Conformation of <Emphasis Type="Italic">Thermus thermophilus</Emphasis> ribosomal protein S1 in solution at different ionic strengths

Small-angle x-ray and neutron scattering were used to study the structure of the ribosomal protein S1 (61 kDa) from Thermus thermophilus in solution at low and moderate ionic strength (0 and 100 mM NaCl). The protein was found to be globular in both cases. Modeling of the S1 structure comprising six homologous domains on the basis of the NMR data for one domain showed that the best fit to scattering data was provided by compact domain packing. The calculated gyration radius was 28–29 Å, as typical of globular proteins about 60 kDa. The protein was prone to self-association, forming mainly dimers and trimers at moderate ionic strength and higher compact associates at low ionic strength. Neutron scattering assays in heavy water at 100 mM NaCl revealed markedly elongated associates. The translational diffusion coefficient calculated for S1 at 100 mM NaCl from dynamic light scattering was markedly lower than the one expected for its globular monomer (D _20,w = (2.7 ± 0.1)·10^?7 versus (5.8–6.0)·10^?7 cm² s^?1), confirming protein association under equilibrium conditions.     

Transient electric birefringence of the bacteriophages T3 and T7

Electric birefringence measurements of suspensions of T3 and T7 bacteriophages in 10^?2 M phosphate buffer, pH 6.9, show that there is a difference in their rotational diffusion coefficient. The values corrected to 25°C and water viscosity are D_25,w = 4630 ± 130 sec^?1 and D_25,w = 5290 ± 260 sec^?1 for T3 and T7, respectively. The value obtained from shell model calculations (according to Filson and Bloomfield) is D_25,w = 4500 ± 600 sec^?1. The apparent permanent dipole moments are 4.5 × 10^?26 C·m and 1.7 × 10^?26 C·m for T3 and T7, respectively. For both phage particles the intrinsic optical anisotropy is +7.2 × 10^?3. It is shown that this anisotropy is mainly due to the DNA molecule inside the head of the phage. Its positive value means that there exists an excess orientation of the DNA helix perpendicular to the symmetry axis of the particle. For T7 an unexpectedly large increase of Δn_s and K_sp occurs at a glycerol concentration of about 30% (v/v). This increase is interpreted as being caused by a change of the shape of the particle and/or a change in the secondary structure of the DNA inside the head of the bacteriophage.     

Purification and Some Properties of β-Xylosidase from Emericella nidulans

β-Xylosidase was purified 662 fold from a culture filtrate by ammonium sulfate fractionation, gel filtration on Biogel P-100, DEAE-Sephadex chromatography, and gel filtration on Sephadex G-200. With isoelectric focusing, the purified β-xylosidase found to be homogeneous on SDS (sodium dodecyl sulfate) polyacrylamide gel electrophoresis. The molecular weight was estimated by gel filtration to be 240,000, and 116,000 by SDS polyacrylamide gel electrophoresis. The purified β-xylosidase had an isoelectric point at pH 3.25, and contained 4% carbohydrate residue. The optimum pH was found to be in the range of 4.5 ~ 5, and the optimum temperature was 55°C. The enzyme activity was inhibited by Hg^{2 +}, SDS, and N-bromosuccinimide at a concentration of 1 × 10^?3 m, and also p-chloromercuribenzoate at a concentration of 1 × 10^?4m. The purified enzyme hydrolyzed phenyl β-d-xyloside (k_o = 302.6 sec^?1),β-nitrophenyl β-d-xyloside (k_o = 438.9 sec^?1), o-nitrophenyl β-d-xyloside (k_o = 431.0 sec^?1), p-chlorophenyl β-d-xyloside (k_o = 207.9 sec^?1), o-chlorophenyl β-d-xyloside (k_o = 211.8 sec^?1), β-methylphenyl β-d-xyloside k_o = 96.5 sec^?1), o-methylphenyl β-d-xyloside (k_o = 83.1 sec^?1), p-methoxyphenyl β-d-xyloside (k_o = 99.3 sec^?1), o-methoxyphenyl β-d-xyloside (k_o= 100.0 sec^?1), xylobiose (k_o = 992A sec^?1), xylotriose (k_o = 1321.9 sec^?1), xylotetraose (k_o = 7S9.1 sec^?1) and xylopentaose (k_o = 508.0 sec^?1). On enzymic hydrolysis of phenyl β-d-xyloside, the reaction product was found to be β-d-xylose with retention of the configuration. The purified β-xylosidase was practically free of a-xylosidase and β-glucosidase activities.     

Purification, characterization and reassembly of the bacteriophage T4D tail sheath protein P18.

P18, the sole component of T4 tail sheath, has been isolated in a monomeric active form from extended sheaths of intact tails which were dissociated at low ionic strength. The molecular weight of P18 is determined to be 65,000 from sedimentation equilibrium and 73,000 from sodium dodecyl sulphate/gel electrophoresis. Combining the diffusion constant (D_20,w = 5·5× 10^?7cm²s^?1)and the sedimentation constant (s⁰_20,w = 4·2 S) a value of 67,000 is obtained. The circular dichroism spectra reveal a striking similarity of the structure of P18 in the monomeric state and in the extended sheath conformation.The purified P18 is found to reassemble into extended sheaths if the core-baseplate complex is present, forming normal length tails. Structures similar to polysheath are formed in the absence of core-baseplates.     

Electric‐induced mortality of newly transformed juvenile fishes in waters of different conductivity

In three experiments, each with three species of newly transformed juvenile fishes, the immediate mortality was determined after electrical exposure to 60 Hz pulsed DC in waters of different conductivity (C_w). With a constant applied power density (D_a; 1·0–4·9 mW cm^?3 depending on species) over a range of C_w(10–1020 μS cm^?1), the results predicted that the highest fish mortality would occur at C_w of 65 μS cm^?1 for bluegill Lepomis macrochirus, 74 μS cm^?1 for largemouth bass Micropterus salmoides and at 140–175 μS cm^?1 for channel catfish Ictalurus punctatus. In experiment 2, the voltage gradient (E) was maintained constant (2·5–8·0 peak V cm^?1 depending on species) over the same range of C_w, and fish mortality increased with current density (J) or D_a, which are directly related to C_w. In experiment 3, fish mortality did not differ when peak E(3 or 8 V cm^?1 depending on species) and mean J(0·09 or 0·24 mA cm^?2 depending on species) were held constant by changing pulse width in waters with different C_w(99, 165 or 495 μS cm^?1). Fish mortality in this experiment was not significantly related to peak or mean transferred power density, and the ‘power transfer theory for electrofishing’ was not useful for predicting electrofishing mortality. Overall, the results of the present study indicated that mortality caused by exposure to electricity can be predicted more accurately with the variables peak E and mean J than with models requiring determination of effective conductivity of the fish.     

Purification and some Properties of β-Xylosidase from Trichoderma viride

β-Xylosidase was purified 25 fold from a culture filtrate by ammonium sulfate fractionation, DEAE-Sephadex chromatography, column electrophoresis, gel filtration on Biogel P-100, and isoelectric focusing. The purified β-xylosidase was found to be homogeneous on SDS (sodium dodecyl sulfate) polyacrylamide gel electrophoresis and on disc electrophoresis. A molecular weight of 101,000 was estimated by chromatography on Sephadex G-200, and 102,000 was obtained by SDS polyacrylamide gel electrophoresis. The purified p-xylosidase had an isoelectric point at pH 4.45, and contained 4.5% carbohydrate residue. The optimum activity for the enzyme was found to be at pH 4.5 and 55°C. The enzyme activity was inhibited by Hg^{2 +}, and N-bromosuccinimide at a concentration of 1 x 10^?3 m. The purified enzyme hydrolyzed phenyl β-d-xyloside (k_o—13.0 sec”¹), p-nitrophenyl β-d-xyloside (k_o=2l.3 sec^?1), o-nitrophenyl β-d-xyloside (k_o = 22.2 sec^?1), o-chlorophenyl β-d-xyloside (k_o = 20.0 sec^?1), p-methylphenyl β-d-xyloside (k_o~9.0 sec^?1), o-methylphenyl β-d-xyloside (k_o= 10.7 sec^?1), p-methoxyphenyl β-d-xyloside (k_o=10.3 sec^?1), o-methoxyphenyl β-d-xyloside (&;_o=10.9 sec^?1), xylobiose (k_o = 36A sec^?1), xylotriose (k_o = 34.5 sec^?1), xylotetraose (k_o~HA sec^?1), and xylopentaose (k_o= 13.0 sec^?1). On enzymic hydrolysis of phenyl β-d-xyloside, the reaction product was found to be β-d-xylose with retention of configuration. The purified p-xylosidase was practically free of α-xylosidase and β-glucosidase activities.     

Effects of pH,Ionic Strength,Temperature, and Complexing Anions on the Sorption Behavior of Cobalt on Hydrous Silica

Sorption of Co(II) on SiO₂.xH₂O (silica gel) has been investigated as a function of time, amount of silica gel (0.10–1.00g), cobalt concentration (5.00 × 10^?5–1.20 × 10^?3 M), ionic strength (0.20–1.40 M NaClO₄), pH (～6.80–10.80), and temperature (273–318 K). Using the sorption kinetics data, the diffusion coefficient of Co(II) was calculated to be 6.86(±0.44) × 10^?12 m²sec^?1 under particle diffusion-controlled conditions. The sorption rate was determined as 2.61(±0.19) × 10^?3 sec^?1 at 298 K, pH 6.70(±0.05) and 0.20 M NaClO₄. The sorption data followed the Freundlich, Langmuir, and Dubinin-Radushkevich (D-R) isotherms. Cobalt sorption decreased with increased ionic strength. A gradual decrease in pH with increased ionic strength supported the sorption of Co(II) by an ion exchange mechanism. The effects of different ligands such as , F^?, and on the sorption of Co(II) were studied in the pH range 6.50 to 8.50. The sorption of cobalt on silica gel increased with increased temperature and had an endothermic enthalpy change (ΔH = 23.60(±0.57) kJ/mol).     

Molecular weight of cytochrome oxidase

The molecular weight of bovine heart mitochondrial cytochrome oxidase in 2% or 3% deoxycholate was determined by the sedimentation velocity method to be 228,000 daltons from an S_20,w=8.44×10^–13 sec, a D_20,w=3.21×10^–7 cm² sec^–1, and the reported by others. The S_20,w value was only slightly concentration-dependent. When the deoxycholate in our preparation was replaced with Tween 80 the S value increased to between 16 and 17. When one preparation in Tween 80 was allowed to stand at room temperature, the S value increased in successive determinations to reach 51.5 at the end of approximately 7 h. The minimum molecular weight of the enzyme as calculated from the heme content (determined from the absorbance at 603 nm) and total protein content (determined from total nitrogen) was 110,000. An amino acid analysis when related to the heme content yielded a minimum molecular weight of 85,000.Deceased.     

Crystallization and Characterization of Polyamine Oxidase from Penicillium chrysogenum

Polyamine oxidase was purified and crystallized with an overall yield of 35% from mycelial extract of Penicillium chrysogenum by a procedure involving ammonium sulfate fractionation, and DEAE-cellulose and Sephadex G-200 column chromatographies. The crystalline enzyme was homogeneous, as judged by disc gel electrophoresis and ultracentrifugation. The sedimentation coefficient (s_{20, w}⁰) of the enzyme was determined to be 6.9S, and diffusion coefficient (D_{20, w}) to be 4.2 × 10^?7 cm² sec^?1. The enzyme showed a molecular weight of about 160,000 by gel filtration method and ultracentrifugal analysis, and it was composed of two identical subunits. The enzyme was a flavoprotein with absorption maxima at 275, 375 and 450 nm. The prosthetic group was identified to be FAD. The enzyme oxidized spermine, and slightly oxidized spermidine. Diamines and monoamines were not oxidized.     

Physicochemical studies on xylinan (acetan). III. Hydrodynamic characterization by analytical ultracentrifugation and dynamic light scattering

A laboratory-made sample of the polysaccharide xylinan (acetan) has been further characterized with respect to (i) purity, (ii) molar mass and polydispersity, and (iii) gross conformation by a combination of hydrodynamic measurements (sedimentation velocity and equilibrium analytical ultracentrifugation, viscometry, and dynamic light scattering) in aqueous NaCl (I = 0.10 mol·L⁻¹). Sedimentation velocity diagrams recorded using Schlieren optics revealed highly pure material sedimenting as a single boundary [s^o_20.w = 9.5 ± 0.7) S; k_s = (273 ± 112) mL/g]. The hypersharp nature of these boundaries is symptomatic of a polydisperse and highly nonideal (in the thermodynamic sense) system. Low speed sedimentation equilibrium in the analytical ultracentrifuge using Rayleigh interference optics and two different types of extrapolation procedure (involving point and whole-cell molar masses) gave a weight average molar mass M_w of (2.5 ± 0.5) × 10⁻⁶ g·mol⁻¹ and also a second virial coefficient, B = (2.8 ± 0.7) × 10⁻⁴ mL·mol·g⁻², both values in good agreement with those from light scattering-based procedures (Part II of this series). A dynamic Zimm plot from dynamic light scattering measurements gave a z-average translational diffusion coefficient D^o_20.w = (3.02 ± 0.05) × 10⁻⁸ cm²·s⁻¹ and the concentration-dependence parameter k_D = (370 ± 15) mL/g. Combination of s^o_20.w with D^o_20.w via the Svedberg equation gave another estimate for M_w of ≅ 2.4 × 10⁶ g/mol, again in good agreement. Both the Wales-van Holde ratio (k_s/[η]) ≅ 0.4 (with [η] = (760 ± 77) mL/g) and the ρ-parameter (ratio of the radius of gyration from static light scattering to the hydrodynamic radius from dynamic light scattering) as ρ > 2.0 all indicate an extended conformation for the macromolecules in solution. These findings, plus Rinde-type simulations of the sedimentation equilibrium data are all consistent with the interpretation in terms of a unimodal wormlike coil model performed earlier. © 1996 John Wiley & Sons, Inc.     

Photon correlation spectroscopy of hemoglobin: Diffusion of Oxy-HbA and Oxy-HbS

Translation diffusion coefficients have been measured for oxyhemoglobin A and oxyhemoglobin S over the concentration range 0.1–37 g/dl by means of photon correlation spectroscopy. The solutions were 0.1 M in KCl and in each case the pH was adjusted to the isoelectric point of the hemoglobin species present. No significant differences were found between the HbA and HbS results; and after correction to water at 20°C, the diffusion coefficients could be described by the equation where D_o = (6.93 ± 0.06) × 10^?7 cm²/sec and c ids the concentration in units of g/dl. No evidence was found for the aggregation of oxy-HbS at high concentrations which was reported by Lindstrom et. al. [(1976) Biophys. J. 16 , 679–689].     

Determination of effective diffusivity of substrate in biological films and particles

Summary Particle supported biofilms of uniform thickness were generated in an aerobic fluidized-bed reactor with phenol as the carbon source. A method was developed for determining the effective diffusivities of oxygen and phenol using trypan blue, a vital stain as the tracer. The effective diffusivities of oxygen and phenol were found to be 2.72×10^–6 cm²/s and 1.12×10^–6 cm²/s respectively.Nomenclature C_i initial solute concentration in bulk, g/cm³ - C_t solute concentration in bulk at time t, g/cm³ - C bulk solute concentration at equilibrium, g/cm³ - D molecular diffusivity, cm²/s - D effective diffusivity, cm²/s - D_o D_p D_tb molecular diffusivity of oxygen, phenol and trypan blue, cm²/s - D_o, D_p, D_tb effective diffusivity of oxygen, phenol and trypan blue, cm²/s - D_s molecular diffusivity of substrate, cm²/s - D_s effective diffusivity of substrate, cm²/s - K partition coefficient - M_t amount of solute in the particle at time t, g - M amount of solute in the particle at equilibrium, g - r particle radius, cm - r _bp radius of the particle with biofilm, cm - S substrate concentration, g/cm³ - S_b substrate concentration in bulk, g/cm³ - S_i initial substrate concentration, g/cm³ - V₁ solute molar volume, cm³/g mol Greek Symbols bf porosity of the biofilm - tortuosity factor     

Static and dynamic light scattering from dilute insulin solutions

Static and dynamic light-scattering measurements are reported on zinc-insulin at room temperature (21 ± l°C) and pH = 6.88 in 0.1M NaCl aqueous solution. The experiments were performed at very low concentration, in the range 0.12 × 10^?4 to 0.90 × 10^?4 g cm^?3. Within experimental error, we find no evidence for a critical micellar concentration in this system. The aggregation phenomenon starts immediately after preparation of the solutions, and takes several days to come to stable equilibrium. The concentration dependence of the diffusion coefficients, D _z, = D_o (1 — k_DC), is negative, and k_D was observed to decrease as a function of time, while the aggregate size was found to increase. The equivalent concentration coefficient, ?2BM _W, obtained from static light scattering, showed a similar behavior, and, within experimental error, was found to be numerically equal to k_D. From the relation found between the diffusion coefficient at infinite dilution and the molecular weight of the aggregates, log D₀ = ?0.240 log M _w ? 5.077, we deduce that the insulin aggregates are compact structures with a characteristic radius of 0.71 Å/(dalton)^1/3, surrounded by a hydration layer of a thickness of 8.0 Å. The equilibrium aggregation number is approximately 10.     

Transient electric birefringence of T-even bacteriophages. I. T4B in the absence of tryptophan and fiberless T4 particles

Measurements of the electric birefringence of suspensions of T4B in the absence of tryptophan and of fiberless T4 particles show that both kinds of particles are hydrodynamically equivalent. Their rotational diffusion coefficients corrected to 25°C and water viscosity (D_25,w) are 280 ± 9 sec^?1 and 295 ± 10 sec^?1, respectively. These corrected rotational diffusion coefficients are almost independent of buffer concentration and temperature. The sedimentation coefficient (s_20,w) of T4 B is equal to 1023 ± 12 S, a value which is likewise independent of buffer concentration. By analysis of the field strength dependence of the steady-state birefringence and by reversing pulse experiments it could be shown that the orientation in an electric field is largely due to a permanent dipole moment. This dipole moment is somewhat dependent on buffer concentration and amounts to about 24,000 debye for T4B and 95,000 debye for fiberless T4. An approximate calculation shows that the difference in dipole moment may be ascribed to positive charges on the fiber tip (at least ten per fiber), to negative charges along the fiber or (and) positive charges on the fiberless particle at those places where the fibers are attached in normal particles.     

Measurement of diffusion coefficient and electrophoretic mobility with a quasielastic light-scattering–band-electrophoresis apparatus

We have constructed an apparatus for the simultaneous measurement of electrophoretic mobility, μ, and diffusion coefficient, D, of macromolecules and cells. It combines band electrophoresis in a vertical, sucrose-gradient stabilized column, with quasielastic laser light-scattering determination of the diffusion coefficient of the species within the band. The entire electrophoresis cell is scanned through the laser beam of the quasielastic laser light-scattering apparatus by a vertical translation stage. Total intensity light-scattering measurement at each point in the cell gives the macromolecular concentration at that point. Solvent viscosity and electrical potential are measured at each point in the cell. Application of this apparatus to resealed red blood cell ghosts and to bovine hemoglobin indicates that measurements of field, viscosity, and migration distance are reliable, and that electroosmosis is insignificant. Application to T4D bacteriophage gives μ_20,w = (?1.05 ± 0.05) × 10^?4 cm²/V sec and D_20,w = (3.35 ± 0.10) × 10^?8 cm²/sec for fiberless particles, and μ_20,w = ?(0.59 ± 0.03) × 10^?4 cm²/V sec and D_20,w = (2.86 ± 0.09) × 10^?8 cm²/sec for whole phage with 6 fibers. Approximate analysis of these results with the Henry electrophoresis theory for spheres in dicates that each fiber contributes about 193 positive charges to the phage particle, compared with 327 from amino-acid analysis. The advantages and disadvantages of this apparatus, relative to conventional electrophoresis and to electrophoretic light scattering, are discussed.     

Certain Properties of Crystalline α-Glucosidase from Mucor javanicus

The crystalline α-glucosidase from Mucor javanicus has a sedimentation constant () of 6.1 S, a diffusion constant (D_{20, w}) of 4.8 × 10^?7 cm² · sec^?1, and an average molecular weight, as determined by two different methods, of 124,600. The α-glucosidase is a glycoprotein containing the following constituents; tryptophan₂₃, lysine₈₁, histidine₃₉, arginine₃₄, aspartic acid₁₀₂, threonine₆₉, serine₄₆, glutamic acid₇₈, proline₅₅, glycine₇₈, alanine₅₅, half cystine₈, valine₅₃, methionine₁₇, isoleucine₅₈, leucine₈₁, tyrosine₅₁, phenylalanine₄₁, glucosamine₁₂, and mannose₃₈.

The low content of half cystine, the high contents of aspartic acid, lysine, and histidine, and the presence of mannose as the sole constituent of neutral sugar are the characteristics of this enzyme.     

The cyanobacterium Synechococcus R-2 (Anacystis nidulans, S. leopoliensis) PCC 7942 has a sodium-dependent chloride transporter

Synechococcus R-2 (PCC 7942) actively accumulated Cl^? in the light and dark, under control conditions (BG-11 media: pH_o, 7·5; [Na⁺]_o, 18 mol m^?3; [Cl^?]_o, 0·508 molm^?3). In BG-11 medium [Cl^?], was 17·2±0·848 mol m^?3 (light), electrochemical potential of Cl^? (ΔμCl^?_i,o) =+211±2mV; [Cl^?]_i= 1·24±0·11 mol m^?3(dark), ΔμCl^?_i,o=+133±4mV. Cl^? fluxes, but not permeabilities, were much higher in the light: ?Cl^?_i,o= 4·01±5·4 nmol m^?2 s^?1, ^PCl^?_i,o= 47±5pm s^?1 (light); ?Cl^?_i,o= 0·395±0·071 nmol m^?2 s^?1, _PCl^?_i,o= 69±14 pm s^?1 (dark). Chloride fluxes are inhibited by acid pH_o (pH_o 5; ?Cl^?_i,o= 0·14±0·04 nmol m^?2 s^?1); optimal at pH_o 7·5 and not strongly inhibited by alkaline pH_o (pH_o 10; ?Cl^?1_i,o= 1·7±0·14 nmol m^?2 s^?1). A Cl^?_in/2H⁺_in coporter could not account for the accumulation of Cl^? alkaline pH_o. Permeability of Cl^? is very low, below 100pm s^?1 under all conditions used, and appears to be maximal at pH_o 7·5 (50–70 pm s^?1) and minimal in acid pH_o (20pm s^?1). DCCD (dicyclohexyl-carbodiimide) inhibited ?Cl^?_i,o in the light about 75% and [Cl^?]_i fell to 2·2±0·26 (4) mol m^?3. Valinomycin had no effect but monensin severely inhibited Cl^? uptake ([Cl^?]_i= 1·02±0·32 mol m^?3; ?Cl^?_i,o= 0·20±0·1 nmol m^?2 s^?1). Vanadate (200 mmol m^?3) accelerated the Cl^? flux (?Cl^?_i,o= 5·28±0·64 nmol m^?2 s^?1) but slightly decreased accumulation of Cl^? ([Cl^?], = 13·9±1·3 mol m^?3) in BG-11 medium but had no significant effect in Na⁺-free media. DCMU (dichlorophenyldimethylurea) did not reduce [Cl^?], or ?Cl^?_i,o to that found in the dark ([Cl^?]_i= 8·41±0·76 mol m^?3; ?Cl^?_i,o= 2·06±0·36 nmol m^?2 s^?1). Synechococcus also actively accumulated Cl^? in Na⁺-free media, [Cl^?]_i was lower but ΔΨ_i,o hyperpolarized in Na⁺-free media and so the ΔμCl^?_i,o was little changed ([Cl^?]_i= 7·98±0·698 mol m^?3; ΔμCl^?_i,o=+203±3 mV). Net Cl^? uptake was stimulated by Na⁺; Li⁺ acted as a partial analogue for Na⁺. Synechococcus has a Na⁺ activated Cl^? transporter which is probably a primary 2Cl^?/ATP pump. The Cl^? pump is voltage sensitive. ΔμCl^?_i,o is directly proportional to ΔΨ_i,o(P»0·01%): ΔμCl^?_i,o= -1·487 (±0·102) ×ΔΨ_i,o, r= -0·983, n= 31. The ΔμCl^?_i,o increased (more positive) as the Δμ_i,o became more negative. The ΔμCl^?_i,o has no known function, but might provide a driving force for the uptake of micronutrients.