Conformational dependence of <Superscript>13</Superscript>C shielding and coupling constants for methionine methyl groups

Methionine residues fulfill a broad range of roles in protein function related to conformational plasticity, ligand binding, and sensing/mediating the effects of oxidative stress. A high degree of internal mobility, intrinsic detection sensitivity of the methyl group, and low copy number have made methionine labeling a popular approach for NMR investigation of selectively labeled protein macromolecules. However, selective labeling approaches are subject to more limited information content. In order to optimize the information available from such studies, we have performed DFT calculations on model systems to evaluate the conformational dependence of ³ J _CSCC, ³ J _CSCH, and the isotropic shielding, σ_iso. Results have been compared with experimental data reported in the literature, as well as data obtained on [methyl-¹³C]methionine and on model compounds. These studies indicate that relative to oxygen, the presence of the sulfur atom in the coupling pathway results in a significantly smaller coupling constant, ³ J _CSCC/³ J _COCC ~ 0.7. It is further demonstrated that the ³ J _CSCH coupling constant depends primarily on the subtended CSCH dihedral angle, and secondarily on the CSCC dihedral angle. Comparison of theoretical shielding calculations with the experimental shift range of the methyl group for methionine residues in proteins supports the conclusion that the intra-residue conformationally-dependent shift perturbation is the dominant determinant of δ¹³Cε. Analysis of calmodulin data based on these calculations indicates that several residues adopt non-standard rotamers characterized by very large ~100° χ³ values. The utility of the δ¹³Cε as a basis for estimating the gauche/trans ratio for χ³ is evaluated, and physical and technical factors that limit the accuracy of both the NMR and crystallographic analyses are discussed.     

Stereospecific assignments of 1H-nmr methyl lines and conformation of valyl residues in the lac repressor headpiece

Two-dimensional ¹H-nmr methods are described to obtain information on the sidechain conformation of valyl residues of the lac repressor headpiece and to assign the resonances of their methyl groups stereospecifically. The spin–spin coupling constants (J_αβ) between C^αand C^β protons are obtained from two-dimensional correlated spectroscopy experiments. Large values for J_αβ(10–12 Hz) corresponding to trans orientations for these protons (g⁺ conformation) are found for all valyl residues in α-helical segments. For these valyl residues, the distance between one methyl group (γ₁)and the valyl amide proton is much shorter than for the other methyl group, so that stereospecific resonance assignments follow from relative intensities of the corresponding cross peaks in a two-dimensional nuclear Overhauser enhancement spectrum. Thus, streospecific assignments could be made for the methyl groups of Val 9, 20, 23, and 38 (of a total of eight valyl residues).     

Kinetic and thermodynamic study of the substituent effect on the amino-Claisen rearrangement of para-substituted N-allyl-N-arylamine: a Hammett study via DFT

In order to find the susceptibility of the amino-Claisen rearrangement and the next proton shift reaction of N-allyl-N-arylamine to the substituent effects in the para position, the kinetic and thermodynamic parameters were calculated at the B3LYP level using the 6-31G^** basis set. The calculated activation energies for the rearrangements and proton shift reactions are close to 44.4 and 49.5 kcal mol^? 1, respectively. The transition states of the rearrangement with electron-donor substituents are more stable than those with electron-withdrawing substituent groups, but for the proton shift reaction, this situation is reversed (with the exception of fluorine atom for the rearrangement and fluorine and chlorine atoms for the proton shift reaction). Negative values for the activation entropy confirm the concerted mechanism for the amino-Claisen rearrangement and proton shift reaction. The Hammett ρ values of ? 2.4172 and ? 1.7791 are obtained for σ_p and σ^?  (enhanced sigma) in the amino-Claisen rearrangement, respectively. The correlation between log(k _X/k _H) and σ_p is weaker than that with σ^?  (enhanced sigma). A negative Hammett ρ value indicates that the electron-donating groups slightly increase the rate of amino-Claisen rearrangement. A positive Hammett ρ value (2.4921) for the proton shift reaction indicates that electron-withdrawing groups increase the rate of reaction.     

C, N CP MAS and high resolution multinuclear NMR study of methyl

Abstract

The fluorinated nucleoside dimers with a 1,2,3-triazole linkage are novel compounds within the field of bioorganic chemistry. We report on the synthesis and properties of two groups of nucleoside dimers analogs possessing a different arrangement of the 1,4-disubstituted 1,2,3-triazole linkage. Based on analysis of the ³J_HH, ³J_H1′C2, and ³J_H1′C6 we estimated conformational preferences of sugar part and orientation around glycosidic bond. These compounds show moderate anticancer activity, with cytostatic studies in three different cancer cell lines.     

Nucleoside dimers analogues with a 1,2,3-triazole linkage: conjugation of floxuridine and thymidine provides novel tools for cancer treatment. Part II

Abstract

The fluorinated nucleoside dimers with a 1,2,3-triazole linkage are novel compounds within the field of bioorganic chemistry. We report on the synthesis and properties of two groups of nucleoside dimers analogs possessing a different arrangement of the 1,4-disubstituted 1,2,3-triazole linkage. Based on analysis of the ³J_HH, ³J_H1′C2, and ³J_H1′C6 we estimated conformational preferences of sugar part and orientation around glycosidic bond. These compounds show moderate anticancer activity, with cytostatic studies in three different cancer cell lines.     

Phosphate accumulation and the occurrence of polyphosphates and cyclic 2,3-diphosphoglycerate in Methanosarcina frisia

Methanosarcina frisia accumulates phosphate up to 14% of its dry weight. The phosphate is stored as long-chain polyphosphates as shown by ³¹P-NMR investigations. Further results show that the accumulation of phosphates is substrate-dependent. In the presence of H₂ and CO₂ as the only carbon and energy source 180 mg of PO _inf4 ^sup3- /g protein were accumulated, whereas 260 mg PO _inf4 ^sup3- /g protein were accumulated in the presence of methanol. This is far more than necessary for the maintenance of essential metabolic pathways. In addition, the ³¹P-NMR studies show the occurrence of cyclic 2,3-diphosphoglycerate in Methanosarcina frisia. The role of the phosphate metabolites in cell metabolism are discussed.Abbreviations M. Methanosarcina - CCP cyclic 2,3-diphosphoglycerate     

NMR study of the structure of short-chain peptides in solution.

The electron-mediated spin–spin coupling constant J between the amide NH and the α-CH protons in the dipeptide fragment C^α? CO(NH? C^αH)R? C′ONH? C^α is dependent on the dihedral angle of rotation (Φ) around the N? C bond. Measurement of J in a series of zwitterionic dipeptides H₃N⁺? CHR₁? CONH? CHR₂? CO₂^? (which is conformationally similar to the dipeptide fragment) in TFA solution shows that J is independent of R₁, but dependent on the steric bulk of R₂. The data are interpreted in terms of a model that assumes that what we measure is an average value of J? a thermal average over all the possible rotamers. The groups R₁ and R₂ are, in most cases, sterically kept apart by the trans and planar amide bonds, and hence the independence of J of R₁. This model is consistent with the theoretical calculations done on the dipeptide fragment. The effect of the structural characteristics of the side chains (e.g., the effect of lengthening and branching the side chains) on the J values in dipeptides is discussed in the light of the existing results of theoretical calculations. Study of 〈J〉 values in tripeptides (C₆H₅CH₂OCONH? CHR₁? CONH? CHR₂? CO₂CH₃, essentially three linked peptide units) shows that electrostatic interaction between the two amide bonds modifies the potential energy surface and the 〈J〉 value of a dipeptide subunit in the tripeptides. Also in some cases, direct steric interaction between the two side chains in the two adjacent dipeptide subunits in the tripeptide affects the potential energy surfaces of the individual dipeptide subunits and hence the 〈J〉 values. The influence of the structural characteristics of the side chains of individual amino acids on structure formation at or beyond the dipeptide level is discussed at various points. The J(NH? ^αCH) values of CH₃CONH? CHR? CONH₂ and CH₃CONH? CHR? CO₂CH₃ with the same R are quite different for R = valine, leucine, phenylalanine, methionine, but equal for R = glycine. This, coupled with the fact that one of the carboxamide NH resonances has a chemical shift different from its counterpart in simple amides like CH₃CONH₂ and the other carboxamide NH has the same chemical shift as its counterpart in CH₃CONH₂, suggest the presence of a hydrogen bond in dipeptide CH₃CONH? CHR? CONH₂ with carboxamide NH as the donor. Theoretical evidence for two seven-membered hydrogen-bonded rings with the carboxamide NH as donor and the acetyl oxygen as acceptor is summarized. Our data cannot suggest the number of such hydrogen-bonded rings, nor can they conclude the relative proportion of these rings in a particular dipeptide. A discussion of the difficulty of interpretation is presented and the data are discussed under certain simplifying assumptions.     

An NMR investigation of putative interresidue H-bonding in methyl α-cellobioside in solution

Methyl α-cellobioside (methyl β-d-glucopyranosyl-(1→4)-α-d-glucopyranoside) was labeled with ¹³C at C4′ for use in NMR studies in DMSO-d₆ solvent to attempt the detection of a trans-H-bond J-coupling (^3hJ_CCOH) between C4′ and OH3. Analysis of the OH3 signal at 600 MHz revealed only the presence of two homonuclear J-couplings: ³J_H3,OH3 and a smaller, longer range J_HH. No evidence for ^3hJ_C4′,OH3 was found. The longer range J_HH was traced to ⁴J_H4,OH3 based on 2D ¹H–¹H COSY data and inspection of the H2 and H4 signal lineshapes. A limited set of DFT calculations was performed on a methyl cellobioside mimic to evaluate the structural dependencies of ⁴J_H2,O3H and ⁴J_H4,O3H on the H3–C3–O3–H torsion angle. Computed couplings range from about −0.7 to about +1.1 Hz, with maximal values observed when the C–H and O–H bonds are roughly diaxial.     

One-bond and two-bond J couplings help annotate protein secondary-structure motifs: J-coupling indexing applied to human endoplasmic reticulum protein ERp18

NMR coupling constants, both direct one‐bond (¹J) and geminal two‐bond (²J), are employed to analyze the protein secondary structure of human oxidized ERp18. Coupling constants collected and evaluated for the 18 kDa protein comprise 1268 values of ¹J_CαHα, ¹J_CαCβ, ¹J_CαC′, ¹J_C′N′, ¹J_N′Cα, ¹J_N′HN, ²J_CαN′, ²J_HNCα, ²J_C′HN, and ²J_HαC′. Comparison with ¹J and ²J data from reference proteins and pattern analysis on a per‐residue basis permitted main‐chain φ,ψ torsion‐angle combinations of many of the 149 amino‐acid residues in ERp18 to be narrowed to particular secondary‐structure motifs. J‐coupling indexing is here being developed on statistical criteria and used to devise a ternary grid for interpreting patterns of relative values of J. To account for the influence of the varying substituent pattern in different amino‐acid sidechains, a table of residue‐type specific threshold values was compiled for discriminating small, medium, and large categories of J. For the 15‐residue insertion that distinguishes the ERp18 fold from that of thioredoxin, the J‐coupling data hint at a succession of five isolated Type‐I β turns at progressively shorter sequence intervals, in agreement with the crystal structure. Proteins 2011. © 2010 Wiley‐Liss, Inc.     

13C-NMR Studies on Halomethylvinyl Chrysanthemic Acids and Esters-Spin-lattice Relaxation Time and 13C-1H Coupling Constants

New types of stable chrysanthemic acids and esters were synthesized, and their ¹³C-NMR were examined and fully analyzed. The configurations of the cyclopropanecarboxylic acid and halomethylvinyl group were reflected on the spin-lattice relaxation time of the substituted methyl carbon involved in their structure. The long-range spin-spin coupling constants (³J_CH) correlated well to the NOE and T₁ measurements, which can generally be used to distinguish the geometry of the substituted double bond.     

Xylem and cell turgor pressure probe measurements in intact roots of glycophytes: transpiration induces a change in the radial and cellular reflection coefficients

Xylem probe measurements in the roots of intact plants of wheat and barley revealed that the xylem pressure decreased rapidly when the roots were subjected to osmotic stress (NaCl or sucrose). The magnitude of the xylem pressure response and, in turn, that of the radial reflection coefficients (σ_r) depended on the transpiration rate. Under very low transpiration conditions (darkness and high relative humidity), σ_r assumed values of the order of about 0·2–0·4. The σ_r values of excised roots were also found to be rather low, in agreement with data obtained using the root pressure probe of Steudle. For transpiring plants (light intensities at least 10 μmol m^?2 s^?1; relative humidity 20–40%) the response was nearly 1:1, corresponding to radial reflection coefficients of σ_r= 1. Further increase of the light intensity to about 400 μmol m^?2 s^?1 resulted in a slight but significant decrease of the σ_r values to about 0·8. Similar measurements on maize roots confirmed our previous results (Zhu et al. 1995, Plant, Cell and Environment 18, 906–912) that, in intact transpiring plants at low light intensities of about 10 μmol m^?2 s^?1 and at relative humidities of 20–40% as well as in excised roots, the xylem pressure response was much less than expected from the external osmotic pressure (σ_r values 0·3–0·5). In contrast to wheat and barley, very high light intensities (about 700 μmol m^?2 s^?1) were needed to shift the radial reflection coefficients of maize roots to values of about 0·9. Osmotically induced xylem pressure changes were apparently linked to changes in turgor pressure in the root cortical parenchyma cells, as shown by simultaneous measurements of xylem and cell turgor pressure. In analogy to the σ_r values of the respective glycophytes, the σ_c values of the root cortical cells of wheat and barley were close to unity, whereas σ_c for maize was significantly smaller (about 0·7) under laboratory conditions. When the light intensity was increased up to about 700 μmol m^?2 s^?1 the cellular reflection coefficient of maize roots increased to about 0·95. In contrast to the σ_r values, the σ_c values of the three species investigated remained almost unchanged when the leaves were exposed to darkness and humidified air or when the roots were cut. The transpiration-dependent (species-specific) pattern of the cellular and radial reflection coefficients of the root compartment of the three glycophytes apparently resulted from (flow-dependent) concentration-polarization and sweep-away effects in the roots of intact plants. The data could be explained straightforwardly terms of theoretical considerations outlined previously by Dainty (1985, Acta Horticulturae 171, 21–31). The far-reaching consequences of this finding for root pressure probe measurements on excised roots, for the occurrence of pressure gradients under transpiring conditions, and for the non-linear flow-force relationships in roots found by other investigators are discussed.     

Influence of extracellular Cl concentration on Cl transport across isolated skin ofRana Pipiens

Summary The effect of changes in Cl concentration in the external and/or serosal bath on Cl transport across short-circuited frog skin was studied by measurements of transepithelial Cl influx (J ₁₃ ^Cl ) and efflux (J ₃₁ ^Cl ), short-circuit current, transepithelial potential, and conductance (G _m).J ₁₃ ^Cl as well asJ ₃₁ ^Cl were found to have a saturating component and a component which is apparently linear with Cl concentration. The linear component ofJ ₃₁ ^Cl appears only upon addition of Cl to external medium, and about 3/4 of this component does not contribute toG _m. The saturating component ofJ ₃₁ ^Cl is only 5% of totalJ ₃₁ ^Cl with 115mm Cl in the serosal medium. Replacement of 115mm Cl^– in external medium by SO ₄ ⁼ , NO ₃ ^– , HCO ₃ ^– or I^– results in 87–97% reduction ofJ ₃₁ ^Cl , whereas replacement with Br^– has no effect. As external Cl concentration is raised in steps from 2 to 115mm,J ₁₃ ^Cl andJ ₃₁ ^Cl increase by the same amount butJ ₁₃ ^Cl is persistently 0.15 eq/cm² hr larger thanJ ₃₁ ^Cl . These results indicate that at least 3/4 of linear components ofJ ₁₃ ^Cl andJ ₃₁ ^Cl proceed via an exchange diffusion mechanism which seems to be located at the outer cell border. The saturating component ofJ ₁₃ ^Cl is involved in active Cl transport in an inward direction, and there is evidence suggesting that Cl uptake across outer cell border, which proceeds against an electrochemical gradient, is electroneutral but not directly linked to Na.Reprinted from The Journal of Membrane Biology, Vol. 54, No. 3, pages 191–202. Our apologies for deleting the author's names on the original version.     

A cohesion/tension model for the gating of aquaporins allows estimation of water channel pore volumes in Chara

The water permeability (hydraulic conductivity; Lp) of turgid, intact internodes of Chara corallina decreased exponentially as the concentration of osmolytes applied in the medium increased. Membranes were permeable to osmolytes and therefore they could be applied on both sides of the plasma membrane at concentrations of up to 2.0 m (5.0 MPa of osmotic pressure). Organic solutes of different molecular size (molecular weight, MW) and reflection coefficients (σ_s) were used [heavy water HDO, MW: 19, σ_s: 0.004; acetone, MW: 58, σ_s: 0.15; dimethyl formamide (DMF), MW: 73, σ_s: 0.76; ethylene glycol monomethyl ether (EGMME), MW: 76, σ_s: 0.59; diethylene glycol monomethyl ether (DEGMME), MW: 120, σ_s: 0.78 and triethylene glycol monoethyl ether (TEGMEE), MW: 178, σ_s: 0.80]. The larger the molecular size of the osmolyte, the more efficient it was in reducing cell Lp at a given concentration. The residual cell Lp decreased with increasing size of osmolytes. The findings are in agreement with a cohesion/tension model of the osmotic dehydration of water channels (aquaporins; AQPs), which predicts both reversible exponential dehydration curves and the dependence on the size of osmolytes which are more or less excluded from AQPs (Ye, Wiera & Steudle, Journal of Experimental Botany 55, 449–461, 2004). In the presence of big osmolytes, dehydration curves were best described by the sum of two exponentials (as predicted from the theory in the presence of two different types of AQPs with differing pore diameters and volumes). AQPs with big diameters could not be closed in the presence of osmolytes of small molecular size, even at very high concentrations. The cohesion/tension theory allowed pore volumes of AQPs to be evaluated, which was 2.3 ± 0.2 nm³ for the narrow pore and between 5.5 ± 0.8 and 6.1 ± 0.8 nm³ for the wider pores. The existence of different types of pores was also evident from differences in the residual Lp. Alternatively, pore volumes were estimated from ratios between osmotic (P_f) and diffusional (P_d) water flow, yielding the number of water molecules (N) in the pores. N-values ranged between 35 and 60, which referred to volumes of 0.51 and 0.88 nm³/pore. Values of pore volumes obtained by either method were bigger than those reported in the literature for other AQPs. Absolute values of pore volumes and differences obtained by the two methods are discussed in terms of an inclusion of mouth parts of AQPs during osmotic dehydration. It is concluded that the mouth part contributed to the absolute values of pore volumes depending on the size of osmolytes. However, this can not explain the finding of the existence of two different types or groups of AQPs in the plasma membrane of Chara.     

Influence of extracellular Cl concentration on Cl transport across isolated skin ofRana pipiens

Summary The effect of changes in Cl concentration in the external and/or serosal bath on Cl transport across short-circuited frog skin was studied by measurements of transepithelial Cl influx (J ₁₃ ^Cl ) and efflux (J ₃₁ ^Cl ), short-circuit current, transepithelial potential, and conductance (G _m).J ₁₃ ^Cl as well asJ ₃₁ ^Cl were found to have a saturating component and a component which is apparently linear with Cl concentration. The linear component ofJ ₃₁ ^Cl appears only upon addition of Cl to external medium, and about 3/4 of this component does not contribute toG _m. The saturating component ofJ ₃₁ ^Cl is only 5% of totalJ ₃₁ ^Cl with 115mm Cl in the serosal medium. Replacement of 115mm Cl^– in external medium by SO ₄ ⁼ , NO ₃ ^– , HCO ₃ ^– or I^– results in 87–97% reduction ofJ ₃₁ ^Cl , whereas replacement with Br^– has no effect. As external Cl concentration is raised in steps from 2 to 115mm,J ₁₃ ^Cl andJ ₃₁ ^Cl increase by the same amount butJ ₁₃ ^Cl is persistently 0.15 eq/cm² hr larger thanJ ₃₁ ^Cl . These results indicate that at least 3/4 of linear components ofJ ₁₃ ^Cl andJ ₃₁ ^Cl proceed via an exchange diffusion mechanism which seems to be located at the outer cell border. The saturating component ofJ ₁₃ ^Cl is involved in active Cl transport in an inward direction, and there is evidence suggesting that Cl uptake across outer cell border, which proceeds against an electrochemical gradient, is electroneutral but not directly linked to Na.     

Alternate promoters direct stress-induced transcription of the Bacillus subtilis clpC operon

clpC ofBacillus subtilis is part of an operon containing six genes. Northern blot analysis suggested that all genes are co-transcribed and encode stress-inducible proteins. Two promoters (P_A and P_B) were mapped upstream of the first gene. P_A resembles promoters recognized by the vegetative RNA polymerase Eσ^A. The other promoter (P_B) was shown to be dependent on σ^B, the general stress σ factor in B. subtilis, suggesting that clpC, a potential chaperone, is expressed in a σ^B-dependent manner. This is the first evidence that σ^B in B, subtilis is involved in controlling the expression of a gene whose counterpart, clpB, is subject to regulation by σ³² in Escherichia coli, indicating a new function of σ^B-dependent general stress proteins. P_B deviated from the consensus sequence of σ^B promoters and was only slightly induced by starvation conditions. Nevertheless, strong induction by heat, ethanol, and salt stress occurred at the σ^B-dependent promoter, whereas the vegetative promoter was only weakly induced under these conditions. However, in a sigB mutant, the σ^A-like promoter became inducible by heat and ethanol stress, completely compensating for sigB deficiency. Only the downstream σ^A-like promoter was induced by certain stress conditions such as hydrogen peroxide or puromycin. These results suggest that novel stress-induction mechanisms are acting at a vegetative promoter. Involvement of additional elements in this mode of induction are discussed.     

Correlation of 2J couplings with protein secondary structure

Geminal two‐bond couplings (²J) in proteins were analyzed in terms of correlation with protein secondary structure. NMR coupling constants measured and evaluated for a total six proteins comprise 3999 values of ²J_CαN′, ²J_C′HN, ²J_HNCα, ²J_C′Cα, ²J_HαC′, ²J_HαCα, ²J_CβC′, ²J_N′Hα, ²J_N′Cβ, and ²J_N′C′, encompassing an aggregate 969 amino‐acid residues. A seamless chain of pattern comparisons across the spectrum datasets recorded allowed the absolute signs of all ²J coupling constants studied to be retrieved. Grouped by their mediating nucleus, C′, N′ or C^α, ²J couplings related to C′ and N′ depend significantly on ?,ψ torsion‐angle combinations. β turn types I, I′, II and II′, especially, can be distinguished on the basis of relative‐value patterns of ²J_CαN′, ²J_HNCα, ²J_C′HN, and ²J_HαC′. These coupling types also depend on planar or tetrahedral bond angles, whereas such dependences seem insignificant for other types. ²J_HαCβ appears to depend on amino‐acid type only, showing negligible correlation with torsion‐angle geometry. Owing to its unusual properties, ²J_CαN′ can be considered a “one‐bond” rather than two‐bond interaction, the allylic analog of ¹J_N′Cα, as it were. Of all protein J coupling types, ²J_CαN′ exhibits the strongest dependence on molecular conformation, and among the ²J types, ²J_HNCα comes second in terms of significance, yet was hitherto barely attended to in protein structure work. Proteins 2010. © 2009 Wiley‐Liss, Inc.     

2-Methylwyosine,a Nucleoside With Restricted Anti Conformation in the East Region Enforced by Nucleobase Moiety Modification: Synthesis and Conformational Analysis by NMR and Molecular Dynamics

GRAPHICAL ABSTRACT

We synthesized a new 2-methyl derivative of wyosine using a multistep procedure starting from guanosine. We examined different synthetic paths and optimized the conditions for each step. Based on MD calculations and analysis of the ³ J _HH and J _C1′H1′ of the ribose moiety, we discovered that the sugar part adopted conformation specific for the East region rarely occurring in solution. This unusual conformational preference is probably due to steric repulsions between the methyl group at position 2 and the 5′-CH₂OH group. We observed that N-glycosidic bond stability weakened 14-fold upon the introduction of the methyl group in position 2 compared with wyosine.     

Effect of FeCl3 on ion transport in isolated frog skin

Summary The effect of addition of FeCl₃ to the media bathing the isolated skin ofRana pipiens was studied by measuring short-circuit current, transepithelial potential, and resistance, and by determining the influx and efflux of sodium (J ₁₃ ^Na andJ ₃₁ ^Na , respectively) and the influx and efflux of chloride (J ₁₃ ^Cl andJ ₃₁ ^Cl , respectively) across the epithelium. With normal Ringer's solution on both sides of the skin, addition of 10^–3 m FeCl₃ to the external medium resulted in nearly complete inhibition of active Na transport (J ₁₃ ^Na decreased from 1.30±0.14 to 0.10±0.04 eq/cm² hr (N=8)) and in appearance of active chloride transport in outward direction due to an 80% increase inJ ₃₁ ^Cl . Average (J ₃₁ ^Cl –J ₁₃ ^Cl ) obtained from means of 8 skins in 6 consecutive control and last 3 experimental periods was –0.17±0.04 and 0.38±0.05 eq/cm² hr, respectively. FeCl₃ added to external medium also induced substantial net chloride movement in outward direction when external medium contained Na-free choline chloride Ringer's or low ionic strength solution. Under the latter condition net Na movement was virtually eliminated by external FeCl₃. After addition of FeCl₃ to serosal medium there was delayed inhibition ofJ ₁₃ ^Na but no change in chloride fluxes. Immediate and profound changes in Na and Cl transport systems seen after external application of FeCl₃ indicate charge effects of Fe³⁺ on surface of apical cell membranes, possibly close to or in ion channels.     

The Osmometer Model of the Root: Water and Solute Relations of Roots of Phaseolus coccineus

Water and solute relations of young roots of Phaseolus coccineus have been measured using the root pressure probe. Biphasic root pressure relaxations were obtained when roots were treated with solutions containing different osmotic test solutes. From the relaxations, the hydraulic conductivity (Lp_r), the permeability coefficient (P_sr), and the reflection coefficient (σ_sr) of the roots could be evaluated. Lp_r was 1.8 to 8.4 . 10^?8 m . s^?1 . MPa^?1 and P_sr (in 10^?10 m . s^?1): methanol, 27–62; ethanol, 44–73; urea, 5–11; mannitol, 1.5; KCl, 7.1–9.2; NaCl, 2.1; NaNO₃, 3.7. The hydraulic conductivity was similar when using osmotic and hydrostatic pressure gradients as driving forces. The hydraulic conductivity of individual root cortex cells (Lp) was by two orders of magnitude larger than Lp_r (Lp = 0.3 to 4.7 . 10^?6 m . s^?1 . MPa^?1) which indicated a predominant cell-to-cell rather than an apoplasmic transport of water in the Phaseolus root. Except for distances shorter than 20 mm from the root apex, the hydraulic resistance of the roots was limited by the radial movement of water across the root cylinder and not by the hydraulic resistance within the xylem. Reflection coefficients were low: methanol: 0.16–0.34; ethanol: 0.15–0.47; urea: 0.41–0.51; mannitol: 0.68; KCl: 0.43–0.54; NaCl: 0.59; NaNO₃: 0.54. The transport coefficients (Lp_r, P_sr, σ_sr) have been critically examined for influences of unstirred layers and active transport. The low σ_sr suggests that the common treatment of the root as a rather perfect osmometer (σ_sr = 1) analogous to plant cells should be treated cautiously. The reasons for the low σ_sr and the possible implications of the absolute values of the transport parameters for the absorption of water and nutrients are discussed.     

Conformational study of ribonucleotides, 2′-deoxyribonucleotides,and arabinonucleotides by carbon-13 nuclear magnetic resonance

The geminal and vicinal ¹³C-³¹P coupling constants have been monitored, as a function of pH, for a series of uracil and cytosine 3′- and 5′-nucleotides with a ribose, arabinose, or 2′-deoxyribose sugar. Data were also obtained for two 3′,5′-diphosphates in the ribose and arabinose series. The geminal J(C5′-P5′) and J(C3′-P3′) couplings show only a small dependence on the ionization state of the phosphate, decreasing by < 0.5 Hz in the pH 5–7 range. For the ribose and arabinose 3′-nucleotides, the vicinal J(C4′-P3′) increase (up to 1.5 Hz) on secondary phosphate ionization in the pH 5–7 range, whereas their J(C2′-P3′) couplings decrease (up to 1.5 Hz) over the same pH range. In contrast for the 2′-deoxyribose molecules, both couplings decrease (～0.5 Hz) on phosphate ionization. The titration curves provide information about the influence of the sugar on the conformation about the C3′? O3′ bond. Some conformational trends could be rationalized by consideration of the sugar-puckerdependent contact interactions between the 3′-phosphate and the substituents on the furanose ring.