Main-chain Dynamics of a Partially Folded Protein:15N NMR Relaxation Measurements of Hen Egg White Lysozyme Denatured in Trifluoroethanol

¹⁵N NMR relaxation measurements have been used to study the dynamic behaviour of the main-chain of hen lysozyme in a partially folded state, formed in a 70% (v/v) trifluoroethanol (TFE)/30% water mixture at 37°C and pH 2. This state is characterised by helical secondary structure in the absence of extensive tertiary interactions. The NMR relaxation data were interpreted by mapping of spectral density functions and by derivation of segmental as well as global order parameters. The results imply that the dynamics of lysozyme in TFE can, at least for the great majority of residues, be adequately described by internal motions which are superimposed on an overall isotropic tumbling of the molecule. Although the dynamic behaviour shows substantial variations along the polypeptide chain, it correlates well with the conformational preferences identified in the TFE state by other NMR parameters. Segments of the polypeptide chain which are part of persistent helical structures are highly restricted in their motion (S²> 0.8, with effective internal correlation times τ_e< 200 ps) but are also found to experience conformational exchange on a millisecond timescale. Regions which are stabilised in less persistent helical structure possess greater flexibility (0.6 <S²< 0.8, 200 ps < τ_e< 1 ns) and those which lack defined conformational preferences are highly flexible (S²< 0.6, τ_e∼1 ns). The dynamic behaviour of the main-chain was found to be correlated with other local features of the polypeptide chain, including hydrophobicity and the position of the disulphide bridges. Despite the absence of extensive tertiary interactions, preferential stabilisation of native-like secondary structure by TFE results in a pattern of main-chain dynamics which is similar to that of the native state.     

Quasi-elastic light scattering from fibrinogen and fibrin intermediate structures

Quasi-elastic light scattering measurements have been carried out for (i) human and bovine fibrinogens under identical conditions, (ii) for a large number of fractionated and unfractionated fibrin intermediates at various pH (10.0 to 10.5), (iii) for three intermediates polymerized at pH 7.4 and stabilized at pH 10.5 and (iv) for a gelled clot. Human and bovine fibrinogen proved to have noticeably different diffusion coefficients (same M_w) indicating a longer rod on the average for the human than for the bovine fibrinogen. This finding is in agreement with measurements of the integrated light scattering from these fibrinogens where a mass per unit length $\text{M}{}_{\mathrm{w}}\text{L}{}_{\mathrm{w}}$ of 3860 and 5460 g mol^?1 nm^?1 were found, respectively. No angular dependence of the apparent diffusion coefficient D_app = Γ/q² was found for the monomers and the fibrin clot; all other samples from (ii) showed an angular dependence if M_w ? 1.76 × 10⁶, were Γ is the first cumulant of the time correlation function and q = (4π/λ) sin?/2. A plot of D_app/D versus q²〈S²〉 gave curves which, for the low molecular weight samples, correspond to ellipsoids. For the longer fibrils a dynamic behaviour in between that of a long rigid rod and random coil was found and indicates a certain flexibility of the fibrils. The diffusion coefficients from (ii) decay with increasing molecular weight and can be described by Perrin's theory for ellipsoids when M₂ < 2 × 10⁶ while a beter agreement with Kirkwood's theory of long rods is obtained for the longer fibrils. The hydrodynamic behaviour of (iii) indicates short and unbranched rods of considerable thickness.     

Anomalies in sedimentation. IV. Decrease in sedimentation coefficients of chains at high fields

A theory is presented for the decrease of sedimentation coefficient at high centrifugal fields recently reported for samples of DNA by Rubenstein and Leighton and others. The theory uses the model of a chain of beads and springs to represent the molecule. Kirkwood's approximation is used for the sedimentation coefficient. The decrease in sedimentation coefficient with field comes about as a result of the uneven frictional forces in the chain, which on the average are less on segments near the center of the chain than on those near the ends. As a result the ends of the chain tend to drag behind the center, and the average intersegment distances are increased; consequently the hydrodynamic shielding of one segment by another is reduced, and the average friction is increased. The effect is thus characteristic of single molecules; intermolecular interaction is not involved. The sedimentation coefficient, S, varies as a power series in a parameter y that measures the distortion produced by the uneven friction: S = S⁰(1?D₂y² + D₄y⁴ ? ·). where S⁰ is the limiting value of S at zero centrifugal field and D₂ and D₄ are constants; y is proportional to the cen speed squared tunes the molecular weight squared divided by S⁰. It has been observed that the effects of centrifuge speed on S are negligible below certain critical values of the speed and molecular weight, but increase dramatically immediately above these values; this follows naturally from the high powers of the speed and molecular weight that appear in the above equation.     

Lyotropic effects of simple anions on the conformation and interactions of kappa-carrageenan

The effect of different co-anions on the formation and aggregation of the ordered structure of the anionic polysaccharide kappa-carrageenan has been investigated by optical rotation, differential scanning calorimetry, and halide-n.m.r. spectroscopy. The mid-point temperature (T_m) of the disorder—order transition increases systematically with the Hofmeister number for the anion through the lyotropic series SO₄^2? < F^? < Cl^? < Br^? < NO₃^? < I^? < SCN^? when salt concentration and cation (Me₄N⁺ or K⁺) are held constant. A corresponding increase is observed in transition enthalpy (ΔH_cal) and entropy (ΔS_cal). Helix—helix aggregation (as indicated by turbidity, gel formation, and hysteresis between heating and cooling scans) also shows a systematic dependence on the Hofmeister number for the anion, but in the opposite sense. Thus, with tetramethylammonium as the sole counterion present, clear solutions with no thermal hysteresis in the order—disorder transition are observed at all temperatures with I^?, Br^?, NO₃^?, or Cl^? as co-anion, whereas weak, turbid gels with significant thermal hysteresis between melting and setting are formed in the presence of SO₄^2?, and to a lesser degree F^?. With K⁺ as counterion, a similar regular progression is observed through the anion lyotropic series from rapid formation of very turbid gels in the presence of F^?, to very slow development of clear gels with I^? or SCN^?. In agreement with previous studies, an increase in ¹²⁷I-n.m.r. linewidth was observed on conformational ordering of kappa-carrageenan (Me₄N⁺ salt form) in the presence of Me₄NI. However, closely similar behaviour was observed for ³⁵Cl and ⁸¹Br, indicating a simple charge-cloud interaction rather than the specific site-binding of I^? which has previously been suggested.     

Characterization of Bacteriophage S13 suN15 Single-Strand and Replicative-Form Deoxyribonucleic Acid

The deoxyribonucleic acid (DNA) of bacteriophage S13 was shown to be single-stranded by the criteria of reactivity with formaldehyde, dependence of optical density on ionic strength, broad temperature-absorbance profile, and lack of molar equivalence of the purine and pyrimidine bases. The DNA has a molecular weight of 1.8 × 10⁶ daltons, an S°₂₀ of 24.6 in SSC (0.15 m NaCl plus 0.015 m sodium citrate), and a buoyant density of 1.726 g/cc in CsCl. Electron microscopy showed the molecule to be circular. S13 replicative-form DNA was shown to be a double-stranded, circular molecule with a molecular weight of 3.5 × 10⁶ daltons, an S_[ill] of 20.7 in SSC, and a buoyant density in CsCl of 1.710 g/cc. The finding that S13 DNA is slightly more pyrimidine-rich than X174 DNA but is indistinguishable by all other parameters supports the close genetic relationship between the two bacteriophages.     

Aggregation of poly(γ-benzyl-α,L-glutamate)

The aggregation of poly(γ-benzyl-α,L -glutamate) and its enantiomer in toluene has been investigated by following the viscosity as a function of temperature, concentration, molecular weight, molecular-weight distribution, helix chirality, and shear rate. The temperature and concentration data for a 138,000-molecular-weight sample was fitted to an open, reversible end-to-end aggregation model. The aggregation numbers resulting from this fit were consistent with the sudden onset in non-Newtonian flow resulting from only a 0.2-wt% increase in concentration. The association equilibrium constant was then used to predict viscosity for comparison with other data, in particular, the effect of molecular weight and molecular-weight distribution. A mixture of right-and left-handed helices showed the aggregation was not chiral selective. The stiffness of end-to-end aggregated (hydrogen-bonded) molecules differed little from their covalent counterparts, at least below a molecular weight of ～10⁶. We conclude that polybenzylglutamate aggregation in toluene can be described by an open end-to-end aggregation model.     

Purification and properties of pyruvate kinase from Mycobacterium smegmatis

Pyruvate kinase (ATP:pyruvate 2-O-phosphotransferase, EC 2.7.1.40) from Mycobacterium smegmatis has been purified to homogeneity through a seven-step procedure with a yield of 16% and specific activity of 220 units/mg protein. The purified enzyme had a molecular weight of 230,700 and was composed of four subunits with identical molecular weights of 57,540. Analysis of amino acid composition revealed a low content of aromatic amino acids. The enzyme exhibited sigmoidal kinetics of varying concentrations of phosphoenolpyruvate, the degree of cooperativity and S_0.5v value for phosphoenolpyruvate being strongly dependent on the pH of the reaction mixture. Among the nucleoside diphosphates acting as substrate for pyruvate kinase, ADP was the best phosphate acceptor, as judged by its lowest K_m value. The enzyme showed an absolute requirement for divalent cations (either Mg²⁺ or Mn²⁺), but monovalent cations were not necessary for activity. Other divalent cations inhibited the Mg²⁺-activated enzyme to varying degrees (Ni²⁺ > Zn²⁺ > Cu²⁺ > Ca²⁺ > Ba²⁺). The differences in the kinetic responses of the enzyme to Mg²⁺ and Mn²⁺ are discussed.     

Solvent Effects on the Shape of a Tetramer

Abstract

The solvent effect on the shape of a tetramer with increasing temperature is analyzed. For this purpose models of an isolated chain and a chain immersed in a solvent have been simulated by Molecular Dynamics. A solvent model represented by stochastic forces has been tested against molecular dynamics results. The behaviour of the mean-square end-to-end distance 〈R ²〉 and 〈l ₁ ³ S ²〉 with increasing temperature are shown. where l ₁ is the longest eigenvalue of the moment of inertia tensor and S is the radius of gyration. All the chain models studied show different behaviour of these quantities at low temperature compared to high temperature where the shape of the tetramer is temperature insensitive. The main solvent effect is to pospone the transition to higher temperature. The stochastic solvent model qualitatively agrees with molecular dynamics results.     

Downstream reaction of cisplatin with methionine-containing peptides: pH-dependent competition between hydrolytic cleavage and macrochelation

The pH- and time-dependent reactions of the antitumor drug cisplatin, cis-[PtCl₂(NH₃)₂], with the methionine-containing peptides Ac-Met-Gly-OH, Ac-Met-Pro-OH, Ac-Met-Pro-Gly-Gly-OH and Ac-Gly-Met-Pro-Gly-Gly-OH (Gly = glycyl, Met = d-methionyl, Pro = L-prolyl) at 313 K have been investigated by high performance liquid chromatography, mass spectrometry and nuclear magnetic resonance. As a result of the strong trans influence of the methionyl S_M atom, initial Pt-S_M binding at pH > 5 is followed by a rapid formation of tridentate machrochelates for the N-acetylated peptides. The site trans to S_M is occupied by a carboxylate O atom in the case of the κ³S_M,N_M,O_G/P macrochelates of the dipeptides and by the C-terminal glycylamide N_G2 atom for the κ³S_M,O_M,N_G2 macrochelate of Ac-Met-Pro-Gly-Gly-OH. Cisplatin simultaneously mediates the rapid hydrolytic cleavage of the Met-X (X = Gly, Pro) amide bond for both dipeptides over the whole range 2.8 ? pH ? 10.0. The released amino acids X react with the resulting κ²S_M, N_M chelate of N-acetylmethionine to afford mixed κS_M:κ²N_x,O_x complexes of the type cis-[Pt(NH₃)(Ac-Met-OH-κS)(H-X-O-κ²N_x,O_x)]⁺ as final products at pH < 5 for X = Gly and pH < 8 for X = Pro. In contrast to the dipeptides, hydrolytic cleavage of the Met-Pro amide bond in Ac-Met-Pro-Gly-Gly-OH at pH > 5 is significantly inhibited by the presence of high concentrations of the macrochelate [Pt(NH₃)(Ac-Met-Pro-Gly-Gly)-κ³S_M,O_M,N_G2]⁺. Downstream hydrolysis of the Met-Gly amide bond is competitive with upstream Ac-Gly cleavage for Ac-Gly-Met-Pro-Gly-Gly-OH at pH < 4.5.     

Alcohol induced structural and dynamic changes in β-lactoglobulin in aqueous solution: A neutron scattering study

Structural and dynamic properties of β-lactoglobulin (β-LG) were revealed as a function of alcohol concentration in ethanol- and trifluoroethanol(TFE)-water mixtures with circular dichroism (CD), small-angle neutron scattering (SANS) and quasi-elastic neutron scattering (QENS). The CD spectra showed that an increase in TFE concentration promotes the formation of the β-sheet structure of β-LG. The SANS-intensities were fitted using form factors for two attached spheres for the native and native-like states of the protein. At higher alcohol concentrations, where aggregation takes place, a form factor modelling diffusion limited colloidal aggregation (DLCA) was employed. The QENS-data were analyzed in terms of internal motions for all alcohol concentrations. While low concentrations of TFE (10% (v/v)) lead to an increase of the mean square amplitudes of vibrations < u²> and a retention of a native-like structure — but not to an increase of the characteristic radius of proton diffusion processes a. Addition of 20% (v/v) of TFE induces aggregation, going along with a further increase of < u²>. Further increase of TFE concentration to 30% (v/v) changes the nanoscale structure of the oligomeric nucleate, but induces no further significant changes in < u²>. The present study underlines the necessity of methods sensitive to the dynamics of a system to obtain a complete picture of a molecular process.     

Transport of ions across peritoneal membrane

The electrical conductance of ions across the peritoneal membrane of young buffalo (approximately 18-24 months old) has been recorded. Aqueous solutions of NaF, NaNO₃, NaCl, Na₂SO₄, KF, KNO₃, KCl, K₂SO₄, MgCl₂, CaCl₂, CrCl₃, MnCl₂, FeCl₃, CoCl₂, and CuCl₂ were used. The conductance values have been found to increase with increase in concentration as well as with temperature (15 to 35 °C) in these cases. The slope of plots of specific conductance, κ, versus concentration exhibits a decrease in its values at relatively higher concentrations compared to those in extremely dilute solutions. Also, such slopes keep on increasing with increase in temperature. In addition, the conductance also attains a maximum limiting value at higher concentrations in the said cases. This may be attributed to a progressive accumulation of ionic species within the membrane. The κ values of electrolytes follow the sequence for the anions: SO₄²⁻>Cl⁻>NO₃⁻>F⁻ while that for the cations: K⁺>Na⁺>Ca²⁺>Mn²⁺>Co²⁺>Cu²⁺>Mg²⁺>Cr³⁺>Fe³⁺. In addition, the diffusion of ions depends upon the charge on the membrane and its porosity. The membrane porosity in relation to the size of the hydrated species diffusing through the membrane appears to determine the above sequence. As the diffusional paths in the membrane become more difficult in aqueous solutions, the mobility of large hydrated ions gets impeded by the membrane framework and the interaction with the fixed charge groups on the membrane matrix. Consequently, the membrane pores reduce the conductance of small ions, which are much hydrated. An increase in conductance with increase in temperature may be due to the state of hydration, which implies that the energy of activation for the ionic transport across the membrane follows the sequence of crystallographic radii of ions accordingly. The Eyring's equation, κ=(RT/Nh)exp[−ΔH*/RT]exp[ΔS*/R], has been found suitable for explaining the temperature dependence of conductance in the said cases. This is apparent from the linear plots of log[κNh/RT] versus 1/T. The results indicate that the permeation of ions through the membrane giving negative values of ΔS* suggest that there may be formation of either covalent linkage between the penetrating ions and the membrane material or else the permeation may not be the rate-determining step. On the one hand, a high ΔS* value associated with the high value of energy of activation, E_a, for diffusion may suggest the existence of either a large zone of activation or loosening of more chain segments of the membrane. On the other hand, low value of ΔS* implies that converse is true in such cases, i.e., either a small zone of activation or no loosening of the membrane structure upon permeation.     

Electrochemical behavior of S,S-bridged adducts of square planar metalladithiolene complexes [M(S2C2Ph2)2](M=Ni, Pd, and Pt)

The electrochemical behavior of the S,S^′-bridged adducts of square planar metalladithiolene complexes was investigated by using cyclic voltammetry and electrochemical spectroscopies (visible, near-IR, and ESR). The norbornene-bridged S,S^′-adduct [Ni(S₂C₂Ph₂)₂(C₇H₈)] (2a; C₇H₈=norbornene) formed by [Ni(S₂C₂Ph₂)₂] (1a) and quadricyclane (Q) was dissociated by an electrochemical reduction, and anion 1a⁻ and norbornadiene (NBD) were formed. Q was isomerized to NBD in the overall reaction. The o-xylyl-bridged S,S^′-adduct [Ni(S₂C₂Ph₂)₂(CH₂)₂(C₆H₄)] (3a; (CH₂)₂(C₆H₄)=o-xylyl) was also dissociated by an electrochemical reduction, and this reaction gave the o-xylyl radical (o-quinodimethane). The reduction of complex 3a in the presence of excess o-xylylene dibromide underwent the catalytic formation of o-quinodimethane. The butylene-bridged S,S^′-adduct [Ni(S₂C₂Ph₂)₂(CH₂)₄] (4a; (CH₂)₄=butylene) was stable on an electrochemical reduction. The lifetimes of reduced species of these adducts 2a-4a were influenced by the stability of the eliminated group (stability: NBD > o-xylyl radical (o-quinodimethane) > butylene radical). Therefore, the reduced species are stable in the sequence 4a⁻ > 3a⁻ > 2a⁻. Although the palladium complex [Pd(S₂C₂Ph₂)₂] (1b) was easier to reduce than the nickel complex 1a or the platinum complex [Pt(S₂C₂Ph₂)₂] (1c), their S,S^′-adducts were easier to reduce in the order of Ni adduct > Pd adduct > Pt adduct.     

Rheological behaviour of high density continuous cultures of Saccharomyces cerevisiae

By culture of Saccharomyces cerevisiae with cell recycle using tangential microfiltration, high cell concentrations are obtained (in the range of 0 to 345 gl⁻¹ dry-weight). The rheological properties of the cell suspension during the cell growth were studied. Over a wide range of biomass concentration (X<275 gl⁻¹D.W.) the power-law model was found adequate to describe the rheological behaviour of the broth. Pronounced non-Newtonian (pseudoplastic) behaviour occurred for X > 75 gl⁻¹. Experimental correlations for apparent viscosity (n_a, mPa.s) and power-law index vs. biomass concentration (X, gl⁻¹) were established: n_a = (1+0.012X)² suitable over the whole range of concentration up to 275 gl⁻¹ D.W. n_a = 1+0.04X in the low concentration range; X<100 gl⁻¹D.W. Beyond the cell concentration of 275 gl⁻¹ D.W. the viscosity increases suddenly.     

Determination of molecular size of O-(2-hydroxyethyl)cellulose (HEC) and its relationship to the mechanism of enzymatic hydrolysis by cellulases

Z-average root mean square end-to-end distance 〈r_o²〉_z^1/2 and radius of gyration 〈s_o²〉_z^1/2 for 13 samples of O-(2-hydroxyethyl)cellulose (HEC) of different molecular weights were derived from Gel Permeation Chromatography and intrinsic viscosity measurements with water as a solvent. At 40 °C and pH 4.5, contraction of chain dimensions is observed, compared with the sizes observed under neutral conditions at room temperature. The effect is lower for samples with higher molecular weights. Values of 〈r_o²〉₄₀/DP_z also indicate that chain flexibility increases at higher temperature and acidic conditions. From the analysis of molecular weight dependence of 〈s_o²〉 _z^1/2, Flory exponent υ was derived at 40 °C and pH 4.5. A value of υ = 0.70 ± 0.02 was recorded, which indicates that a relatively stiff chain is present under these conditions. Finally, different equations to calculate persistence length L_p were evaluated. Values in the range of 260-400 Å were derived for persistence length. Implications of chain conformation in the enzymatic action of cellulases are also discussed.     

Comparative thermodynamics of opioid receptor ligand interaction in the bovine adrenal medulla membranes—Evidence of opioid site heterogeneity

1. A marked dependence on temperature of agonist binding δ, μ and κ₁₋₃, opioid sites in the bovine adrenal medulla was observed, at the range of 0 to 37°C. These changes concern kinetic (k₁) and equilibrium constants (K_d), but not binding capacities (B_max).2. These dependences are different for each ligand and each opioid receptor, suggesting their molecular heterogeneity.3. The comparative thermodynamics indicates that the interaction of opioid agonists with their receptor is exergonic (ΔG° < 0) and entropy driven (ΔS° > 0).4. The comparison of Van't Hoff and Arrhenius plots indicates a discrete mechanism in the binding of each opioid receptor.     

GC–MS analysis of S-nitrosothiols after conversion to S-nitroso-N-acetyl cysteine ethyl ester and in-injector nitrosation of ethyl acetate

S-Nitrosothiols from low-molecular-mass and high-molecular-mass thiols, including glutathione, albumin and hemoglobin, are endogenous potent vasodilators and inhibitors of platelet aggregation. By utilizing the S-transnitrosation reaction and by using the lipophilic (pK_L 0.78) and strong nucleophilic synthetic thiol N-acetyl cysteine ethyl ester (NACET) we have developed a GC–MS method for the analysis of S-nitrosothiols and their ¹⁵N- or ²H–¹⁵N-labelled analogs as S-nitroso-N-acetyl cysteine ethyl ester (SNACET) and S¹⁵NACET or d₃-S¹⁵NACET derivatives, respectively, after their extraction with ethyl acetate. Injection of ethyl acetate solutions of S-nitrosothiols produced two main reaction products, compound X and compound Y, within the injector in dependence on its temperature. Quantification was performed by selected-ion monitoring of m/z 46 (i.e., [NO₂]^?) for SNACET and m/z 47 (i.e., [¹⁵NO₂]^?) for S¹⁵NACET/d₃-S¹⁵NACET for compound X, and m/z 157 for SNACET and m/z 160 for d₃-S¹⁵NACET for compound Y. In this article we describe the development, validation and in vitro and in vivo applications of the method to aqueous buffered solutions, human and rabbit plasma. Given the ester functionality of SNACET/S¹⁵NACET/d₃-S¹⁵NACET, stability studies were performed using metal chelators and esterase inhibitors. The method was found to be suitable for the quantitative determination of various S-nitrosothiols including SNACET externally added to human plasma (0–10 μM). Nitrite contamination in ethyl acetate was found to interfere. Our results suggest that the concentration of endogenous S-nitrosothiols in human plasma does not exceed about 200 nM in total. Oral administration of S¹⁵NACET to rabbits (40–63 μmol/kg body weight) resulted in formation of ALB-S¹⁵NO, [¹⁵N]nitrite and [¹⁵N]nitrate in plasma.     

Segmental conformational disorder and dynamics in the intrinsically disordered protein α-synuclein and its chain length dependence

Conformational ensembles of fully disordered natural polypeptides represent the starting point of protein refolding initiated by transfer to folding conditions. Thus, understanding the transient properties and dimensions of such peptides under folding conditions is a necessary step in the understanding of their subsequent folding behavior. Such ensembles can also undergo alternative folding and form amyloid structures, which are involved in many neurological degenerative diseases. Here, we performed a structural study of this initial state using time-resolved fluorescence resonance energy transfer analysis of a series of eight partially overlapping double-labeled chain segments of the N-terminal and NAC domains of the α-synuclein molecule. The distributions of end-to-end distance and segmental intramolecular diffusion coefficients were simultaneously determined for eight labeled chain segments. We used the coefficient of variation, C_v, as a measure of the conformational heterogeneity (i.e., structural disorder). With the exception of two segments, the C_vs were characteristic of a fully disordered state of the chain. Subtle deviations from this behavior at the segment labeled in the NAC domain and the segment at the N termini reflected subtle conformational bias that might be related to the initiation of transition to amyloid aggregates. The chain length dependence of the mean segmental end-to-end distance followed a power law as predicted by Flory, but the dependence was steeper than previously predicted, probably due to the contribution of the excluded volume effect, which is more dominant for shorter-chain segments. The observed intramolecular diffusion coefficients (< 10 to ∼ 25 ?²/ns) are only an order of magnitude lower than the common diffusion coefficients of low molecular weight probes. This diffusion coefficient increased with chain length, probably due to the cumulative contributions of minor bond rotations along the chain. These results gave us a reference both for characteristics of a natural unfolded polypeptide at the moment of initiation of folding and for detection of possible initiation sites of the amyloid transition.     

Dynamic viscoelastic properties of solutions of shear-degraded deoxyribonucleic acid

Calf thymus and salmon sperm deoxyribouncleie acid were degraded by high-shear stirring to molecular weights M in the range of 1.3–3.2 × 10⁶ and purified by chromatography on methylated bovine serum albumin. Dynamic viscoelastic properties of the fragmented products, in aqueous glycerol solutions in the concentration range of c = 0.003–0.01 g./ml., were investigated with the apparatus of Birnboim and Ferry. At values of the product cM higher than 4 × 10³, the frequency dependence of the components of the complex shear modulus, G′ and G″, displayed a plateau region in which G′ > G″ – ων₁η_S, similar to that observed in concentrated solutions of coiling polymers where it is attributed to an entanglement network (ω is radian frequency, ν₁ volume fraction of solvent, and η₈, solvent viscosity). The width of this plateau region on the logarithmic frequency scale is given by Δ = 3.8 (log cM – 3.56). At lower values of cM, the frequency dependence is intermediate between those predicted by the theory of Zimm for flexible coiled macromolecules and by the theory of Kirkwood and Auer for rods. Fitting to the Zimm theory gives highly discrepant values for molecular weights, while fitting the low-frequency end of the dispersion to the Kirkwood-Auer theory gives reasonable agreement for both molecular weight and rotary diffusion coefficient. It is concluded that the helical fragments appear as nearly rigid rods in their behavior at very low frequencies, but at higher frequencies reveal substantial bending flexibility.     

Further evidence and biological significance for non-random localization of the centromere on mammalian chromosomes

A further quantitative analysis of the localization of the centromere on chromosomes was made using 16,817 individual chromosomes obtained from 723 mammalian species. Centromeric position was expressed quantitatively by the size of short arms as per cent weight (S_w) relative to the X-containing haploid set. When the class interval of S_w was 0·1 instead of the previous 0·2 (Imai, 1975), the frequency distribution of S_w showed an uneven (W-shaped) pattern with two distinct antimodes lying at S_w 0·6 (reconfirmation) and 0·1 (new finding). Two hypotheses, that are not mutually exclusive, are proposed to explain the non-random distribution of centromere position. One is that there are three structurally different short arms consisting of (1) centromere, (2) constitutive heterochromatin (as determined by C-banding), and (3) euchromatin, each arm-type being approximately characterized by the size of short arms (S_w) as S_w < 0·1, 0·1 ? S_w ? 0·6 and S_w > 0·6. The other possibility is concerned with an “orthogenetical” change of chromosome morphologies. When the chromosomes with S_w < 0·1, 0·1 ? S_w ? 0·6 and S_w > 0·6 are denoted as telocentric (T), acrocentric (A), and meta-, submeta- and subtelocentric (M, SM & ST), it was suggested that the chromosome morphologies tend to change orthogenetically (in a statistical sense) from T to M, SM & ST via A-chromosomes by rearrangements such as tandem growth of constitutive heterochromatic, pericentric inversions, and centric fusions.     

The Role of Allosteric Coupling on Thermal Activation of Thermo-TRP Channels

Thermo-transient receptor potential channels display outstanding temperature sensitivity and can be directly gated by low or high temperature, giving rise to cold- and heat-activated currents. These constitute the molecular basis for the detection of changes in ambient temperature by sensory neurons in animals. The mechanism that underlies the temperature sensitivity in thermo-transient receptor potential channels remains unknown, but has been associated with large changes in standard-state enthalpy (ΔH^o) and entropy (ΔS^o) upon channel gating. The magnitude, sign, and temperature dependence of ΔH^o and ΔS^o, the last given by an associated change in heat capacity (ΔC_p), can determine a channel’s temperature sensitivity and whether it is activated by cooling, heating, or both, if ΔC_p makes an important contribution. We show that in the presence of allosteric gating, other parameters, besides ΔH^o and ΔS^o, including the gating equilibrium constant, the strength- and temperature dependence of the coupling between gating and the temperature-sensitive transitions, as well as the ΔH^o/ΔS^o ratio associated with them, can also determine a channel’s temperature-dependent activity, and even give rise to channels that respond to both cooling and heating in a ΔC_p-independent manner.