M^?1 s^?1 without camphor, and 0.15, 0.1, 1.8 and 110 × 10⁷ M^?1 s^?1 for the camphor complex. Hydrated electrons react with cytochrome P450 with a rate constant of 3.0 × 10¹⁰ M^?1 s^?1 whether camphor is bound or not, but little of the reduction takes place at the haem iron. No reduction of the haem iron by $\text{CO}{}_2{}^{\mathrm{?-}}$ or $\text{O}{}_2{}^{\mathrm{?-}}$ could be detected, whether camphor is bound or not.     

Analysis of the kinetics of electron transfer reactions of hemoglobin and myoglobin with inorganic complexes.

The kinetics of methemoglobin reduction by Fe(EDTA)^2? have been studied and found to follow a second order rate law with k = 29.0 $\text{M}$^?1 s^?1 [25°C, μ = 0.2 $\text{M}$, pH 7.0 (phosphate)], $\text{ΔH}{}^3\text{= 5.5 ± 0.7 kcal/mol}$, and $\text{ΔS}{}^2\text{= ?33 ± 2 e.u.}$. The electrostatics-corrected self-exchange rate constant (k₁₁^corr) for hemoglobin based on the Fe(EDTA)^2? cross-reaction is 2.79×10^?3$\text{M}$^?1 s^?1. This rate constant is compared with others reported for a water-soluble iron porphyrin and calculated from published data for the reactions of myoglobin and hemoglobin with Fe(EDTA)^2? and Fe(CDTA)^2?/?. The k₁₁^corr values for these systems range over ten orders of magnitude with heme ? myoglobin > hemoglobin.     

Cobalt cytochrome c: enzymic assays.

An improved synthesis for cobalt-cytochrome $\text{c}$ has been developed; its half reduction potential is ?140 ± 20mV. Reduced ^Cocyt-$\text{c}$³ is oxidized by bovine heart cytochrome $\text{c}$ oxidase at a rate ～45% that of the native cytochrome $\text{c}$. It is not reduced by mitochondrial NADH or succinate cytochrome $\text{c}$ reductase nor by microsomal NADH or NADPH cytochrome $\text{c}$ reductase.     

Bound nucleotides and phosphorylation in Rhodospirillum rubrum.

Chromatophores from $\text{Rhodospirillum rubrum}$ contain 12 × 10^?3 mol ATP and 8.3 × 10^?3 mol ADP per mol chlorophyll, tightly bound to the coupling ATPase. Under energised conditions, these exchange slowly with added nucleotide. Using single turnover light flashes, it is demonstrated that the release of bound ATP is too slow to be on the direct pathway of photophosphorylation.     

Kinetic parameters for the binding of p-nitrophenyl alpha-d-mannopyranoside to concanavalin A.

Binding of the chromogenic ligand p-nitrophenyl α-d-mannopyranoside to concanavalin A was studied in a stopped-flow spectrometer. Formation of the protein-ligand complex could be represented as a simple one-step process. No kinetic evidence could be obtained for a ligand-induced change in the conformation of concanavalin A, although the existence of such a conformational change was not excluded. The entire change in absorbance produced on ligand binding occurred in the monophasic process monitored in the stopped-flow spectrometer. The value of the apparent second-order rate constant (k_a) for complex formation (k_a = 54,000 s^?1m^? at 25 °C, pH 5.0, Γ/2 0.5) was independent of the protein concentration when the protein was in the range of 233–831 μm in combining sites and in excess of the ligand. The apparent first-order rate constant (k_?a) for dissociation of the complex was obtained from the rate constant for the decomposition of the complex upon the addition of excess methyl α-d-mannopyranoside (k_?a = 6.2 s^?1 at 25 °C, pH 5.0, Γ/2 0.5). The ratio $\text{k}{}_{\mathrm{<mtext>a</mtext>}}{}_{\mathrm{<mtext>?</mtext><mtext>a</mtext>}}$ (0.9 × 10₄m^?1) was in reasonable agreement with value of 1.1 ± 0.1 × 10⁴m^?1 determined for the equilibrium constant for complex formation by ultraviolet difference spectrometry. Plots of ln($\text{k}{}_{\mathrm{<mtext>a</mtext>}}\text{T}$) and ln($\text{k}{}_{\mathrm{<mtext>a</mtext>}}\text{T}$) vs $\text{1}\text{T}$ were linear (T is temperature) and were used to evaluate activation parameters. The enthalpies of activation for formation and dissociation of the complex are 9.5 ± 0.3 and 16.8 ± 0.2 kcal/mol, respectively. The unitary entropies of activation for formation and dissociation of the complex are 2.8 ± 1.1 and 1.3 ± 0.7 entropy units, respectively. These entropy changes are much less than those usually associated with substantial changes in the conformation of proteins.     

Halogenated biphenyls as AHH inducers: Effects of different halogen substituents

4′-Iodo-, 4′-bromo-, 4′-chloro- and 4′-fluoro-2,3,4,5-tetrachlorobiphenyl were administered to immature male Wistar rats and the effects of this homologous series of 4′-halo-2,3,4,5-tetrachlorobiphenyls on the microsomal drug-metabolizing enzymes were determined. All the halogenated biphenyls increased microsomal benzo[a]pyrene hydroxylase (or aryl hydrocarbon hydroxylase, AHH), ethoxyresorufin (ER) O-deethylase and dimethylaminoantipyrine (DMAP) N-demethylase. The effects of the 4′-halo-2,3,4,5-tetrachlorobiphenyls on the microsomal enzyme activities and on the relative peak intensities and spectral shifts of the reduced cytochrome P-450:CO and ethylisocyanide (EIC) binding difference spectra were similar to those observed after coadministration of phenobarbitone (PB) and 3-methylcholanthrene (MC). The relative activities of the halogenated biphenyls were determined using two $\text{in}$$\text{vitro}$ assays; namely cytochrome P-448 associated induction in rat hepatoma H-4-II E cells in culture and competitive binding to the hepatic cytosolic $\text{Ah}$ receptor protein from male Wistar rats. Dose-response experiments for the iodo, bromo, chloro and fluoro analogs gave EC₅₀(M) values of 8.5×10^?9, 6.6×10^?8, 5.7×10^?7, and 3.3×10^?5, and 1.5×10^?6, 2.5×10^?6, 4.1×10^?6 and 2.5×10^?5 for the ER O-deethylase induction and receptor binding assays respectively. The relative potencies of the 4′-halo-2,3,4,5-tetrachlorobiphenyls followed the order I>Br>Cl>F for both assays and differences in the EC₅₀ values for the iodo and fluoro analogs were greater than three orders of magnitude for ER O-deethylase induction in rat hepatoma cells in culture. One possible explanation for these effects may be associated with differences in the polarizability of the laterally substituted halogen groups. However, other differences in the physico-chemical properties of the halogen atoms may also be important.     

Studies on two isozymes of aconitase from Bacillus cereus T. III. Enzymatic properties.

The present communication describes a comparative study of some enzymatic properties of an early and a late aconitase (EC.4.2.1.3) present in $\text{Bacillus}\text{cereus}$ T cells of 5 and 12 hr culture age, respectively. The activity of both enzymes increased linearly with increase in enzyme concentration. They demonstrated similar pH *7.5) and temperature (30 C) optima, but differed in their activation energy and affinity for substrate. Late aconitase had higher activation energy (16,100 cal) as compared to early aconitase (9,200 cal). Early aconitase showed a Km value of 100 × 10^?4M for sodium citrate and 33.3 × 10^?4M for isocitrate. Late aconitase exhibited 5 to 7 times greater affinity for citrate and isocitrate yielding Km values 14 × 10^?4M and 7 × 10^?4M, respectively. On the basis of available evidence, it is suggested that early and late aconitase present in 5 and 12 hr aged cells of $\text{Bacillus}\text{cereus}$ T behave as isozymes, and may be designated as aconitase (EC.4.2.1.3) isozyme I and aconitase (EC.4.2.1.3) isozyme II, respectively. The significance of their plausible role during growth and sporulation has been discussed.     

Oxidation of ascorbic acid with superoxide anion generated by the xanthine-xanthine oxidase system.

Ascorbic acid was found to be oxidized by O₂^$\text{?}$ which was generated by the xanthine-xanthine oxidase system. From a kinetic analysis of the inhibition of this reaction by superoxide dismutase, the second-order rate constant for the reaction between ascorbic acid and O₂^? at pH 7.4 was estimated to be 2.7 × 10⁵ M^?1 sec^?1. A function of ascorbic acid as a defense against O₂^? is presented.     

Reversible redox titrations of cytochrome c and cytochrome c oxidase using detergent solubilized electrochemically generated mediator-titrants

The repetitive, $\text{reversible}$ equilibrium redox cycling of cytochrome $\text{c}$, cytochrome $\text{c}$ oxidase, or mixtures thereof has been made possible by the use of the oxidant, ferricinium ion. This ion is electrochemically generated by the use of non-ionic detergent solubilized ferrocene which is apparently incorporated as micelles and readily electron transfers with an electrode. The $\text{ferricinium-ferrocene}$ couple equilibrates rapidly with these heme proteins. Electrochemically generated benzylviologen radical cations are used as the reductant. The E^O′ values for cytochrome $\text{c}$ oxidase at pH 7.0 are 209 ± 15 mv (2e^?) and 340 ± 15 mv (2e^?).     

Actions of morphine on prostate gland fructose metabolism

Varying doses of morphine sulfate (10, 20 or 40 mg/kg daily × 10) were observed to suppress metabolic activities in the mouse prostate gland. Prostate gland fructose, an index of androgenic activity, was significantly reduced by these dose regimes of morphine (P < 0.01). Injections of morphine sulfate (20 mg/kg daily × 10) led to an inhibitition in the $\text{in vitro}$ synthesis of both fructose^?14C and sorbitol^?14C from glucose^?14C by the prostate gland, part of which may have been due to decreased uptake of glucose by the gland. The $\text{in vitro}$ assimilation of 2-deoxyglucose^?14C by the prostate was also reduced by morphine treatment. The $\text{in vitro}$ actions of morphine (2 × 10^?3M) on the metabolism of radioactive glucose by the mouse prostate gland likewise revealed a significant reduction in the formation of sorbitol^?14C, but no decrease in fructose^?14C formation. These results indicate that both the $\text{in vitro}$ and $\text{in vivo}$ actions of morphine can inhibit fructose metabolism in the prostate gland.     

Structural dynamics of bacterial ribosomes: V. Magnesium-dependent dissociation of tight couples into subunits: Measurements of dissociation constants and exchange rates

The magnesium ion-dependent equilibrium of vacant ribosome couples with their subunits

$\text{70}\text{S}\text{?}\text{k}{}_{\mathrm{?1}}\text{k}{}_1\text{50}\text{S}\text{+30}\text{S}$

has been studied quantitatively with a novel equilibrium displacement labeling method which is more sensitive and precise than light-scattering. At a concentration of 10^?7m, tight couples (ribosomes most active in protein synthesis) dissociate between 1 and 3 mm-Mg²⁺ at 37 °C with a 50% point at 1.9 mm. The corresponding association constants K_a′ are 5.1 × 10⁵m^?1 (1 mm-Mg²⁺), 3.5 × 10⁷m^?1 (2 mm), and 1.2 × 10⁹m^?1 (3 mm), about five orders of magnitude higher than the K_a′ value of loose couples studied by Spirin et al. (1971) and Zitomer & Flaks (1972).In this range of Mg²⁺ concentrations ($\text{37 °C, 50 mm-}\text{NH}{}_4{}^{\mathrm{+}}$) the rate constants depend exponentially and in opposite ways on the Mg²⁺ concentration: k₁ = 2.2 × 10^?3s^?1, k_?1 = 7.7 × 10⁴m^?1s^?1 (2mm-Mg²⁺); k₁ = 1.5 × 10^?4s^?1, k_?1 = 1.7 × 10⁷m^?1s^?1 (5 mm-Mg²⁺). Under physiological conditions (Mg²⁺ ～- 4 mm, ribosome concn ～- 10^?7m), the equilibrium strongly favors association and the rate of exchange is slow ($\text{t}{}_{\mathrm{<mtext>1</mtext><mtext>2</mtext>}}\text{～- 10}\text{min}$). In the range of dissociation (2 mm-Mg²⁺), association of subunits proceeds without measurable entropy change and hence ΔG^O = ΔH^O. The negative enthalpy change of ΔH^O = ? 10 kcal suggests that association of subunits involves a shape change.Below a critical Mg²⁺ concentration (～- 2 mm), the 50 S subunits are converted irreversibly into the b-form responsible for the transition to loose couples. The results are compatible with two classes of binding sites, one class binding Mg²⁺ non-co-operatively and contributing to the free energy of association by reduction of electrostatic repulsion, and another class probably consisting of hydrogen bonds between components at opposite interfaces whose critical spatial alignment rapidly denatures in the absence of stabilizing magnesium ions.     

Cytochalasin B inhibition and temperature dependence of 3-O-methylglucose transport in fat cells

The transport of $\text{3-O-}\text{methylglucose}$ in white fat cells was measured under equilibrium exchange conditions at $\text{3-O-}\text{methylglucose}$ concentrations up to 50 mM with a previously described method (Vinten, J., Gliemann, J. and Østerlind, K. (1976) J. Biol. Chem. 251, 794–800). Under these conditions the main part of the transport was inhibitable by cytochalasin B. The inhibition was found to be of competitive type with an inhibition constant of about 2.5 · 10^?7 M, both in the absence and in the presence of insulin (1μM). The cytochalasin B-insensitive part of the $\text{3-O-}\text{methylglucose}$ permeability was about 2 · 10^?9 cm · s^?1, and was not affected by insulin. As calculated from the maximum transport capacity, the half saturation constant and the volume/ surface ratio, the maximum permeability of the fat cell membrane to $\text{3-O-}\text{methylglucose}$ at 37°C and in the presence of insulin was 4.3 · 10^?6 cm · s^?1. From the temperature dependence of the maximum transport capacity in the interval 18–37°C and in the presence of insulin, an Arrhenius activation energy of $\text{14.8 ± 0.44}\text{kcal/mol}$ was found. The corresponding value was $\text{13.9 ± 0.89}$ in the absence of insulin. The half saturating concentration of $\text{3-O-}\text{methylglucose}$ was about 6 mM in the temperature interval used, and it was not affected by insulin, although this hormone increased the maximum transport capacity about ten-fold to 1.7 mmol · s^?1 per 1 intracellular water at 37°C.     

Cytochrome c interaction with membranes. I. Use of a fluorescent chromophore in the study of cytochrome c interaction with artificial and mitochondrial membranes

Quenching of 12-(9-anthroyl) stearic acid (AS) fluorescence by cytochrome c occurs through an energy-transfer mechanism and can be used to measure the binding of the cytochrome to artificial and mitochondrial membranes. The quenching of AS³ fluorescence is biphasic ($\text{t}{}_{\mathrm{<mtext>1</mtext><mtext>2</mtext>}}$ below 25 msec and above 500 msec) and its extent diminishes at high salt concentration or at high pH and increases in the presence of negatively charged lipids.Addition of cytochrome c to cytochrome c-depleted mitochondria results in binding of the cytochrome to the membrane and quenching of AS fluorescence. The affinity of oxidized cytochrome c for cytochrome c-depleted mitochondria is 1.8 × 10⁶m, while the affinity constant for reduced cytochrome c is 0.5 × 10⁶m. The lower affinity of the reduced cytochrome c for mitochondrial membranes is in accordance with midpoint potential differences between the bound and free forms.     

Alteration of thermal stability of glucose oxidase associated with the redox states

By the method of differential scanning calorimetry, it was found that thermal stability of glucose oxidase was dependent on its redox states. The oxidized form showed an apparent denaturation temperature at 76°C and the denaturation enthalpy was approximately 865 kcal/mol. On reduction of the enzyme, the denaturation temperature increased by about 10°, but no significant change was seen in the denaturation enthalpy. The activation energies of the denaturation of the oxidized and the reduced enzymes were about 89 and 103 kcal/mol, respectively. These results may imply conformational changes in the catalytic turnover of this enzyme.     

Catalytic effect of sulfhydryl oxidase on the formation of three-dimensional structure in chymotrypsinogen A

The effect of sulfhydryl oxidase on the rate of disulfide bond formation and polypeptide chain folding in reductively denatured chymotrypsinogen A has been investigated using an immobilized zymogen preparation and a cylindrical quartz flow-through fluorescence cell. Enzymatic oxidation of the 10 sulfhydryl groups in reduced chymotrypsinogen followed first order kinetics at pH 7.0 with an apparent first order rate constant governing sulfhydryl group disappearance of 4.2 × 10^?2 min^?1. This provides a $\text{t}\text{1}\text{2}$ of 16.3 min for the sulfhydryl oxidase-catalyzed oxidation, whereas 165 min are required for nonenzymatic aerobic oxidation of one-half the sulfhydryl groups. Refolding of the reductively denatured polypeptide chains, monitored by changes in protein fluorescence, did not follow first order kinetics characteristic of a simple two-state mechanism, nor did the return of trypsin activatability. It appears that at least one intermediate must exist in such refolding, in both the uncatalyzed and sulfhydryl oxidase-catalyzed processes. Estimation of the rate constants governing refolding, assuming a single intermediate between the denatured and native states, provided values of 3 × 10^?2 min^?1 and 7 × 10^?3 min^?1 for uncatalyzed autoxidation and 4 × 10^?2 min^?1 and 1.1 × 10^?2 min^?1 for the sulfhydryl oxidase-catalyzed transition. Thus, enzymic catalysis of disulfide bond formation can lead to apparent catalysis of protein refolding as monitored both by fluorescence and by acquisition of biological function.     

EPR study of the effect of formate on cytochrome C oxidase

The addition of formate to oxidized cytochrome $\text{c}$ oxidase (ferrocytochrome $\text{c}$: oxygen oxidoreductase, EC 1.9.3.1) causes the appearance of a high spin heme signal at g = 6 and a splitting of g = 3 signal to g = 2.98 and 3.07. When formate-cytochrome $\text{c}$ oxidase is reduced, the g = 2.98 signal decreases significantly. The spectrophotometric studies showed that formate is a specific ligand to cytochrome $\text{a}$₃. Data suggest that binding of formate to oxidized cytochrome $\text{c}$ oxidase produces a ligand-$\text{a}$₃ interaction leading to the splitting of g = 3 signal hitherto considered as due to cytochrome $\text{a}$. Thus both cytochrome $\text{a}$ and $\text{a}$₃ contribute to the resonance of g = 3 signal of cytochrome $\text{c}$ oxidase.     

Evidence for the existence of a ubiquinone protein and its radical in the cytochromes b and c1 region in the mitochondrial electron transport chain.

A highly purified cytochrome $\text{b}\text{?}\text{c}{}_1$ complex which is free of QP_s (see BBRC $\text{78}\text{, 259 and}\text{79}$, 939) yields 20–30% of semiubiquinone (based on the total Q content in the system) in the presence of catalytic amounts of QP_s and succinate dehydrogenase (at or lower than 2 × 10^{?9 M}. The radical shows typical $\text{g}\text{= 2.00}$ signals with line widths of 8 and 9 gauss, respectively, at about 22°C and 77°K. The appearance of the radicals approximately parallels that of $\text{b}$ reduction but not its disappearance. However, addition of theonytrifluoroacetone or antimycin A immediately abolishes both radical formation and $\text{b}$ reduction. These and other observations indicate that the true carrier property of Q is through its binding with proper proteins but not the protei-free form.     

Fungal proteases and the mammalian kinin system. II. Kinin hydrolysis by brinolase.

Brinolase, a thrombolytic fungal protease capable of forming vasoactive kinins, has been shown to hydrolyze kinins after their formation. Using synthetic bradykinin as a substrate, the kinetics and mechanism of hydrolysis have been elucidated, evidently explaining the apparently low kinin formation $\text{in vivo}$, Bradykinin hydrolysis proceeded rapidly $\text{in vitro}$ with a pH optimum of 7.0–7.5, and a half-life of 5.1 min, using 250 ng/ml bradykinin and 50 μg/ml brinolase. The K_m was 3.2×10^?6 M and the V_max was 4.6 × 10^?8 mol/liter/min, using 5 μg/ml brinolase. Two-dimensional paper fractionation of the brinolase-bradykinin digest revealed the presence of free arginine amongst the five peptide fragment spots.