Electric birefringence of bacterial flagellar protein filaments: evidence for field-induced interactions.

The time course for the build-up and decay of birefringence induced by a rectangular voltage pulse was measured on solutions of flagellar filaments from Salmonella equi-abortus (strain SJ25). These filaments are tubular polymers of protein (degree of polymerization ≈ 10³) constituted by non-covalent linkage of flagellin monomers of molecular weight 4 × 10⁴. The effect on electro-optical properties of solutions of filaments due to variations in temperature, concentration and mean length of protein filaments, and the duration and intensity of the applied electric field is described. Analysis of the field intensity dependence of the birefringence and comparison of the build-up and decay processes indicate that orientation in the field is due primarily to the existence of a permanent dipole moment in the filaments. At 18 °C the following values were obtained for a solution of filaments with mean length and standard deviation of 0.39 and 0.30 μm: specific Kerr constant (K_sp) = 6.14 × 10⁻³ electrostatic units; optical anisotropy factor (g₁ — g₂) = 5.66 × 10⁻³; dipole moment (μ) = 1.01 × 10⁵ Debye units; and mean relaxation time ($\text{}\text{̄}$gt) = 9.20 ms. At temperatures below 20 °C there is a marked increase in the optical anisotropy factor of the filaments which may be due to a change in their flexibility. The large values of K_sp obtained indicate the highly responsive nature of these filaments to an electric field. The birefringence decay curves were decomposed by computer into a specified number of exponential terms from which both the mean length and the size distribution of these polydisperse filaments were calculated. The results obtained were in substantial agreement with the values of these parameters observed by electron microscopy. A cumulative field effect dependent on field intensity and filament concentration was observed. Repeated pulsing of electric field, above threshold values of field intensity and filament concentration, produced decreases in the birefringence near 60% of its initial value. The effect was reversible with a time constant greater than two minutes. No appreciable change in the relaxation time for decay of birefringence was observed on multiple pulsing of these solutions. These results are interpreted consistently to arise from the sidewise aggregation of filaments induced by electrical impulses of sufficient intensity and duration. These properties appear relevant to bacterial motility: variations in electric potential along the membrane of the bacterium might serve first to orient these organelles and then to induce their coalescence to “bundles” of filaments. The latter structures are commonly observed in vivo. In this way the activity of flagella might be co-ordinated.     

Electro-optical properties of carboxymethylcellulose. I. Concentration and ionic strength dependence

The experimental conditions for studying the electro-optical properties of a natural, modified polyelectrolyte, carboxymethylcellulose (DS 1.3; DP 180) were determined. The transient Kerr effect was found to be a function of CMC concentration, field strength, and ionic strength, I. If the concentration and I were low enough (c < 20 mg.l^?1), saturation was obtained for field strengths of approximately 15 kV.cm^?1. The optical anisotropy was shown to be independent of I; the electrical anisotropy decreased sharply when I increased. These results are discussed in connection with polarization theories of polyelectrolytes. The molecular dimensions of carboxymethylcellulose, calculated from the birefringence kinetics, suggest that the molecule is a rigid rod.     

Electric and optical anisotropy and their osmotically induced changes of photoreceptor disk membrane vesicles

Electro-optical characterization of the photoreceptor disk membrane vesicle is performed by examining the electric field and concentration dependence of the steady-state birefringence of aqueous suspensions of the vesicles. The electric polarizability anisotropy is found to be negative and of large magnitude: α₁ ?α₂ = ?(1?3) × 10^?10 cm³. The optical anisotropy is determined to be also negative but of small magnitude: g₁ ?g₂ = ?1 × 10^?7. The specific Kerr constant deduced from the concentration dependence of the Kerr constant is found to be very large: K_sp = 7 × 10^?4 c.s.u. Upon deforming the vesicles osmotically from the spherical shell to the disk structure, the steady-state birefringence increases by an order of magnitude which is attributed solely to the increase in optical anisotropy attending the corresponding change in the geometric eccentricity of the vesicle. A plausible birefringence mechanism based on the known structural features of the vesicles is proposed, which would account for these findings.     

Stretching and overstretching of DNA in pulsed field gel electrophoresis. I. A quantitative study from the steady state birefringence decay

Using a sensitive birefringence instrument, the birefringence arising from the orientation of the DNA chain during electrophoretic transport has been recorded. This birefringence is shown to proceed both from the alignment (stretching) of the molecule in the direction of the electric field and from the extension of the length of its primitive path (overstretching). The contribution of these two processes can be separated in the decay of the birefringence after the end of the application of the electric field. The fast relaxation of the overstretching occurs first and is demonstrated to be the main contribution to the birefringence. The orientation factor of the remaining stretched state and its decay can be quantitatively understood using the biased reptation model. It provides, in addition, a high value for the tube diameter or gel pore size a (4500 ± 450 Å for a 0.7% agarose gel with a c^?0.6_g dependence in the agarose concentration c_g) and a low value for the effective charge per base pair (0.2e as compared to 0.5e using the condensation hypothesis). The contribution of overstretching to the birefringence is also quantitatively interpreted in term of the change in the mean length l of DNA inside a pore size a. The dynamics of decay of this overstretching is well represented by a stretched exponential with a stretching exponent α = 0.44. The mean decay time decreases slightly with increasing fields and scales with the overall DNA length close to N²₀. © 1993 John Wiley & Sons, Inc.     

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.     

Reversing-pulse electric birefringence of poly(γ-benzyl-L-glutamate). III. The temperature dependence of the transient and steady-state behavior in cyclohexanone

The reversing-pulse electric birefringence (RPEB) technique was applied to the study of the temperature effect on the electrooptical and hydrodynamic properties of a fractionated [Glu(OBzl)]_n sample, which is molecularly dissolved in cyclohexanone. The aim was to develop a standard analytical method for thermal denaturation and temperature-induced conformational transitions. The field-on reverse and steady-state signal, and the field-off decay signal, were measured at 535 nm and at a constant low field strength (ca. 3 kV/cm) over a wide temperature range (5–90°C). The steady-state birefringence and the relaxation time in the decay process were also measured at two constant temperatures (5 and 70°C) over a wide field strength range (E ≤ 20 kV/cm). By the combination of these two different sets of RPEB measurements, the unwanted effect of the high pulse field on polymer conformation at elevated temperatures could be minimized. Together with the density and viscosity of cyclohexanone measured between 5 and 95°C, the following quantities could be evaluated: the weight-average permanent dipole moment and polarizability anisotropy, the reduced optical anisotropy factor (Δg/n), the weight-average length, and the degree of polydispersity. All these quantities, except for Δg/n, were found to be almost independent of temperature (5–90°C) and concentration (1.54–4.27 mM).     

Kinetic comparisons of mesophilic and thermophilic aerobic biomass

Kinetic parameters describing growth and decay of mesophilic (30°C) and thermophilic (55°C) aerobic biomass were determined in continuous and batch experiments by using oxygen uptake rate measurements. Biomass was cultivated on a single soluble substrate (acetate) in a mineral medium. The intrinsic maximum growth rate (μ _max) at 55°C was 0.71±0.09 h⁻¹, which is 1.5 times higher than the μ _max at 30°C (0.48±0.11 h⁻¹). The biomass decay rates increased from 0.004 h⁻¹ at 30°C to 0.017 h⁻¹ at 55°C. Monod constants were very low for both types of biomass: 9±2 mg chemical oxygen demand (COD) l⁻¹at 30°C and 3±2 mg COD l⁻¹at 55°C. Theoretical biomass yields were similar at 30 and 55°C: 0.5 g biomass COD (g acetate COD)⁻¹. The observed biomass yields decreased under both temperature conditions as a function of the cell residence time. Under thermophilic conditions, this effect was more pronounced due to the higher decay rates, resulting in lower biomass production at 55°C compared to 30°C. Electronic Publication     

Orientational interactions in low-concentration DNA solutions

The rotational relaxation tiem τ₃ of DNA molecules (M_w ? 5 × 10⁶) in solution has been determined by the transient electric birefringence method. The analysis of the birefringence decay makes it possible to study only the higher-molecular-weight fraction, the molecules being considered as rigid elongated particles in a short time scale. A marked concentration dependence of the relaxation time has been observed for DNA in low ionic strengths. Above a critical concentration c*, τ₃ increases with the DNA concentration, c. The value of c* increases with the ionic strength. For 10^?3 ionic strength (with NaCl), c* is about 10 μg/mL; then we observe the same strong concentration dependence of rotational relaxation times as recently reported for rodlike M-13 viruses [Maguire, J. F., McTague, J. P. & Rondelez, F. (1980) Phys. Rev. Lett. 45 , 1891–1894]. These results may be discussed in terms of the Doi-Edwards theory for rotational relaxation time of rigid macromolecules [Doi, M. (1975) J. Phys. 36 , 607–611; Doi, M. & Edwards, S. F. (1978) J. Chem. Soc. Faraday Trans. 74 , 918–932] and the critical concentration above which the interactions between the molecules begin to appear allows determining the corresponding molecular length. We observe a very good agreement between the DNA lengths obtained from the c* values and by using the infinite dilution value of τ₃ and Broersma's equation. Therefore, only highly diluted solutions can be used if intrinsic molecular properties based on the rotational diffusion of high-molecular-weight elongated molecules are studied.     

Cold-adapted Features of Arginine Kinase from the Deep-sea Clam <Emphasis Type="Italic">Calyptogena kaikoi</Emphasis>

The heterodont clam Calyptogena kaikoi, which inhabits depths exceeding 3,500 m where low ambient temperatures prevail, has an unusual two-domain arginine kinase (AK) with molecular mass of 80 kDa, twice that of typical AKs. The purpose of this work is to investigate the nature of the adaptations of this AK for functioning at low temperatures. Recombinant C. kaikoi AK constructs were expressed, and their two-substrate kinetic constants (k _cat, K _a, and K _ia) were determined at 10°C and 25°C, respectively. When measured at 25°C, the K _ia values were tenfold larger than those for corresponding K _a values, while at 10°C, the K _ia values decreased remarkably, but the K _a values were almost unchanged. The Calyptogena two-domain enzyme has threefold higher catalytic efficiency, calculated by k _cat/(K _a^ARG·K _ia^ATP), at 10°C, than that at 25°C, reflecting adaptation for function at reduced ambient temperatures. The activation energy (E _a) and thermodynamic parameters were determined for Calyptogena two-domain enzyme and compared with those of two-domain enzymes from mesophilic Corbicula and Anthopleura. The value for E _a of Calyptogena enzyme were about half of those for mesophilic enzymes, and a larger decrease in entropy was observed in Calyptogena AK reaction. Although large decrease in entropy increases the ΔG ^o‡ value and consequently lowers the k _cat value, this is compensated with its lower E _a value thereby minimizing the reduction in its k _cat value. These thermodynamic properties, together with the kinetic ones, are also present in the separated domain 2 of the Calyptogena two-domain enzyme.     

Binding of the Bovine and Porcine Pancreatic Secretory Trypsin Inhibitor (Kazal) To Human Leukocyte Elastase: A Thermodynamic Study

Abstract

The effect of pH and temperature on the apparent association equilibrium constant (K_a) for the binding of the bovine and porcine pancreatic secretory trypsin inhibitor (Kazal-type inhibitor, PSTI) to human leukocyte elastase has been investigated. At pH8.0, values of the apparent thermodynamic parameters for human leukocyte elastase: Kazal-type inhibitor complex formation are: bovine PSTT – K_a = 6.3 × 10⁴M^?1, δ5G° = -26.9kJ/mol, δH° = +11.7kJ/mol, and δS° = +1.3 × 10² entropy units; porcine PSTI –K_a = 7.0 × 10³M^?1,δG° = -21.5kJ/mol, δH° = +13.0kJ/mol, and δS° = +1.2 × 10² entropy units (values of K_a δG° and δS° were obtained at 21.0°C; values of δH° were temperature independent over the range (between 5.0°C and 45.0°C) explored). On increasing the pH from 4.5 to 9.5, values of K_a for bovine and porcine PSTI binding to human leukocyte elastase increase thus reflecting the acidic pK-shift of the His57 catalytic residue from ?7.0, in the free enzyme, to ?5.1, in the serine proteinase: inhibitor complexes. Thermodynamics of bovine and porcine PSTI binding to human leukocyte elastase has been analyzed in parallel with that of related serine (pro)enzyme/Kazal-type inhibitor systems. Considering the known molecular models, the observed binding behaviour of bovine and porcine PSTI to human leukocyte elastase was related to the inferred stereochemistry of the serine proteinase/inhibitor contact region(s).     

Transient electric birefringence of T-even bacteriophages. II. T4B in the presence of tryptophan and T4D.

Measurements of electric birefringence, sedimentation velocity, and biological adsorption rate are used to study the properties of bacteriophage T4B in the presence of excess tryptophan. The adsorption rate determined in borate buffer pH 9 (at 25°C) increases from 0.003 × 10^?8 ml min^?1 (0.025 M) to 0.130 × 10^?8 ml min^?1 (0.150 M). The Kerr coefficient, rotational diffusion coefficient, and the sedimentation coefficient of the phage are also dependent on buffer concentration and reach plateau values above 0.12 M given by K_sp = ?(275 ± 18) × 10^?9 OD^?1 cm² statvolt^?2, D_25,w = 133 ± 4 sec^?1, and s_20,w = 818 ± 11 S. From a comparison of electric birefringence measurements of T4B and T4D it is concluded that T4D and T4B (in the presence of excess tryptophan) exhibit a similar hydrodynamic behavior. The change in physical parameters is solely due to a shift in fiber configuration. At high buffer concentrations the fibers make an angle of approximately 3π/4 with the sheath and the permanent dipole moment is about 200,000 D. This dipole moment is roughly ten times as large as that of a phage particle with nonextended fibers. This difference may be due to a change in hydrodynamic center upon fiber extension or to the presence of positive charges on the fiber tips, or both. At intermediate buffer concentrations the phage population behaves as if it were monodisperse. Probably not all six fibers are extended under such conditions.     

Reversible phosphorylation regulation of NADPH-linked polyol dehydrogenase in the freeze-avoiding gall moth, Epiblema scudderiana: role in glycerol metabolism

Larvae of the goldenrod gall moth, Epiblema scudderiana, use a freeze avoidance strategy of cold hardiness to survive the winter. A key metabolic adaption that supports subzero survival is the accumulation of large amounts of glycerol as a colligative antifreeze. Production of glycerol relies on polyol dehydrogenase (PDH) which catalyzes the NADPH‐dependent conversion of glyceraldehyde into glycerol. Kinetic analysis of PDH from E. scudderiana revealed significant changes in properties as a result of subzero temperature acclimation; the K_m for glyceraldehyde in 5°C‐acclimated larvae was 7.0 mM and doubled in ? 15°C‐exposed larvae. This change suggested that PDH is regulated by a state‐dependent covalent modification. Indeed, high and low K_m forms could be interconverted by incubating larval extracts in vitro under conditions that stimulated either endogenous protein kinases or protein phosphatases. Protein kinase incubations doubled the K_m glyceraldehyde of the 5°C enzyme, whereas protein phosphatase incubations decreased the K_m of the ? 15°C enzyme by about 50%. PDH was purified by ion exchange and affinity chromatography steps and then subjected to electrophoresis. Staining with ProQ Diamond phosphoprotein stain showed a much higher phosphate content of PDH from ? 15°C‐acclimated larvae, a result that was further confirmed by immunoblotting that showed a much greater phosphoserine content on the ? 15°C enzyme. These experiments established that PDH is regulated by state‐dependent reversible phosphorylation in E. scudderiana and suggest that this regulatory mechanism makes a significant contribution to controlling the synthesis, maintenance, and degradation of glycerol pools over the winter months. © 2011 Wiley Periodicals, Inc.     

Purification and characterization of carbonic anhydrase from bovine stomach and effects of some known inhibitors on enzyme activity

Carbonic anhydrase (CA) was purified from four different cell localisation (outer peripheral, cytosolic, inner peripheral and integral) in bovine stomach using affinity chromatography with Sepharose-4B-l-tyrosine sulphanilamide. During the purification steps, the activity of the enzyme was measured using p-nitrophenyl acetate at pH 7.4. Optimum pH and optimum temperature values for all CA samples were determined, and their K_m and V_max values for the same substrate by Lineweaver–Burk graphics. The extent of purification for all CA localizations was controlled by SDS-PAGE. The K_m values at optimum pH and 20°C were 0.625?mM, 0.541?mM, 0.785?mM and 0.862?mM with p-nitro phenyl acetate, for all CA localizations. The respective V_max values at optimum pH and 20°C were 0.875?μmol/L?min, 0.186?μmol/L?min, 0.214?μmol/L?min and 0.253?μmol/L?min with the same substrate. The K_i and I₅₀ values for the inhibitors sulphanilamide, KSCN, NaN₃ and acetazolamide were determined for all the CA localizations.     

Energy budget for the larval development ofElminius modestus (Crustacea: Cirripedia)

Biomass (CHN), respiration rate and food uptake were estimated for the larval development ofElminius modestus at three temperatures (12, 18, 24°C). Mean values of dry weight, elemental composition and energy equivalents increased exponentially with the development from nauplius II to VI. Dry weight, elemental composition and energy content exhibited the highest values at 18°C. Respiration rates increased with the larval stages expressed by a power function, but increased logarithmically with the dry weight of the larvae. The cypris larvae showed a reduced respiration rate compared with nauplius VI. The ingestion rate was measured at a concentration of 100 cells ofSkeletonema costatum μl⁻¹. At 12 and 18°C ingestion rates increased exponentially and at 24°C by a logarithmic function. The fittings were used to estimate the energy budget ofE. modestus during larval development. The energy content of the larvae increased during the development from nauplius II to VI by a factor of 21 at 12°C, 25 at 24°C and 31 at 18°C. The estimated energy content of the freshly metamorphosed barnacle is 100 mJ (12°C), 130 mJ (24°C) and 150 mJ (18°C). The assimilation- (A/I) and gross growth efficiencies (K₁) increased strongly during the development from nauplius II to VI (A/I: 6–14% in nauplius II to 50–90% in nauplius VI; K₁: 4% in nauplius II to 75% in nauplius VI). The net growth efficiency (K₂) showed a relatively constant level ranging between 57 and 83%.     

Kinetics of lipase catalyzed isoamyl acetate synthesis at high hydrostatic pressure in hexane

Lipase-catalyzed synthesis of isoamyl acetate in hexane at 10–250 MPa at 80°C and 1–100 MPa at 40°C resulted in activation volumes of −12.9 ± 1.7 and −21.6 ± 2.9 cm³ mol⁻¹, respectively. Increasing pressure from 10 to 200 MPa resulted in approximately 10-fold increase in V _max at both 40 and 80°C. Pressure increased the K _m from 2.4 ± 0.004 to 38 ± 0.78 mM at 40°C. In contrast, at 80°C the pressure did not affect the K _m.     

Kinetics and characteristics of 70 °C, VFA-grown, UASB granular sludge

We studied in batch reactors the kinetics and characterization of 70 °C, volatile fatty acids (VFAs)-grown, upflow anaerobic sludge blanket granular sludge with 55 and 35 °C sludge as reference. The half-saturation constant (K _s), the inhibition constant (K _i), the maximum specific methane production rate (μ_CH4max), and the inhibition response coefficient (n) of the 70 °C sludge were 6.15 mM, 48.2 mM, 0.132 h⁻¹, and 2.48, respectively, while no inhibition occurred at 55 and 35 °C, where the K _s was 3.67 and 3.82 mM, respectively. At 70 °C, the highest initial specific methanogenic activity (ISMA, 0.311 gCH₄-COD per gram volatile solids per day) on VFAs was about 12–15% lower than that on acetate and three to four times less than the ISMA for the 55 and 35 °C sludge. In the acetate conversion study, residual acetate (79 mg l⁻¹) at 70 °C was three to five times higher than that at 55 and 35 °C. Further, the methane produced as percentage of the acetate consumed at 70 °C (89%) was lower than that at 55 (95%) and 35 °C (97%). At 70 °C, 10% of the ISMA remained after 15 days of starvation as compared to 26% (55 °C) and 92% (35 °C) after 30 days of starvation. Thus, the kinetics of the 70 °C granular sludge seem to differ from those at 55 and 35 °C. Received: 1 February 1999 / Accepted: 20 March 1999     

Modeling of the pH-and the temperature-dependant deviations of the free to total PSA (Prostate Specific Antigen) ratios for clinical predictability of prostate cancer and benign prostate hyperplasia

Both the free and total PSA had been reported to be pH-and temperature-labile. We have introduced two submodels to describe the decay of PSAs. The overall pH-dependent decay model (OPDDM) describes the ratio of free to true scientific total PSA including both the immunoreactive and the nonimmunoreactive. We elucidated four pH-dependent formation constants for the free PSA with hydronium ion [H⁺], the PSA-ACT (α ₁-antichymotrypsin), the PSA-API (α ₁-protease inhibitor), and the nonimmunoreactive PSA-AMG (α ₂-macroglobulin) complexes, respectively, to model the stability of the free to total PSA ratios. Model simulation indicated a highly pH-sensitive behavior of the free to total PSA ratios. While the temperature dependent decay model (TDDM) indicated that kinetically, free PSA revealed the most rapid decay rates due to its low activation energy, total PSA was shown to be relatively more thermally stable. The decay of all PSA species could be more accelerated at temperatures higher than −4°C, while it is kept below −70°C throughout. It is thus recommended to use the preparation fresh (better within 8 h) after venipuncture. And it is advisable that all the results are advised to perform corrections for each determination by calculating back to the data at the original physiological pH prior to clinical diagnostic interpretation.