13C-nuclear magnetic resonance studies of 85% 13C-enriched amino acids and small peptides: pH effects on the chemical shifts,coupling constants,kinetics of cis-trans isomerisation and conformation aspects

The ¹³C chemical shifts of several 85% ¹³C-enriched amino acids and small peptides were studied as a function of pH. The results show that the chemical shifts of carbon atoms of ionizable groups vary significantly within the zone of their pK. Generally with the pH going from 7 to 1 all the δC are shifted more or less upfield with the exception of the carbonyl group of the second last residue which is shifted slightly downfield. This suggests the formation of an hydrogen bond at acid pH involving in a seven-membered ring the C=O in question and the COOH terminal.The percentage of cis and trans conformers of glycyl-l-proline and glycyl-l-prolylglycine were studied as a function of pH. The trans form is always preponderant whatever the pH. The accessibility of the carbonyl group to protonation of the proline residue strongly influences the cis-trans equilibrium. Thus, with the pH varying from 7 to 1, the trans isomer changes from 61 to 85% for glycyl-l-proline and only from 77 to 80% for glycyl-l-prolylglycine.The proton NMR studies underline the important differences existing between the two molecular forms of glycyl-l-proline. The cis conformation is characterized with regard to the trans form by the non-equivalence of the α-protons of the glycine residue, by a lower pK₁ and by a larger ΔδH_α of the proline residue as a function of pH. These results could suggest an end-to-end interaction in the cis form of the glycyl-l-proline molecule.The ¹³C-¹³C coupling constants were also studied as a function of pH. The results show that J_Co-Cα of a C-terminal residue, varying from 5 to 6 Hz and reflecting the pK of the carboxylate group, is a linear function of δ_Co and δ_Cα as in the case of the amino acids. The total variation of the electron density of those two carbons in an amino acid is approximately 40% weaker than in a C-terminal residue. The charge distribution along the C_α−C_o bond, however, is practically the same in both cases.Finally the ratios of the conversion rate constants of the two isomers cis-trans of glycyl-proline were calculated at different pH values; the relations between the isomer percentages and δ_Co, δ_Cα on the one hand and the J_Co-Cα on the other were established.     

Linear analysis of carbon-13 chemical shift differences and its application to the detection and correction of errors in referencing and spin system identifications

Statistical analysis reveals that the set of differences between the secondary shifts of the α- and β-carbons for residues i of a protein (Δδ¹³C^α_i- Δδ¹³C^β_i) provides the means to detect and correct referencing errors for ¹H and ¹³C nuclei within a given dataset. In a correctly referenced protein dataset, linear regression plots of Δδ¹³C^α_i,Δδ¹³C^β_i, or Δδ¹H^α_i vs. (Δδ¹³C^α_i- Δδ¹³C^β_i) pass through the origin from two directions, the helix-to-coil and strand-to-coil directions. Thus, linear analysis of chemical shifts (LACS) can be used to detect referencing errors and to recalibrate the ¹H and ¹³C chemical shift scales if needed. The analysis requires only that the signals be identified with distinct residue types (intra-residue spin systems). LACS allows errors in calibration to be detected and corrected in advance of sequence-specific assignments and secondary structure determinations. Signals that do not fit the linear model (outliers) deserve scrutiny since they could represent errors in identifying signals with a particular residue, or interesting features such as a cis-peptide bond. LACS provides the basis for the automated detection of such features and for testing reassignment hypotheses. Early detection and correction of errors in referencing and spin system identifications can improve the speed and accuracy of chemical shift assignments and secondary structure determinations. We have used LACS to create a database of offset-corrected chemical shifts corresponding to nearly 1800 BMRB entries: 300 with and 1500 without corresponding three-dimensional (3D) structures. This database can serve as a resource for future analysis of the effects of amino acid sequence and protein secondary and tertiary structure on NMR chemical shifts.Supplementary material to this paper is available in electronic form at http://dx.doi.org/10.1007/s10858-005-1717-0     

C magnetic resonance study of the ionization of N-acetyl-dl-proline in aqueous solution

NMR titration curves have been recorded for all the ¹³C resonances of cis and transN-acetyl-dl-proline in ²H₂O. the measured pK_2H values are 3.4 ± 0.8 and 4.13 ± 0.08 respectively; the free energy of ionization for the trans isomer being (3.8 kJ/mole) greater than for the cis. The ionization shifts of the two isomers differ significantly only at the acetyl carbonyl and C_γ positions. It is suggested that these are related to conformational changes which stabilize the trans form at low p²H.     

Synthesis and conformation of the cyclic octapeptides cyclo(Phe-Pro)4, cyclo(Leu-Pro)4, and cyclo[Lys(Z)-Pro]4

Cyclic octapeptides, cyclo(X-Pro)₄, where X represents Phe, Leu, or Lys(Z), were synthesized and their conformations investigated. A C₂-symmetric conformer containing two cis peptide bonds was found in all of these cyclic octapeptides. The numbers of available conformations due to the cis–trans isomerization of Pro peptide bonds depended on the nature of the solvent and X residue: they decreased in the following order: cyclo[Lys(Z)-Pro]₄ > cyclo(Leu-Pro)₄ > cyclo(Phe-Pro)₄ in CDCl₃. ¹³C spin-lattice relaxation times (T₁) of these cyclic octapeptides were measured, and the contribution of segmental mobility to T₁ was found to vary with the nature of the X residue.     

Synthesis of 5,6-dihydro-4H-1,3-oxazines from neutral and cationic platinum(II) nitrile complexes. X-ray structure of trans-[Pt(CF3){ H2CH2}(PPh3)2]BF4

The 1,3-oxazine complexes cis- and trans-[PtCl₂{ C(R)OCH₂CH₂C}H₂₂] (cis: R=CH₃ (1a), CH₂CH₃ (2a), (CH3)₃C (3a), C₆H₅ (4a); trans:R =CH₃ (1b), C₆H₅ (4b)) were obtained in 51-71% yield by reaction in THF at 0 °C of the corresponding nitrile complexes cis- and trans-[PtCl2(NCR)₂] with 2 equiv. of ⁻OCH₂CH₂CH₂Cl, generated by deprotonation of 3-chloro-1-propanol with n-BuLi. The cationic nitrile complexes trans-[Pt(CF₃)(NCR)(PPh₃)₂]BF₄ (R=CH3, C₆H₅) react with 1 equiv, of ⁻ OCH2CH2CH2Cl to give a mixture of products, including the corresponding oxazine derivatives trans-[Pt(CF₃){ CH₂}(PPh₃)₂]BF₄ (5 and 6), the chloro complex _trans- [Pt(CF₃)Cl(PPh₃)₂] and free oxazine H2. For short reaction times (c. 5–15 min) the oxazine complexes 5 and 6 could be isolated in modest yield (37–49%) from the reaction mixtures and they could be separated from the corresponding chloro complex (yield 40%) by taking advantage of the higher solubility of the latter derivative in benzene. For longer reaction times (> 2 h), trans-[Pt(CF₃)Cl(PPh₃)₂] was the only isolated product. Complex 6 was crystallographically characterized and it was found to contain also crystals of trans- [PtCl{ H₂}(PPh₃)₂]BF₄, which prevented a more detailed analysis of the bond lengths and angles within the metal coordination sphere. The 1,3-oxazine ring, which shows an overall planar arrangement, is characterized by high thermal values of the carbon atoms of the methylene groups indicative of disordering in this part of the molecule in agreement with fast dynamic ring processes suggested on the basis of ¹H NMR spectra. It crystallizes in the trigonal space group P , with a=22.590(4), b=15.970(3) Å, γ=120°, V=7058(1) ų and Z=6. The structure was refined to R=0.059 for 3903 unique observed (I3σ(I)) reflections. A mechanism is proposed for the conversion of nitrile ligands to oxazines in Pt(II) complexes.     

The stereochemistry of hexahydroprenol, ubiquinone and ergosterol biosynthesis in the mycelium of Aspergillus fumigatus Fresenius

1. The mycelium of Aspergillus fumigatus has been shown to incorporate mevalonate into squalene, ubiquinone, ergosterol and hexahydroprenol. 2. The ³H/¹⁴C ratio in ubiquinone, biosynthesized from [2-¹⁴C-(4R)-4-³H₁]mevalonate, is the same as in the squalene; essentially no ³H was incorporated from [2-¹⁴C-(4S)-4-³H₁]mevalonate, indicating the biosynthesis of biogenetically trans-isoprene units. 3. The ³H/¹⁴C ratio for ergosterol (from `4R-mevalonate') was 3:5, showing that the proton at C-24 is not lost during alkylation of the side chain; it probably migrates to C-25. 4. As ³H from both mevalonates was incorporated into the hexahydroprenols the biosynthesis of both cis- and trans-isoprene units must occur. 5. The saturated ω- and ψ-isoprene units are shown to be biogenetically trans, as are two of the unsaturated residues. 6. The saturated α- and unsaturated β-isoprene residues are both biogenetically cis. 7. An inexplicable loss of approximately half of the olefinic protons from the cis-portion of hexahydroprenol occurs; possible reasons for this loss are discussed. 8. Increase in chain length of the hexahydroprenols is by a cis addition. 9. A biosynthesis of hexahydroprenols by addition of cis-isoprene units to all-trans-geranylgeranyl pyrophosphate, or a dihydro or tetrahydro derivative thereof, is suggested.     

Two Structurally Different Dienelactone Hydrolases (TfdEI and TfdEII) from Cupriavidus necator JMP134 Plasmid pJP4 Catalyse Cis- and Trans-Dienelactones with Similar Efficiency

In this study, dienelactone hydrolases (TfdEI and TfdEII) located on plasmid pJP4 of Cupriavidus necator JMP134 were cloned, purified, characterized and three dimensional structures were predicted. tfdEI and tfdEII genes were cloned into pET21b vector and expressed in E. coli BL21(DE3). The enzymes were purified by applying ultra-membrane filtration, anion-exchange QFF and gel-filtration columns. The enzyme activity was determined by using cis-dienelactone. The three-dimensional structure of enzymes was predicted using SWISS-MODEL workspace and the biophysical properties were determined on ExPASy server. Both TfdEI and TfdEII (M_r 25 kDa) exhibited optimum activity at 37°C and pH 7.0. The enzymes retained approximately 50% of their activity after 1 h of incubation at 50°C and showed high stability against denaturing agents. The TfdEI and TfdEII hydrolysed cis-dienelactone at a rate of 0.258 and 0.182 µMs⁻¹, with a K_m value of 87 µM and 305 µM, respectively. Also, TfdEI and TfdEII hydrolysed trans-dienelactone at a rate of 0.053 µMs⁻¹ and 0.0766 µMs⁻¹, with a K_m value of 84 µM and 178 µM, respectively. The TfdEI and TfdEII k_cat/K_m ratios were 0.12 µM⁻¹s⁻¹and 0.13 µM⁻¹s⁻¹ and 0.216 µM⁻¹s⁻¹ and 0.094 µM⁻¹s⁻¹ for for cis- and trans-dienelactone, respectively. The k_cat/K_m ratios for cis-dienelactone show that both enzymes catalyse the reaction with same efficiency even though K_m value differs significantly. This is the first report to characterize and compare reaction kinetics of purified TfdEI and TfdEII from Cupriavidus necator JMP134 and may be helpful for further exploration of their catalytic mechanisms.     

Recycling ofd-glucose in collagenous cuticle: A means of nutrient conservation?

Summary Transport by an epithelium, possessing an accumulating, saturable transport system in the apical membrane as well as a finite Fick permeability to the transported solute, was considered in the steady state in the case of zerocis concentration, and in the presence of a peripheral diffusion resistance in a layer apposing thecis face of the tissue (unstirred solution or structural coating). Under suitable conditions, the combination of peripheral diffusion resistance and accumulating epithelial transport may lead to recycling of solute at thecis face of the epithelium. This causes a decrease of the effective permeability to diffusionaltrans-cis flow across the tissue. The phenomenon is discussed in terms of epidermald-glucose transport by the integument of aquatic animals with a collagenous cuticle, such as the seawater-acclimated polychaete wormNereis diversicolor. The recycling phenomenon may be of significance to other epithelia with the function of maintaining large concentration gradients of permeating substances.List of Symbols and Fixed Parameter Values C _m Bulk medium solute concentration,cis face of epidermisC _m=0 mol cm^–3 - C _i Concentration of solute at interface between cuticle and unstirred medium (mol cm^–3) - C _s Concentration of solute atcis face of apical epidermal membrane (mol cm^–3) - C _e Concentration of solute in extracellular fluid,trans-side of epidermisC _e=1.0×10^–6 mol cm^–3 - D _m Diffusion coefficient of solute in outside mediumD _m=6.7×10^–6 cm² sec^–1 - D _c Diffusion coefficient of solute in cuticleD _c=7.4×10^–9 cm² sec^–1 - _m Operative thickness of unstirred medium layer - _c Thickness of cuticle - J Steady-state net flux of solute through cuticle or unstirred layer (flux is positive indirectioncis-trans) (mol cm^–2 sec^–1) - J _i ^max Maximal influx through saturable transport system in apical membraneJ _i ^max =2.0×10^–12 mol cm^–2 sec^–1 - K _t Transport constant, saturable systemK _t=1.0×10^–7 mol cm^–3 - P Epithelial permeability (cm sec^–1)     

Specific growth rate as a determinant of the carbon isotope composition of the temperate seagrass Zostera marina

The seasonal variability of specific growth rate and the carbon stable isotope ratio (δ¹³C) of leaf blades (δ¹³C_leaf) of a temperate seagrass, Zostera marina (within 10 days old) were measured simultaneously, together with the δ¹³C of dissolved inorganic carbon (δ¹³C_DIC) at three sites in the semi-closed Akkeshi estuary system, northeastern Japan, in June, September, and November 2004. The δ¹³C_leaf ranged from −16.2 to −6.3‰ and decreased from summer to winter. The simultaneous measurement of the δ¹³C_leaf, growth rate, and morphological parameters (mean leaf length and width, mean number of leaves per shoot, and sheath length) of the seagrass and δ¹³C_DIC in the surrounding water allowed us to compare directly the δ¹³C_leaf and specific growth rate of seagrass. The difference in the δ¹³C of seagrass leaves relative to the source DIC (Δδ¹³C_{leaf − DIC}) was the least negative (−11 to −7‰) in June at all three sites and became more negative (−17 to −8‰) as the specific growth rate decreased. This positive correlation between Δδ¹³C_{leaf − DIC} and specific growth rate can be used to diagnose the growth of seagrasses. Δδ¹³C_{leaf − DIC} changed by −1.7 ± 0.2‰ when the leaf specific growth rate decreased by 1% d⁻¹.     

The metabolism of methylcyclohexane

1. When [U-¹⁴C]methylcyclohexane is fed to rabbits (dose 2–2·5m-moles/kg. body wt.), 65% of the radioactivity is excreted in the urine as metabolites, 0·5% appears in the faeces and about 15% in the expired air, some 4–5% remaining in the body in about 60hr. after dosing. The 15% of the dose appearing in the expired air consists of unchanged methylcyclohexane (10%) and ¹⁴CO₂ (5%). The low output of ¹⁴CO₂ shows that reactions leading to complete oxidation of methylcyclohexane are of minor importance. 2. The main metabolite found in the urine was the glucuronide of trans-4-methylcyclohexanol which was isolated. Seven methylcyclohexanols were found in the urine as conjugated glucuronides. The amounts of these were determined by isotope dilution to be as follows: cis-2-, 0·6%; trans-2-, 1·2%; cis-3-, 11·5%; trans-3-, 10·5%; cis-4-, 2·4%; trans-4-methylcyclohexanol, 14·7%, cyclohexylmethanol, 0·3%. No 1-methylcyclohexanol was found. There was evidence also that a small amount (approx. 1%) of the hydrocarbon aromatized to benzoic acid, probably via cyclohexylmethanol and cyclohexane-carboxylic acid. 3. The pattern of hydroxylation and the various amounts of the isomers found suggest that the hydroxylation in vivo of methylcyclohexane is dependent on steric factors in the molecule, hydroxylation occurring to the greatest extent at the carbon atom furthest away from the methyl group.     

Purification and characterization of 9-hexadecenoic acid cis-trans isomerase from Pseudomonas sp. strain E-3

A 9-hexadecenoic acid cis-trans isomerase (9-isomerase) that catalyzed the cis-to-trans isomerization of the double bond of free 9-cis-hexadecenoic acid [16:1(9c)] was purified to homogeneity from an extract of Pseudomonas sp. strain E-3 and characterized. Electrophoresis of the purified enzyme on both incompletely denaturing and denaturing polyacrylamide gels yielded a single band of a protein with a molecular mass of 80 kDa, suggesting that the isomerase is a monomeric protein of 80 kDa. The 9-isomerase, assayed with 16:1(9c) as a substrate, had a specific activity of 22.8 μmol h^–1 (mg protein)^–1 and a K _m of 117.6 mM. The optimal pH and temperature for catalysis were approximately pH 7–8 and 30° C, respectively. The 9-isomerase catalyzed the cis-to-trans conversion of a double bond at positions 9, 10, or 11, but not that of a double bond at position 6 or 7 of cis-mono-unsaturated fatty acids with carbon chain lengths of 14, 15, 16, and 17. Octadecenoic acids with a double bond at position 9 or 11 were not susceptible to isomerization. These results suggest that 9-isomerase has a strict specificity for both the position of the double bond and the chain length of the fatty acid. The enzyme catalyzed the cis-to-trans isomerization of fatty acids in a free form, and in the presence of a membrane fraction it was also able to isomerize 16:1(9c) esterified to phosphatidylethanolamine. The 9-isomerase was strongly inhibited by catecholic antioxidants such as α-tocopherol and nordihydroguaiaretic acid, but was not inhibited by 1,10-phenanthroline or EDTA or under anoxic conditions. Based on these results, the possible mechanism of catalysis by this enzyme is discussed. Received: 21 May 1997 / Accepted: 5 September 1997     

Platinum cross-linking of adenines and guanines on the quadruplex structures of the AG3(T2AG3)3 and (T2AG3)4 human telomere sequences in Na+ and K+ solutions

The quadruplex structures of the human telomere sequences AG₃(T₂AG₃)₃ I and (T₂AG₃)₄ II were investigated in the presence of Na⁺ and K⁺ ions, through the cross-linking of adenines and guanines by the cis- and trans-[Pt(NH₃)₂(H₂O)₂](NO₃)₂ complexes 1 and 2. The bases involved in chelation of the cis- and trans-Pt(NH₃)₂ moieties were identified by chemical and 3′-exonuclease digestions of the products isolated after denaturing gel electrophoresis. These are the four adenines of each sequence and four out of the 12 guanines. Two largely different structures have been reported for I: A from NMR data in Na⁺ solution and B from X-ray data of a K⁺-containing crystal. Structure A alone agrees with our conclusions about the formation of the A1–G10, A13–G22, A1–A13 platinum chelates at the top of the quadruplex and A7–A19, G4–A19 and A7–G20 at the bottom, whether the Na⁺ or K⁺ ion is present. At variance with a recent proposal that structures A and B could be the major species in Na⁺ and K⁺ solutions, respectively, our results suggest that structure A exists predominantly in the presence of both ions. They also suggest that covalent platinum cross-linking of a human telomere sequence could be used to inhibit telomerase.     

Stereochemical aspects of the metabolism of the isomeric methylcyclohexanols and methylcyclohexanones

1. The seven isomeric optically inactive forms of methylcyclohexanol (i.e. 1-, and cis- and trans-2-, -3- and -4-) are excreted by rabbits mainly as glucuronides of the thermodynamically more stable forms of the alcohols. The eighth isomer, cyclohexylmethanol, however, undergoes aromatization in vivo, giving rise to benzoic acid and hippuric acid. The (±)-2-, (±-3- and 4-methylcyclohexanones are reduced in the rabbit and excreted mainly as the glucuronides of the thermodynamically more stable forms of the corresponding methylcyclohexanols. 2. Racemic cis- and trans-2-methylcyclohexanol and 2-methylcyclohexanone are all excreted as conjugated trans-2-methylcyclohexanol. However, when the (±)-cis-alcohol or the (±)-ketone is fed, (+)-trans-2-methylcyclohexanol is excreted, whereas when the (±)-trans-alcohol is fed it is excreted as the (±)-trans-alcohol. 3. Racemic cis- and trans-3-methylcyclohexanol and 3-methylcyclohexanone are all excreted as conjugated racemic cis-3-methylcyclohexanol. cis- and trans-4-Methylcyclohexanol and 4-methylcyclohexanone are all excreted as conjugated trans-4-methylcyclohexanol. 4. The metabolic differences between the various methylcyclohexanols are explicable in terms of their conformations and of Vennesland's (1958) hypothesis of the role of NADH in dehydrogenation reactions.     

Regular left-handed fragment of amylose: crystal and molecular structure of methyl-α-maltotrioside, 4H2O

The crystal structure of methyl-α-maltotrioside tetrahydrate C₁₆H₃₄O₁₆, 4H₂O), has been established by direct methods from 2269 independent reflections and refined to a final R value of 0.054. The crystal belongs to the orthorhombic system, space group P2₁2₁2₁ and has a unit cell of dimensions: a = 1.037 (1), b = 2.439 (1) and c = 1.065 (1) nm. The three glucose residues have the ⁴C₁ pyranose conformation and are α-(1–4)-linked. The conformation of the glycosidic linkage is characterized by torsion angles (φ, ψ) which take the values (82.2, −148.9) between the non-reducing and the middle residue and (82.8, −151.8) between the middle residue and the reducing one. The primary hydroxyl groups exist in a gauche-gauche conformation. This structure is also characterized by the lack of intramolecular hydrogen bonding between secondary hydroxyl groups belonging to contiguous residues. The molecules are held together by a complicated network of hydrogen bonds involving all the hydroxylic groups and the water molecules. the three dimensional arrangement corresponds to a regular alternation of antiparallel bilayers strongly linked by water molecules. A survey of the distribution of the glycosidic torsion angles in all known linear α-(1–4)-linked d-glucose residues, discloses the existence of three stable conformers. This crystal structure provides the first experimental evidence of a regular left-handed fragment of the amylosic chain in a highly hydrated neighbourhood. Furthermore, the helical conformation adopted by the trisaccharide gives rise to helical parameters which are close to those found experimentally for native A and B amyloses. The relevance of the present results to the rationalization of the polymorphic transformation of amylose, along with its crystallization habits is also discussed.     

Determination of metabolites of pyrethroids in human urine using solid-phase extraction and gas chromatography–mass spectrometry

The described method permits the determination of the five most important metabolites of the pyrethroids permethrin, cypermethrin, deltamethrin, λ-cyhalothrin, fenvalerate, phenothrin and β-cyfluthrin in human urine in one run. The major urinary metabolites of these substances are cis-3-(2,2-dichlorovinyl)-2,2-dimethylcyclopropane-1-carboxylic acid (cis-Cl₂CA), trans-3-(2,2-dichlorovinyl)-2,2-dimethylcyclopropane-1-carboxylic acid (trans-Cl₂CA), cis-3-(2,2-dibromovinyl)-2,2-dimethylcyclopropane-1-carboxylic acid (Br₂CA), fluoro-3-phenoxybenzoic acid (F-PBA) and 3-phenoxybenzoic acid (3-PBA). After acidic hydrolysis to release the conjugated carboxylic acid metabolites, the analytes were separated from the matrix by means of solid-phase extraction using a reversed-phase column. The components of the eluate were converted to their methyl esters and extracted in hexane. Separation and quantitative analysis of the pyrethroid metabolites was carried out by capillary gas chromatography and mass selective detection. 2-Phenoxybenzoic acid served as an internal standard. The detection limits lay between 0.3 and 0.5 μg per litre urine. The relative standard deviations of the within-series imprecision were between 1% and 6%. The relative recovery rates ranged between 90% and 98%. Using this method we determined the elimination of pyrethroid metabolites in 24-h urine samples from eight pest controllers after indoor application of permethrin. The detected concentrations ranged from 1 to 70 μg g⁻¹ creatinine.     

NMR evidence for a type I β-turn in (Pro2)-tetrapeptides and interdependence of cis:Trans isomerism,ring flexibility,and backbone conformation

Tetrapeptides with proline in position 2, asparagine or leucine in position 3, and glycine in positions 1 and 4, with end groups free or blocked on the N-terminal side, were studied in their various ionic states in ²H₂O and in Me₂SO-d₆ by ¹H- and ¹³C-nmr. In order to clarify or refine some details, successive substitutions of the residues in these peptides with amino acids enriched to 85% in ¹³C, or to 85% ¹³C plus 97% ²H were carried out. The ¹H and ¹³C chemical shifts as well as the ¹H-¹H, ¹³C-¹³C, and ¹³C-¹H coupling constants and the signal intensities show strong similarity of behavior between the tetrapeptides of asparagine and leucine. The main conformational characteristics are (1) the almost total stabilization of the trans conformer in the type I β-turn structure when the peptide is in the zwitterion state dissolved in Me₂SO. This is deduced from the ³J and the ³J coupling constants, which both furnish a dihedral angle of ?³ = ?90°, and from the positive value of the temperature coefficient of the glycine-4 amide protons, which suggests a type 4 → 1 hydrogen bond; (2) the evolution of cis and trans isomer fractions which change with the ionic state of the peptides in Me₂SO, whereas they remain constant in aqueous solution; and (3) the conformation of the pyrrolidine ring as it follows the variations in cis:trans isomer populations together with the side-chain rotamer fractions of the residue in position 3. In the β-turn conformation the isomer cis is less abundant and the pyrrolidine ring is more flexible; this explains the perfect accommodation of the proline residue in position 2 of a bend. The interdependence of these phenomena where interactive forces play a predominant role underlines the importance of cooperative effects in the molecule. The results also suggest that the cis isomer of proline can adapt itself just as well as the trans isomer to position 2 of a type I β-turn.     

Stable carbon isotope variations in surface bloom scum and subsurface seston among shallow eutrophic lakes

Carbon stable isotope analysis of surface bloom scum and subsurface seston samples was conducted in shallow eutrophic lakes in China during warm seasons from 2003 to 2004. δ¹³C values of bloom scum were always higher (averaged 5‰) than those of seston in this study, and the possible reasons were attributed to (i) direct use of atmospheric CO₂ at the air–water interface, (ii) decrease in ¹³C fractionation due to higher carbon fixation, (iii) active CO₂ transport, and/or (iv) HCO₃⁻ accumulation. Negative correlation between δ¹³C_scum − δ¹³C_seston and pH in the test lakes indicated that phytoplankton at the subsurface water column increased isotopic enrichment under the carbon limitation along with the increase of pH, which might in turn decreased the differences in δ¹³C between the subsurface seston and the surface scums. Significant positive correlations of seston δ¹³C with total concentrations of nitrogen and phosphorus in water column suggested that the increase in δ¹³C of seston with trophic state was depending on nutrient (N or P, or both) supply. Our study showed that δ¹³C of phytoplankton was indicative of carbon utilization, primary productivity, and nutrient supply among the eutrophic lakes.     

pH-Modulation of Chloride Channels from the Sarcoplasmic Reticulum of Skeletal Muscle

The understanding of the role of cytoplasmic pH in modulating sarcoplasmic reticulum (SR) ion channels involved in Ca²⁺ regulation is important for the understanding of the function of normal and adversely affected muscles. The dependency of the SR small chloride (SCl) channel from rabbit skeletal muscle on cytoplasmic pH (pH _cis ) and luminal pH (pH _trans ) was investigated using the lipid bilayer-vesicle fusion technique. Low pH _cis 6.75–4.28 modifies the operational mode of this multiconductance channel (conductance levels between 5 and 75 pS). At pH _cis 7.26–7.37 the channel mode is dominated by the conductance and kinetics of the main conductance state (65–75 pS) whereas at low pH _cis 6.75–4.28 the channel mode is dominated by the conductance and kinetics of subconductance states (5–40 pS). Similarly, low pH _trans 4.07, but not pH _trans 6.28, modified the activity of SCl channels. The effects of low pH _cis are pronounced at 10⁻³ and 10⁻⁴ m [Ca²⁺] _cis but are not apparent at 10⁻⁵ m [Ca²⁺] _cis , where the subconductances of the channel are already prominent. Low pH _cis -induced mode shift in the SCl channel activity is due to modification of the channel proteins that cause the uncoupling of the subconductance states. The results in this study suggest that low pH _cis can modify the functional properties of the skeletal SR ion channels and hence contribute, at least partly, to the malfunction in the contraction-relaxation mechanism in skeletal muscle under low cytoplasmic pH levels. Received: 20 May 1998/Revised: 24 September 1998     

Transformation of steroids by Bacillus strains isolated from the foregut of water beetles (Coleoptera:Dytiscidae): I. Metabolism of androst-4-en-3,17-dione (AD)

Two Bacillus strains were isolated from the foregut of the water beetle Agabus affinis (Payk.) and tested for their steroid transforming ability. After incubation with androst-4-en-3,17-dione (AD), 13 different transformation products were detected. AD was hydroxylated at C₆, C₇, C₁₁ and C₁₄, resulting in formation of 6β-, 7α-, 11α- and 14α-hydroxy-AD. One strain also produced small amounts of 6β,14α-dihydroxy-AD. Partly, the 6β-hydroxy group was further oxidized to the corresponding 6-oxo steroids. In addition, a specific reduction of the Δ⁴-double bond was observed, leading to the formation of 5α-androstane derivatives. In minor yields the carbonyl functions at C₃ and C₁₇ were reduced leading to the formation of 3ξ-OH or 17β-OH steroids. EI mass spectra of the trimethylsilyl and O-methyloxime trimethylsilyl ether derivatives of some transformation products are presented for the first time.