Ordinate Intercept Research Articles

Metrics of color-difference formula improvement.

Metrics of color-difference formula improvement (i.e., standardized residual sum of squares and Pearson product moment correlation) are shown to convey the same information. Furthermore, each metric has two computational forms that assume different linear data models, specifically, with or without an ordinate intercept. It is essential to choose a computational form that matches the data model. We recommend explicitly declaring whether or not the data have been centered, i.e., by subtracting the mean value from each datum, to match the intercept-free data model. Statistical testing of the metrics assumes independent, normally distributed randomness of residuals from the data model, and homogeneous variance. Procedures consistent with these assumptions include robust statistical tests, homogenizing data transformations, and meta-analysis.

Finite Heterogeneous Rate Constants for the Electrochemical Oxidation of VO2+ at Glassy Carbon Electrodes

The electrochemical oxidation of VO2+ at planar glassy carbon electrodes is investigated via stationary and rotating linear sweep voltammetry as well as via chronoamperometry. It is demonstrated that introducing finite kinetic rate constants into the Butler-Volmer equation captures the experimentally observed concentration dependence of the ordinate intercept in Koutecky-Levich plots, that cannot be explained by using the classical model. This new concept leads to a three-term Koutecky-Levich equation considering mass transport limitations, Butler-Volmer kinetics as well as finite heterogeneous kinetics simultaneously. Based on these findings it is pointed out that stationary linear sweep voltammetry followed by an irreversible Randles-Sevcik analysis is not sufficient for deducing the electrode kinetics of the VO2+-oxidation. In contrast, it is verified experimentally and theoretically that a Tafel analysis will still provide reasonable values of k0 = 1.35 10-5 cm/s and a = 0.38, respectively. Finally, it is shown that introducing the concept of finite heterogeneous kinetics into the theory of stationary linear sweep voltammetry also explains the failure of the irreversible Randles-Sevcik relation leading to an extension of the classical model and providing insights into the electrochemical oxidation reaction of VO2+.

Process inference from topographic fractal characteristics in the tectonically active Northwest Himalaya, India

AbstractTopography evolves under the coupled effect of exogenic and endogenic governing factors, and their scale‐(in)variant dynamics. This results in a self‐affine topography across a finite range, with a characteristic fractal dimension. Fractal analysis has been used to classify geological terrains having distinct litho‐tectonic settings. However, process‐based understanding of the fractal behaviour of a natural landscape is still limited. The current study aims to substantiate and expand upon the present knowledge of topographic response to the complex actions of the governing factors using fractal characteristics. We examined the association between the litho‐tectonic, climatic settings and the fractal characteristics of the topography in the tectonically active Northwest Himalaya. Our analysis was carried out in three separate sectors having diverse litho‐tectonic settings. We used the roughness–length method to calculate the fractal parameters (fractal dimension, D; ordinate intercept, q). The Higher and the Lesser Himalaya were found to be characterized by low D and high q, while the tectonically active Sub Himalaya was found to have moderate D and low q. The southernmost foreland alluvial plains were characterized by high D and low q. Clusters of the fractal parameters were found to be consistent in spatial pattern across the three sectors. Our results showed that the geological–geomorphological settings and the associated processes (e.g. uplift, erosion and diffusion) can be well inferred using the fractal characteristics of the topography. Further, our results implied first‐order control of lithology in sustaining and shaping the topographic geometry (both its amplitude and texture) in the tectonically active Northwest Himalaya. The spatial distribution of the fractal parameters also suggested the secondary control of tectonic uplift and, to a much lesser extent, mean annual rainfall on the topographic geometry. These results collectively point to the role of complex actions of the governing factors in the landscape evolution process. © 2020 John Wiley & Sons, Ltd.

The synthesis of 4,4′-(9,9-dioctyl-9H-fluorene-2,7-diyl)bis(N,N-diphenylaniline) organic semiconductor and use of it as an interlayer on Au/n-Si diode

In the present study, firstly, a 4,4′-(9,9-dioctyl-9H-fluorene-2,7-diyl)bis(N,N-diphenylaniline) (FC) organic compound was synthesized and its structural and optical characterization were carried. Then, the effect on the device of the FC thin film prepared between n-type silicon substrate and gold metal by the spin coating technique was reported. The ideality factor (n), barrier height ( $$ \Phi_{B} $$ ) and series resistance ( $$ R_{s} $$ ) values of the prepared structure from the I-V data have been found at 1.08, 0.78 eV and 240 Ω at room temperature (300 K), respectively. According to the Gaussian distribution of the barrier height obtained from the various temperature ranges (220–380 K), the $$ \Phi_{b0} $$ and A* values from the ordinate intercept and the slope of the modified Richardson curve of $$ \ln \left( {I_{0} /T^{2} } \right) - \left( {q^{2} \sigma_{s}^{2} /2k^{2} T^{2} } \right) $$ versus 1/T plot which has been found to be 0.97 eV and 114 A/cm2K2, respectively. Results indicate that the high barrier height is achieved for the Au/FC/n-Si metal–organic layer-semiconductor diode as compared to the Au/n-Si metal–semiconductor (MS) diode.

Analysis of the substrate inhibition of complete and partial types.

A simple graphical method was described for determining the kinetic parameters of substrate inhibition of complete and partial types. The method consists of plotting experimental data as v/left( {V_{max} - v} right) versus the reciprocals of the substrate concentrations, where Vmax represents the maximal velocity. The reaction rate constant of enzyme–substrate–inhibitor complex (k^{prime } /k) can be calculated from the ordinate intercept of the linear relationship between v/left( {V_{max} - v} right) and the reciprocal of the substrate concentrations at the higher and inhibitory concentrations of the substrate: partial type (k^{prime } /k < 1) of the substrate inhibition gives straight lines intersecting with the ordinate at (k^{prime } /k)/( 1- k^{prime } /k), whereas complete substrate inhibition (k^{prime } = 0) yields straight lines converging on the origin. The K_{i}^{prime } value also can be calculated from the slope by using the k^{prime } /k value determined. Validity of the method was confirmed by analyzing the substrate inhibition of phosphofructokinase II from E. coli. The present method provides a simple way for determining kinetic parameters of the substrate inhibition irrespective of complete and partial types.

Engineering science aspects of the Hall–Petch relation

Hall and Petch had established in the early 1950s a linear inverse square root of grain diame- ter dependence for yielding and cleavage of polycrystalline iron and steel materials, with ordinate intercept stress, σ0, and slope value, k. Petch and colleagues extended the relationship in 1962 to the full stress- strain behavior of a diverse number of metals and alloys. Connection with other mechanical properties such as the hardness, fatigue and strain rate sensitivity properties was demonstrated in 1970. In 1983, Weng incorporated the dependence into a micromechanical analysis of material strength by building onto earlier Taylor-initiated work on multiply-coupled grain deformations. More recently, Armstrong, Weng and col- leagues have applied dislocation and continuum mechanics models of the H-P relationship to predict order-of- magnitude increases in strength properties of nanopolycrystalline materials, especially including description of the strain rate sensitivity dependence on average grain diameter. These topics are assessed from a dislo- cation mechanics viewpoint in the present report that provides H-P connection with the Taylor dislocation density-based theory of strength properties, in σ0e, and with the Griffith brittle fracture theory by way of point- ing to the H-P slope value, ke, being a microstructural stress intensity analogous to the fracture mechanics parameter, K.

Influence of composition and storage conditions on chocolate hardness and heat resistance

The EU Chocolate Directive 2000/36/EC allows the use of the vegetable fats CBEs and CBIs up to a maximum of 5% in chocolate. Manufacturers and users must know how this has an influence on the properties of chocolate. The objective of the work reported here was to find out by systematic investigations, which effect CBEs/CBIs have on the quality parameters, hardness and heat resistance of chocolate. The influence on the hardness was tested also under extreme practical storage conditions. The quality monitoring was performed up to one year. For the determination of the heat resistance the penetrometric method was used in the temperature range 25–32 °C measuring the maximum loading force, occurring during the penetration of a cylindrical probe of 2 mm diameter with 4 mm penetration. The correlation between the average maximum loading force, relevant to the hardness of chocolate, and the temperature can be described by a linear regression at 95% confidence level. Statistical analyses (correlation analysis, residual analysis, Durban-Watson statistic) showed that it is possible to define the heat resistance of solid chocolate in the temperature range of 25–32 °C by the slope and the ordinate intercept of the regression line of the loading force vs. temperature for given parameters (composition, storage, experimental layout, etc.). For the determination of the hardness of the chocolate also the penetrometric method was used to measure the maximum loading force occurring during the penetration of a needle probe with 2 mm deformation. The hardness of the chocolate samples determined with the penetrometric method and statistical analysis (One-Way, Two-Way Analysis of Variance, Dunnett’s comparisons) is significantly dependent on the composition and storage conditions, where the storage conditions are the dominant factor. The results show that the differences in hardness between the chocolate samples with CBE/CBI and those without CBE/CBI, both stored in the cellar (cold storage), are marginal. After one week of storage the sample with CBI has nearly the same hardness as the standard sample with CB, whereas the sample with CBE was slightly softer. The differences are slightly clearer for the northern storage room (moderate temperature) and for the southern room (warm temperature). After a definite storage time the hardness of all samples increased and was in the case of the southern storage room (warm temperature) up to twice as high. The quality monitoring up to one year showed that the reason for this increase in hardness is not a special storage time but the increasing temperatures with the beginning of the warm season and the cyclic change of the temperature during day and night. So an explanation for this unexpected increase in hardness can be a thermocyclic hardening of the chocolate samples under these storage conditions.

Oxygen isotope anomaly not present in water vapor from Alert, Canada

Lin et al. (1) claim to have discovered a positive 17O anomaly in water vapor from Alert, Canada, indicative of a component of stratospheric origin. However, their data (Tables S1−S5) do not support that assertion. Their finding of Δ17O = 76 ± 16 ppm (2 SE) in the Alert samples is relative to a reference fractionation line [designated Chicago local precipitation (CLP)] of much lower precision [slope 0.529 ± 0.003, ordinate axis intercept 70 ± 33 ppm from Vienna Standard Mean Ocean Water (VSMOW) on the logarithmic form of the oxygen triple-isotope plot] than is the norm for investigations that characterize Δ17O values at the parts per million …

Kinetic Characterization of the ATPase and Actin-activated ATPase Activities of Acanthamoeba castellanii Myosin-2

Phosphorylation of Ser-639 in loop-2 of the catalytic motor domain of the heavy chain of Acanthamoeba castellanii myosin-2 and the phosphomimetic mutation S639D have been shown previously to down-regulate the actin-activated ATPase activity of both the full-length myosin and single-headed subfragment-1 (Liu, X., Lee, D. Y., Cai, S., Yu, S., Shu, S., Levine, R. L., and Korn, E. D. (2013) Proc. Natl. Acad. Sci. U.S.A. 110, E23-E32). In the present study we determined the kinetic constants for each step in the myosin and actomyosin ATPase cycles of recombinant wild-type S1 and S1-S639D. The kinetic parameter predominantly affected by the S639D mutation is the actin-activated release of inorganic phosphate from the acto myosin·ADP·Pi complex, which is the rate-limiting step in the steady-state actomyosin ATPase cycle. As consequence of this change, the duty ratio of this conventional myosin decreases. We speculate on the effect of Ser-639 phosphorylation on the processive behavior of myosin-2 filaments.

Fractal properties of landforms in the Ordos Block and surrounding areas, China

This paper investigates the fractal properties of landforms in the Ordos Block and surrounding areas within China and discusses their geological and geomorphological implications. The Ordos Block is tectonically stable, but the surrounding areas are much more active and are affected by shear-extensional structures. We utilized the variogram method and the cellular fractal model to estimate parameters such as the fractal dimension (D), ordinate intercept (γ), and range (R) from the SRTM3 DEM using a moving window operation. We suggest that the fractal dimension reflects the frequency of variation in elevation, while the ordinate intercept reflects the amplitude of relief. The fractal properties range from 90m to 30km. Geomorphological zones can be delineated using the fractal dimension and ordinate intercept. These zones are consistent with known qualitative types of topography. Some basins are characterized by high fractal dimensions and low ordinate intercepts, in contrast to mountainous areas with low fractal dimensions and high ordinate intercepts. Other areas such as the Loess Plateau and deserts also have unique values of fractal properties. The effects of geological and geomorphological processes on the fractal properties are also discussed.

Ileal endogenous amino acid losses: Response of broiler chickens to fiber and mild coccidial vaccine challenge

The effects of mild coccidial vaccine (5× dose) challenge and fiber levels on ileal endogenous amino acid (EAA) losses was determined in 26-d-old broiler chickens using the regression method. Birds were fed semipurified diets containing 3 levels of casein (40, 80, or 120 g/kg of diet) as the only source of dietary protein at 2 levels of fiber (purified cellulose at 25 or 75 g/kg of diet). One half of the experimental birds were gavaged with coccidial vaccine on d 20. The 3 levels of casein were used to determine EAA losses for each replicate cage of birds. The design of the study was a randomized complete block employing a factorial arrangement of treatments with 2 levels of coccidial challenge (control or unchallenged, and challenged) and 2 levels of fiber, with 6 replicate cages per treatment and 8 birds per cage. Birds were killed on d 26 and contents from the entire ileum were flushed with distilled water and stored at -20°C until processed. Ileal EAA losses were determined from the ordinate intercept, at zero amino acid (AA) intake, of the regression of ileal digesta AA concentration in milligrams per kilogram of DM intake against dietary AA intake in milligrams per kilogram of DM. The effect of fiber level on EAA losses, expressed in milligrams per kilogram of DM intake, were higher (P < 0.05) for most of the AA in birds fed 25 g of fiber/kg of diet. Coccidial vaccine challenge decreased (P < 0.05) EAA losses for 8 of the 18 AA. A significant interaction between fiber level and coccidial vaccine challenge was obtained for all AA except His and Lys. Coccidial vaccine challenge resulted in increased (P < 0.05) NaPi-IIb expression. The results suggest that ileal EAA loss is higher in challenged birds fed the diet containing 25 g of fiber/kg of diet. Changes in EAA losses as a result of dietary fiber level and coccidial infection are important in amino acid nutrition of the broiler chicken.

A reappraisal of the Ca2+ dependence of fast inactivation of Ca2+ release in frog skeletal muscle

Two procedures to inhibit Ca(2+) release designed to differentiate between local and common pool mechanisms for the Ca(2+) dependent, fast inactivation of Ca(2+) release in skeletal muscle of the frog were compared. Inhibition by voltage dependent inactivation of Ca(2+) release, without modification of the single channel current of the Ryanodine Receptor (RyR) and the [Ca(2+)] close to the open pore, produced a reduction in the rate of inactivation linearly related to the reduction in the peak of Ca(2+) release flux. Linear fits in the individual fibers were performed, giving average values (+/-SEM, N = 8) of the best fit parameters of 5.75 x 10(-3) +/- 7.35 x 10(-4 )microM(-1) for the slope and 0.07 +/- 0.015 ms(-1) for the ordinate intercept. Inhibition of Ca(2+) release by reducing the Ca content of the sarcoplasmic reticulum (SR) involves reduction of the Ca(2+) current through the single RyR. The reduction in rate of inactivation also followed linearly the reduction in Ca(2+) peak release flux. The average values (+/-SEM) of the best fit parameters of linear fits were 14 x 10(-3) +/- 3.76 x 10(-3 )microM(-1) and 0.019 +/- 0.006 ms(-1) (N = 7) for slope and ordinate intercept respectively. The differences between both parameters were statistically significant (by t test, at P = 0.05). The extent of inactivation, measured by the peak/final Ca(2+) release flux ratio, was differentially affected by the two procedures. Inhibition by voltage dependent inactivation, despite slowing down the fast inactivation, increased the peak/final Ca(2+) release flux ratio. In contrast, depletion of the SR reticulum reduced it. If the fast inactivation is driven by the high [Ca(2+)] attained locally, close to the open pore of the RyR, the inhibition of Ca(2+) release due to voltage dependent inactivation should not modify the rate of inactivation while inhibition by SR Ca(2+) depletion should reduce it. A process driven by [Ca(2+)] in a common pool should depend on the overall Ca(2+) release independently of how it was modified. In this case both inhibitory procedures should reduce the inactivation rate similarly. Our findings are generally consistent with a common pool process. The differences between the two protocols could be understood if the organization of RyR in junctional and parajunctional release units is considered.

Effect of humidity and temperature on polymer electrolyte membrane (Nafion 117) studied by positron annihilation spectroscopy

Polymer electrolyte fuel cells (PEMFC) have emerged as portable power source for future transportation and home based systems. Over the past years perfluorosulfonated cation exchange membranes have been used as solid polymer electrolyte in fuel cells, however, humidity control and water management have remained major issues in the area of stack development. Local drying that results in the changes in the local conductivity behavior and physical failure of the membrane is one of the key issues to be addressed in detail. It is therefore necessary to estimate these changes precisely to evaluate the membrane behavior during the fuel cell operation. Positron annihilation spectroscopy offers a very sensitive probe to the determination of polymer free-volume as well as electronic state of the polymer. In the present study we studied Nafion 117 with varying temperature and humidity using positron annihilation spectroscopy. The ortho-positronium lifetime ( o-Ps, i.e., τ 3) which gives the size of the free-volume holes inside the polymer was determined. It was found that the o-Ps lifetime increases for relative humidity between 40% and 60% and thereafter it decreases in the temperature range of 62–95 °C. When the inversion point at which the o-Ps lifetime is the maximum was plotted with actual vapor pressure, it was observed that it has a logarithmic behavior with the ordinate intercept of 1 ns. This indicates that at higher temperature the membrane has larger free-volume holes; and also at higher temperature a larger vapor pressure is essential to saturate the membrane. Above this point the clusters expand and the free-volume hole size decreases. This also indicates the optimum vapor in the cell to be maintained during the fuel cell operation. The studies also indicated that there is a correlation between o-Ps lifetime and the hydrogen crossover current density under the similar temperature and humidity condition.

Exploring scaling laws in surface topography

Surface topography affects many soil properties and processes, particularly surface water storage and runoff. Application of fractal analysis helps understand the scaling laws inherent in surface topography at a wide range of spatial scales and climatic regimes. In this research, a high resolution digital elevation model with a 3 mm resolution on one side of the spectrum and large scale DEMs, with a 500 m spatial resolution on the other side were used to explore scaling laws in surface topography. With appropriate exploratory spatial data analysis of both types of data sets, two conventional computational procedures – variogram and Box Counting Methods (BCM) – address scaling laws in surface topography. The results respect scaling laws in surface topography to some extent as neither the plot treatment nor the direction treatment has a significant impact on fractal dimension variability. While in the variogram method, the change in slope in Richardson’s plots appears to be the norm rather than the exception; Richardson’s plots resulting from box counting implementation lack such mathematical behavior. These breaks in slope might have useful implications for delineating homogeneous hydrologic units and detecting change in trend in hydrologic time series. Furthermore, it is shown that fractal dimension cannot be used to capture anisotropic variabilities both within and among micro-plots. In addition, its numerical value remains insignificant at the 5% level in moving from one direction to another and also from one spatial scale to another while the ordinate intercept could discriminate the surface roughness variability from one spatial scale to another.

Comment on “Record of δ18O and 17O‐excess in ice from Vostok Antarctica during the last 150,000 years” by Amaelle Landais et al.

Comment on “Record of <i>δ</i>¹⁸O and ¹⁷O‐excess in ice from Vostok Antarctica during the last 150,000 years” by Amaelle Landais et al.

Molecular Basis of the Bohr Effect in Arthropod Hemocyanin

Flash photolysis and K-edge x-ray absorption spectroscopy (XAS) were used to investigate the functional and structural effects of pH on the oxygen affinity of three homologous arthropod hemocyanins (Hcs). Flash photolysis measurements showed that the well-characterized pH dependence of oxygen affinity (Bohr effect) is attributable to changes in the oxygen binding rate constant, k(on), rather than changes in k(off). In parallel, coordination geometry of copper in Hc was evaluated as a function of pH by XAS. It was found that the geometry of copper in the oxygenated protein is unchanged at all pH values investigated, while significant changes were observed for the deoxygenated protein as a function of pH. The interpretation of these changes was based on previously described correlations between spectral lineshape and coordination geometry obtained for model compounds of known structure (Blackburn, N. J., Strange, R. W., Reedijk, J., Volbeda, A., Farooq, A., Karlin, K. D., and Zubieta, J. (1989) Inorg. Chem., 28, 1349-1357). A pH-dependent change in the geometry of cuprous copper in the active site of deoxyHc, from pseudotetrahedral toward trigonal was assigned from the observed intensity dependence of the 1s --> 4p(z) transition in x-ray absorption near edge structure (XANES) spectra. The structural alteration correlated well with increase in oxygen affinity at alkaline pH determined in flash photolysis experiments. These results suggest that the oxygen binding rate in deoxyHc depends on the coordination geometry of Cu(I) and suggest a structural origin for the Bohr effect in arthropod Hcs.

The Homotetrameric Phosphoseryl-tRNA Synthetase from Methanosarcina mazei Exhibits Half-of-the-sites Activity

Synthesis of cysteinyl-tRNA(Cys) in methanogenic archaea proceeds by a two-step pathway in which tRNA(Cys) is first aminoacylated with phosphoserine by phosphoseryl-tRNA synthetase (SepRS). Characterization of SepRS from the mesophile Methanosarcina mazei by gel filtration and nondenaturing mass spectrometry shows that the native enzyme exists as an alpha4 tetramer when expressed at high levels in Escherichia coli. However, active site titrations monitored by ATP/PP(i) burst kinetics, together with analysis of tRNA binding stoichiometry by fluorescence spectroscopy, show that the tetrameric enzyme binds two tRNAs and that only two of the four chemically equivalent subunits catalyze formation of phosphoseryl adenylate. Therefore, the phenomenon of half-of-the-sites activity, previously described for synthesis of 1 mol of tyrosyl adenylate by the dimeric class I tyrosyl-tRNA synthetase, operates as well in this homotetrameric class II tRNA synthetase. Analysis of cognate and noncognate reactions by ATP/PP(i) and aminoacylation kinetics strongly suggests that SepRS is able to discriminate against the noncognate amino acids glutamate, serine, and phosphothreonine without the need for a separate hydrolytic editing site. tRNA(Cys) binding to SepRS also enhances the capacity of the enzyme to discriminate among amino acids, indicating the existence of functional connectivity between the tRNA and amino acid binding sites of the enzyme.

A general algorithm for the 17O abundance correction to 13C/ 12C determinations from CO 2 isotopologue measurements, including CO 2 characterised by ‘mass-independent’ oxygen isotope distributions

It is widely recognised that a significant limitation to the ultimate precision of carbon stable isotope ratio measurements, as obtained from dual-inlet mass spectrometric measurements of CO 2 isotopologue ion abundances at m/ z 44, 45, and 46, is the correction for interference from 17O-bearing molecular ions. Two long-established, alternative procedures for determining the magnitude of this correction are in widespread use (although only one has IAEA approval); their differences lead to small but potentially significant discrepancies in the magnitude of the resulting correction. Furthermore, neither approach was designed to accommodate oxygen three-isotope distributions which do not conform to terrestrial mass-dependent behaviour. Stratospheric CO 2, for example, contains a strongly ‘mass-independent’ oxygen isotope composition. A new strategy for determining the 17O-bearing ion correction is presented, for application where the oxygen three-isotope characteristics of the analyte CO 2 are accurately known (or assigned) in terms of the slope λ of the three-isotope fractionation line and the ordinate axis intercept 10 3 ln(1 + k) on a 10 3 ln(1 + δ 17O) versus 10 3 ln(1 + δ 18O) plot. At the heart of the approach is the relationship between 17R, which is the 17O/ 16O ratio of the sample CO 2, and other assigned or empirically determined parameters needed for the δ 13C evaluation: 2 18 R ref 17 R ( 1 + k ) 17 R ref 1 λ - 3 ( 17 R ) 2 + 2 17 R 45 R - 46 R = 0 With 45R and 46R as the respective 45/44 and 46/44 ion abundance ratios of the sample, as obtained by measurement of δ 45(CO 2) and δ 46(CO 2) values reported relative to a reference material (usually VPDB-CO 2), and 17R ref and 18R ref being the respective 17O/ 16O and 18O/ 16O ratios in the same reference material, 17R can readily be obtained by numerical methods, for given λ and k values. The correction procedure involves no approximations, in principle, and is equally applicable to CO 2 of terrestrial, mass-dependent oxygen isotopic composition, as well as to more ‘exotic’ sources. Besides isotopic characterisation of stratospheric CO 2, potential applications include high precision δ 13C measurements of CO 2 derived from the oxidation of tropospheric CO (also characterised by significantly ‘mass-independent’ oxygen isotopic composition); high precision isotopic monitoring of atmospheric CO 2; the metrology of carbonate isotopic reference materials; and the isotopic characterisation of CO 2 which has been equilibrated with waters artificially ‘labelled’ with known enrichments of 17O and 18O.

Role of Regulatory F-domain in Hepatocyte Nuclear Factor-4α Ligand Specificity

The F-domain of rat HNF-4alpha1 has a crucial impact on the ligand binding affinity, ligand specificity and secondary structure of HNF-4alpha. (i) Fluorescent binding assays indicate that wild-type, full-length HNF-4alpha (amino acids 1-455) has high affinity (Kd=0.06-12 nm) for long chain fatty acyl-CoAs (LCFA-CoA) and low affinity (Kd=58-296 nm) for unesterified long chain fatty acids (LCFAs). LCFA-CoA binding was due to close molecular interaction as shown by fluorescence resonance energy transfer (FRET) from full-length HNF-4alpha tryptophan (FRET donor) to bound cis-parinaroyl-CoA (FRET acceptor), which yielded an intermolecular distance of 33 A, although no FRET to cis-parinaric acid was detected. (ii) Deleting the N-terminal A-D-domains, comprising the AF1 and DNA binding functions, only slightly affected affinities for LCFA-CoAs (Kd=0.9-4 nm) and LCFAs (Kd=93-581 nm). (iii) Further deletion of the F-domain robustly reduced affinities for LCFA-CoA and reversed ligand specificity (i.e. high affinity for LCFAs (Kd=1.5-32 nm) and low affinity for LCFA-CoAs (Kd=54-302 nm)). No FRET from HNF-4alpha-E (amino acids 132-370) tryptophan (FRET donor) to bound cis-parinaroyl-CoA (FRET acceptor) was detected, whereas an intermolecular distance of 28 A was calculated from FRET between HNF-4alpha-E and cis-parinaric acid. (iv) Circular dichroism showed that LCFA-CoA, but not LCFA, altered the secondary structure of HNF-4alpha only when the F-domain was present. (v) cis-Parinaric acid bound to HNF-4alpha with intact F-domain was readily displaceable by S-hexadecyl-CoA, a nonhydrolyzable thioether analogue of LCFA-CoAs. Truncation of the F-domain significantly decreased cis-parinaric acid displacement. Hence, the C-terminal F-domain of HNF-4alpha regulated ligand affinity, ligand specificity, and ligand-induced conformational change of HNF-4alpha. Thus, characteristics of F-domain-truncated mutants may not reflect the properties of full-length HNF-4alpha.

Interaction between the Catalytic Site and the A-M3 Linker Stabilizes E2/E2P Conformational States of Na+,K+-ATPase

The consequences of mutations Ile(265) --> Ala, Thr(267) --> Ala, Gly(271) --> Ala, and Gly(274) --> Ala for the partial reaction steps of the Na(+),K(+)-ATPase transport cycle were analyzed. The mutated residues are part of the long loop ("A-M3 linker") connecting the cytoplasmic A-domain with transmembrane segment M3. It was found that mutation Ile(265) --> Ala displaces the E(1)-E(2) and E(1)P-E(2)P equilibria in favor of E(1)/E(1)P, whereas mutations Thr(267) --> Ala, Gly(271) --> Ala, and Gly(274) --> Ala displace these conformational equilibria in favor of E(2)/E(2)P. The mutations affect both the rearrangement of the cytoplasmic domains (seen by changes in phosphoenzyme properties and apparent ATP/vanadate affinities) and the membrane sector (indicated by change in K(+)/Rb(+) deocclusion rate). Destabilization of E(2)/E(2)P in Ile(265) --> Ala, as well as a direct effect on the intrinsic affinity of the E(2) form for vanadate, may be explained on the basis of the E(2) crystal structures of the Ca(2+)-ATPase, showing interaction of the equivalent isoleucine with conserved residues near the catalytic region of the P-domain. The rate of phosphorylation from ATP was unaffected in Ile(265) --> Ala, indicating a lack of interference with the catalytic function in E(1)/E(1)P. The effects of mutations Thr(267) --> Ala, Gly(271) --> Ala, and Gly(274) --> Ala provide the first evidence in the literature of a relative stabilization of E(2)/E(2)P resulting from perturbation of the A-M3 linker region. These mutations may lead to increased strain of the A-M3 linker in E(1)/E(1)P, increased stability of the A3 helix of the A-M3 linker in E(2)/E(2)P, and/or a change of the orientation of the A3 helix, facilitating its interaction with the P-domain.