Fast-HMQC using Ernst angle pulses: An efficient tool for screening of ligand binding to target proteins

The application of Ernst angle pulses in multidimensional NMR spectroscopy is theoretically and experimentally investigated. Theory shows that only for a few pulse sequences employed at high repetition rate, a remarkable gain in sensitivity is possible using Ernst angle pulses. As an example, a new variant of the heteronuclear multiple quantum coherence (HMQC) experiment, the fast-(¹H,¹⁵N)-HMQC, is described. This sequence allows, with a 1 mM protein sample in H₂O, the acquisition of a highly resolved two-dimensional (¹H,¹⁵N) correlated spectrum within 37 s. The high efficiency of the fast-HMQC to detect ligand binding to a target protein is demonstrated.     

<Superscript>15</Superscript>N and <Superscript>13</Superscript>C- SOFAST-HMQC editing enhances 3D-NOESY sensitivity in highly deuterated,selectively [<Superscript>1</Superscript>H,<Superscript>13</Superscript>C]-labeled proteins

The ongoing NMR method development effort strives for high quality multidimensional data with reduced collection time. Here, we apply ‘SOFAST-HMQC’ to frequency editing in 3D NOESY experiments and demonstrate the sensitivity benefits using highly deuterated and ¹⁵N, methyl labeled samples in H₂O. The experiments benefit from a combination of selective T ₁ relaxation (or L-optimized effect), from Ernst angle optimization and, in certain types of experiments, from using the mixing time for both NOE buildup and magnetization recovery. This effect enhances sensitivity by up to 2.4× at fast pulsing versus reference HMQC sequences of same overall length and water suppression characteristics. Representative experiments designed to address interesting protein NMR challenges are detailed. Editing capabilities are exploited with heteronuclear ¹⁵N,¹³C-edited, or with diagonal-free ¹³C aromatic/methyl-resolved 3D-SOFAST-HMQC–NOESY–HMQC. The latter experiment is used here to elucidate the methyl-aromatic NOE network in the hydrophobic core of the 19 kDa FliT-FliJ flagellar protein complex. Incorporation of fast pulsing to reference experiments such as 3D-NOESY–HMQC boosts digital resolution, simplifies the process of NOE assignment and helps to automate protein structure determination.     

Gradient and sensitivity enhanced multiple-quantum coherence in heteronuclear multidimensional NMR experiments

Recent studies have indicated that the relaxation rate of the ¹H-¹³C multiple-quantum coherence is much slower than that of the ¹H-¹³C single-quantum coherence for non-aromatic methine sites in ¹³ C labeled proteins and in nucleic acids at the slow tumbling limit. Several heteronuclear experiments have been designed to use a multiple-quantum coherence transfer scheme instead of the single-quantum transfer method, thereby increasing the sensitivity and resolution of the spectra. Here, we report a constant time, gradient and sensitivity enhanced HMQC experiment (CT-g/s-HMQC) and demonstrate that it has a significant sensitivity enhancement over constant time HMQC and constant time gradient and sensitivity enhanced HSQC experiments (CT-g/s-HSQC) when applied to a ¹³C and ¹⁵ N labeled calmodulin sample in D₂O. We also apply this approach to 3D NOESY-HMQC and doubly sensitivity enhanced TOCSY-HMQC experiments, and demonstrate that they are more sensitive than their HSQC counterparts.     

Resolution and sensitivity enhancement of heteronuclear correlation for methylene resonances via 2H enrichment and decoupling

Summary Monodeuterated methylene positions exhibit substantially superior spectral characteristics in ¹H–¹³C correlation experiments as compared to diprotio signals. A combination of ²H decoupling and multiplet editing of HMQC and HSQC experiments provides resolution enhancement for both stereoselective and random fractionally deuterated samples. For HMQC experiments with [2-²H_r, 2-¹³C]glycine-labeled E. coli thioredoxin (11.7 kDa), 3-fold increases in both ¹H and ¹³C resolution result in a complementary 9-fold enhancement in sensitivity. Owing to a smaller improvement in ¹³C resolution, the corresponding enhancements for the HSQC experiment are 2-fold less.     

Optimization of <Superscript>1</Superscript>H decoupling eliminates sideband artifacts in 3D TROSY-based triple resonance experiments

TROSY-based triple resonance experiments are essential for protein backbone assignment of large biomolecular systems by solution NMR spectroscopy. In a survey of the current Bruker pulse sequence library for TROSY-based experiments we found that several sequences were plagued by artifacts that affect spectral quality and hamper data analysis. Specifically, these experiments produce sidebands in the ¹³C(t ₁) dimension with inverted phase corresponding to ¹H_N resonance frequencies, with approximately 5% intensity of the parent ¹³C crosspeaks. These artifacts originate from the modulation of the ¹H_N frequency onto the resonance frequency of ¹³Cα and/or ¹³Cβ and are due to 180° pulses imperfections used for ¹H decoupling during the ¹³C(t ₁) evolution period. These sidebands can become severe for CA_i, CA_i?1 and/or CB_i, CB_i?1 correlation experiments such as TROSY-HNCACB. Here, we implement three alternative decoupling strategies that suppress these artifacts and, depending on the scheme employed, boost the sensitivity up to 14% on Bruker spectrometers. A class of comparable Agilent/Varian pulse sequences that use WALTZ16 ¹H decoupling can also be improved by this method resulting in up to 60–80% increase in sensitivity.     

Comparison of <Superscript>13</Superscript>CH<Subscript>3</Subscript>, <Superscript>13</Superscript>CH<Subscript>2</Subscript>D,and <Superscript>13</Superscript>CHD<Subscript>2</Subscript> methyl labeling strategies in proteins

A comparison of three labeling strategies for studies involving side chain methyl groups in high molecular weight proteins, using ¹³CH₃,¹³CH₂D, and ¹³CHD₂ methyl isotopomers, is presented. For each labeling scheme, ¹H–¹³C pulse sequences that give optimal resolution and sensitivity are identified. Three highly deuterated samples of a 723 residue enzyme, malate synthase G, with ¹³CH₃,¹³CH₂D, and ¹³CHD₂ labeling in Ile δ1 positions, are used to test the pulse sequences experimentally, and a rationalization of each sequence’s performance based on a product operator formalism that focuses on individual transitions is presented. The HMQC pulse sequence has previously been identified as a transverse relaxation optimized experiment for ¹³CH₃-labeled methyl groups attached to macromolecules, and a zero-quantum correlation pulse scheme (¹³CH₃ HZQC) has been developed to further improve resolution in the indirectly detected dimension. We present a modified version of the ¹³CH₃ HZQC sequence that provides improved sensitivity by using the steady-state magnetization of both ¹³C and ¹H spins. The HSQC and HMQC spectra of ¹³CH₂D-labeled methyl groups in malate synthase G are very poorly resolved, but we present a new pulse sequence, ¹³CH₂D TROSY, that exploits cross-correlation effects to record ¹H–¹³C correlation maps with dramatically reduced linewidths in both dimensions. Well-resolved spectra of ¹³CHD₂-labeled methyl groups can be recorded with HSQC or HMQC; a new ¹³CHD₂ HZQC sequence is described that provides improved resolution with no loss in sensitivity in the applications considered here. When spectra recorded on samples prepared with the three isotopomers are compared, it is clear that the ¹³CH₃ labeling strategy is the most beneficial from the perspective of sensitivity (gains ≥2.4 relative to either ¹³CH₂D or ¹³CHD₂ labeling), although excellent resolution can be obtained with any of the isotopomers using the pulse sequences presented here.     

An Endocytosed TGN38 Chimeric Protein Is Delivered to the TGN after Trafficking through the Endocytic Recycling Compartment in CHO Cells

To examine TGN38 trafficking from the cell surface to the TGN, CHO cells were stably transfected with a chimeric transmembrane protein, TacTGN38. We used fluorescent and ¹²⁵I-labeled anti-Tac IgG and Fab fragments to follow TacTGN38''s postendocytic trafficking. At steady-state, anti-Tac was mainly in the TGN, but shortly after endocytosis it was predominantly in early endosomes. 11% of cellular TacTGN38 is on the plasma membrane. Kinetic analysis of trafficking of antibodies bound to TacTGN38 showed that after short endocytic pulses, 80% of internalized anti-Tac returned to the cell surface (t _1/2 = 9 min), and the remainder trafficked to the TGN. When longer filling pulses and chases were used to load anti-Tac into the TGN, it returned to the cell surface with a t _1/2 of 46 min. Quantitative confocal microscopy analysis also showed that fluorescent anti-Tac fills the TGN with a 46-min t _1/2. Using the measured rate constants in a simple kinetic model, we predict that 82% of TacTGN38 is in the TGN, and 7% is in endosomes. TacTGN38 leaves the TGN slowly, which accounts for its steady-state distribution despite the inefficient targeting from the cell surface to the TGN.     

Kinetics of NAD reduction in the nucleus and the cytoplasm

Summary The kinetics of the nuclear and cytoplasmic fluorescence response to glycolytic substrate were studied in ascites cells in culture (EL2 cells) and radiation giants (EL2G) maintained under a variety of conditions, using a beam-splitter supplemented microfluorimeter which allows fluorescence recording simultaneously with microelectrophoretic addition of substrate. A sequence of fluorescence pulses which resemble closely the curve of formation and disappearance of the enzyme-substrate complex were obtained upon repetitive additions of substrate. The pulses were analyzed in terms of peak fluorescence response (PFR), duration of steady state, halftime of fluorescence rise and decay (tin1/2off), number of consecutive cycles elicited, etc. There is a considerable parallelism in the kinetics of the nuclear and cytoplasmic fluorescence after addition of glycolytic substrate, over the whole time course of consecutive pulses. However in untreated, Amytal- or Rotenone-perfused cells the peak magnitudes of the cytoplasmic fluorescence are significantly lower and the cytoplasmic pulses are damped earlier than the nuclear upon repeated additions of substrate. In Amytal-grown EL2 cells there is a drop of PFR and a prolongation of t _1/2off in the nucleus and cytoplasm, which persists when the cells are transferred to an Amytal-free medium. However, if the cells are maintained for longer periods in Amytal, the nuclear fluorescence tends to return to control level, while the cytoplasmic pulses remain small and are easily damped. In T₃- or Amytal + T₃ -grown EL2 cells the cytoplasmic fluorescence instead of dropping like in the controls, follows the nuclear level over the whole time course of repeated pulses, and can even exceed the nuclear. Comparable phenomena are observed in T₃ -and Insulin-grown radiation giants When the amount of substrate is varied, starting from levels which can barely elicit a response the magnitude of the fluorescence response (integrated fluorescence pulse) in the cytoplasm and nucleus follows a sigmoid curve which can be interpreted as a function of allosteric enzymes.List of Abbreviations EL2 mouse Ehrlich ascites cells in tissue culture - EL2G giant tissue culture mouse Ehrlich ascites cells obtained by X-irradiation - EL2T giant tissue culture EL2 cells obtained by treatment with Trenimon (2.3.5-Tris aethyleniminobenzochinon-1.4) - G6P glucose-6-phosphate - FDP fructose-1.6-diphosphate - 6 PG 6-phospho-gluconate - UDPG uridine-5-diphosphoglucose - AMP adenosine-5-monophosphate - ADP adenosine-5-diphosphate - G1P D-glucose-1-phosphate - PFR peak fluorescence response - t _1/2off halftime of fluorescence rise and decay - T3 triidothyronine     

Refocusing revisited: An optimized,gradient-enhanced refocused HSQC and its applications in 2D and 3D NMR and in deuterium exchange experiments

Summary 2D ¹⁵N-¹H correlation spectra are ideal for measuring backbone amide populations to determine amide exchange protection factors in studies of protein folding or other structural features. Most protein NMR spectroscopists use HSQC, which has been shown to be generally superior to HMQC in both resolution and sensitivity. The refocused HSQC experiment is intrinsically less sensitive than the regular HSQC, due to T₂ relaxation during the refocusing delays. However, we show here that, when high ¹⁵N resolution is needed, an optimized refocused HSQC sequence that utilizes a semi-constant time evolution period and pulsed field gradients has better signal-to-noise ratio and resolution, and integrates more accurately, than a similar HSQC. The differences are demonstrated on a 20 kDa protein. The technique can also be applied to 3D NOESY experiments to eliminate strong NH₂ geminal peaks and their truncation artefacts at a modest cost in sensitivity.     

Melanin Granule Models for the Processes of Laser-Induced Thermal Damage in Pigmented Retinal Tissues. I. Modeling of Laser-Induced Heating of Melanosomes and Selective Thermal Processes in Retinal Tissues

The computer modeling was applied for investigation of the processes of laser-induced tissue damage. The melanin granule models for the processes of laser-induced thermal damage and the results of computer modeling of the optical, thermophysical, and thermochemical processes during selective laser interaction with melanoprotein granules (melanosomes) in retinal pigment epithelium are presented in this paper. Physical-mathematical model and system of equations are formulated which describe thermal interaction processes for “short” laser pulses of duration t _p<10⁻⁶ s and for “ long’ pulses of duration t _p10⁻⁶ s. Results of numerical simulation of the processes give the space–time distributions of temperature and degrees of thermodenaturation of the protein molecules inside and around melanosomes and in the volume of irradiated tissues. Energy absorption, heat transfer and thermochemical (thermodenaturation, coagulation) processes occurring during the interaction of laser pulses with pigmented spherical and spheroidal granules in heterogeneous tissues are theoretically investigated. The possibility for selective interaction of short laser pulses with pigmented granules is discussed which results in the formation of denaturation microregions inside and near the pigmented granules (granular thermodenaturation) without origination of a continuous macroscopic thermodenaturation lesion in tissue. Analytical model of heating of single spherical and spheroidal granule under laser pulse is presented. Simple equations for time dependencies of particle temperature are obtained. The presented results are of essential interest for laser applications in and can be used for investigation of laser interaction with pigmented tissues in different fields of laser medicine.     

Total Relaxivities of Material Content in Various Cysts and Ameloblastoma: Implications for Discriminating Different Fluids

Nuclear magnetic resonance T ₁ and T ₂ relaxivities (r ₁ and r ₂) exhibit efficiency of a material to alter the relaxation rates (1/T ₁ and 1/T ₂), and they are being used for diagnostic purposes. The determination of total relaxivities (r _1t and r _2t) of cystic fluid content and ameloblastoma may therefore be useful for discriminative purposes. In order to determine what makes total relaxivities of hemorrhagic cysts, four sets of tubes containing pooled cyst were doped with increasing concentrations of iron, copper, albumin, and γ-globulins. These sets were replaced in a phantom together with six individual cysts and one ameloblastoma. The relaxation times were measured by magnetic resonance imaging operating at 1.5 T. The relaxivities of individual ions and proteins were determined from the slope of the relation between relaxation rates and concentration, while total relaxivities were determined by using the increases in relaxation rates and material content of cystic fluid (MC). Iron, copper, albumin, and γ-globulins were found to be the sources of r _1t and r _2t. Each of r _1t, r _2t, r _1tMC, r _2tMC, and r _2t/r _1t are distinctive parameters for each cystic category and ameloblastoma. Except for MC, the parameters measured for ameloblastoma are significantly smaller than those of cysts. The similarity of the present results to those used in clinical applications suggests that each of r _1t, r _2t, r _1tMC, r _2tMC, and r _2t/r _1t has an ability to discriminate various fluids and masses. The present work also suggests that r _1tMC, r _2tMC, and r _2t/r _1t can be determined in vivo.     

Characterization of a putative thioredoxin peroxidase prx1 of <Emphasis Type="Italic">Candida albicans</Emphasis>

In this study, we characterized a putative peroxidase Prx1 of Candida albicans by: 1) demonstrating the thioredoxin-linked peroxidase activity with purified proteins, 2) examining the sensitivity to several oxidants and the accumulation of intracellular reactive oxygen species with a null mutant (prx1Δ), a mutant (C69S) with a point mutation at Cys69, and a revertant, and 3) subcelluar localization. Enzymatic assays showed that Prx1 is a thioredoxin-linked peroxidase which reduces both hydrogen peroxide (H₂O₂) and tert-butyl hydroperoxide (t-BOOH). Compared with two other strong H₂O₂ scavenger mutants for TSA1 and CAT1, prx1Δ and C69S were less sensitive to H₂O₂, menadione and diamide at all concentrations tested, but were more sensitive to low concentration of t-BOOH. Intracellular reactive oxygen species accumulated in prx1Δ and C69S cells treated with t-BOOH but not H₂O₂. These results suggest that peroxidase activity of Prx1 is specified to t-BOOH in cells. In both biochemical and physiological cases, the evolutionarily conserved Cys69 was found to be essential for the function. Immunocytochemical staining revealed Prx1 is localized in the cytosol of yeast cells, but is translocated to the nucleus during the hyphal transition, though the significances of this observation are unclear. Our data suggest that PRX1 has a thioredoxin peroxidase activity reducing both t-BOOH and H₂O₂, but its cellular function is specified to t-BOOH.     

Experimental investigation of X-ray lasing in a capillary argon discharge

Results are presented from experiments on the laser generation of X-ray radiation at the wavelength λ=469 ? (ε=26.4 eV) on the 3p(J=0)−3s(J=1) transition of Ne-like Ar ions. Experiments were carried out on the SIGNAL electrophysical facility with a 3.1-mm-diameter 157-mm-long Al₂O₃ ceramic capillary filled with argon at a pressure of 0.2–1.0 Torr. The discharge current amplitude was I ∼ 25–40 kA, the current rise rate being dI/dt ∼ 10¹² A/s. By a vacuum X-ray diode tuned to detect X-ray photons with energies in the range 10–40 eV, laser pulses with a duration of t ₁ ∼ 1 ns and maximum energy of E _1,max ∼ 1 μJ were recorded. The pulses were generated 35 ns after the discharge current was switched on. The line spectra in the wavelength range of 150–500 ? showed the bright λ=469 ? line. The angular divergence of the generated X-ray laser beam was estimated to be Δϑ ∼ 2 mrad. Original Russian Text ? O.N. Gilev, V.I. Afonin, V.I. Ostashev, V.Yu. Politov, A.M. Gafarov, A.L. Zapysov, A.V. Andriyash, é.P. Magda, L.N. Shamraev, A.A. Safronov, A.V. Komissarov, N.A. Khavronin, N.A. Pkhaĭko, L.V. Antonova, L.N. Shushlebin, 2006, published in Fizika Plazmy, 2006, Vol. 32, No. 2, pp. 160–165.     

Temporal profile of the singlet oxygen emission endogenously produced by photosystem II reaction centre in an aqueous buffer

The temporal profile of the phosphorescence of singlet oxygen endogenously photosensitized by photosystem II (PSII) reaction centre (RC) in an aqueous buffer has been recorded using laser excitation and a near infrared photomultiplier tube. A weak emission signal was discernible, and could be fitted to the functional form a[exp( - t/t₂ ) - exp( - t/t₁ )] a[\exp ( - t/\tau_{2} ) - \exp ( - t/\tau_{1} )] , with $ a > 0 $ a > 0 and $ \tau_{2} > \tau_{1} $ \tau_{2} > \tau_{1} . The value of t₂ \tau_{2} decreased from 11.6 ± 0.5 μs under aerobic conditions to 4.1 ± 0.2 μs in oxygen-saturated samples, due to enhanced bimolecular quenching of the donor triplet by oxygen, whereas that of t₁ \tau_{1} , identifiable with the lifetime of singlet oxygen, was close to 3 μs in both cases. Extrapolations based on the low amplitude of the emission signal of singlet oxygen formed by PSII RC in the aqueous buffer and the expected values of t₁ \tau_{1} and t₂ \tau_{2} in chloroplasts indicate that attempts to analyse the temporal profile of singlet oxygen in chloroplasts are unlikely to be rewarded with success without a significant advance in the sensitivity of the detection equipment.     

The Matched Pairs Problem with Exponential Variates

Two classes of tests for the hypothesis of bivariate symmetry are studied. For paired exponential survival times (t_1j, t_2j), the classes of tests are those based on t_1j-t_2j and those based on log t_1j–log t_2j. For each class the sign, signed ranks, t and likelihood ratio tests are compared via Pitman's criterion of asymptotic relative efficiency (ARE). For tests based on t_1j — t_2j, it is found that: (1) the efficacy of the paired t depends on the coefficient of variation (CV) of the pair means, (2) the signed rank test has the same ARE to the sign test as for the usual location problem. For tests based on log t_1j — log t_2j, the ARE comparisons reduce to the well-known results for the one-sample location problem for samples from a logistic density. Hence, the signed rank test is asymptotically efficient. Furthermore, analyses based on log t_1j — log t_2j are not complicated by the underlying pairing mechanism.     

Sap flow in Scots pines growing under conditions of year-round carbon dioxide enrichment and temperature elevation

Starting in rrrr, individual trees of Scots pine (Pinus sylvestris L.) aged 30 years were grown in closed-top chambers and exposed to normal ambient conditions (CON), elevated CO₂ (Elev. C), elevated temperature (Elev. T) and a combination of elevated CO₂ and temperature (Elev. C + T). Using the constant-power heat balance method, sap flow was monitored simultaneously in a total of 16 trees, four for each treatment, over a 32 d period (after the completion of needle expansion and branch elongation in 1997). An overall variation in diurnal sap flow totals (F_t) was evident during the period of measurement (days 167–198, 1997) regardless of the treatments, with a range from 0·15 to 2·82 kg tree^–1 d^–1. Elev. C reduced F_t by 4·1–13·7% compared with CON on most days (P varies from 0·042 to 0·108), but slightly increased it on some days (P≥ 0·131), depending on the weather conditions. Although the decrease in F_t caused by Elev. C was statistically significant on only a few days (P≤ 0·042), the cumulative F_t for the 32 d decreased by 14·4% (P = 0·047), indicating that Elev. C may have an important influence on seasonal water use of the Scots pine. Analysis of the diurnal courses of sap flow combined with corresponding weather factors indicated that the CO₂-induced decrease in F_t could be largely attributed to an increase in stomatal sensitivity to vapour pressure deficit (VPD), whereas the CO₂-induced increase in F_t related to an increase in stomatal sensitivity to low light levels. Elev. T increased F_t by 11·2–35·6% throughout the measuring period and the cumulative F_t for the 32 d by 32·5% (P = 0·019), which could be largely attributed to the temperature-induced increase in current-year needle area and decrease in stomatal sensitivity to high levels of VPD. There were no significant interactive effects of CO₂ and temperature on sap flow, so that Elev. C + T had approximately the same F_t as Elev. T and similar diurnal patterns of sap flow, suggesting that the temperature factor played a dominant role in the case of Elev. C + T.     

Linear prediction enhancement of 2D heteronuclear correlated spectra of proteins

Summary Linear prediction has been used to extrapolate the t₁ domain of natural abundance¹H–¹³C correlated two-dimensional (2D) FIDs of insulin. The FIDs were obtained by two different heteronuclear correlation experiments, one that utilizes heteronuclear multiple-quantum coherence during t₁, and one that utilizes¹³C single-quantum coherence. It is shown that the enhancement of the resolution and sensitivity in the F₁ dimension of the Fourier transform spectrum that results from the linear prediction extrapolation allows the t₁ domain to be confined to a relatively short time period where the signal intensity is at maximum. In particular, it is found that the enhancement thus obtained is sufficiently good to allow an observation of the difference between the F₁ line widths in the single-quantum and double-quantum coherence spectra.     

Killing of microorganisms by pulsed electric fields

 Lethal effects of pulsed electric fields (PEF) on suspensions of various bacteria, yeast, and spores in buffer solutions and liquid foodstuffs were examined. Living-cell counts of vegetative cell types were reduced by PEF treatment by up to more than four orders of magnitude (>99.99%). On the other hand, endo- and ascospores were not inactivated or killed to any great extent. The killing of vegetative cell types depends on the electrical field strength of the pulses and on the treatment time (the product of the pulse number and the decay time constant of the pulses). For each cell type, a specific critical electric field strength (E _c) and a specific critical treatment time (t _c) were determined. Above these critical values, the fractions of surviving cells were reduced drastically. The “limits”E _c and t _c depend on the cell characteristics as well as on the type of medium in which the cells are suspended. Especially in acid media living-cell counts were sufficiently decreased at very low energy inputs. In addition to the inactivation of microorganisms, the effect of PEF on food components such as whey proteins, enzymes and vitamins, and on the taste of foodstuffs was studied. The degree of destruction of these food components by PEF was very low or negligible. Moreover, no significant deterioration of the taste of foodstuffs was detected after PEF treatment. Disintegration of cells by PEF treatment in order to harvest intracellular products was also studied. Yeast cells, suspended in buffer solution, were not disintegrated by electric pulses. Hence, PEF treatment is an excellent process for inactivation of microorganisms in acid and in thermosensive media, but not for complete disintegration of microbial cells. Receivced: 1 June 1995 / Received last revision: 13 September 1995 / Accepted: 20 September 1995     

Orientation ofFucus egg polarity by electric a. c. and d. c. fields

Summary Zygotes of the marine brown alga,Fucus serratus, have been subjected to the different modes of electric fields. 1) The result of a former study with conductive d.c. fields has been confirmed using electrostatic d.c. fields of 0.5 to 4 V/cm: the zygotes develop the cell polarity axis parallel to the imposed field with the rhizoid pole toward the cathode. 2) The frequency response to both, conductive and electrostatic, a.c. fields represents an optimum curve. The response,i.e. rhizoid formation at either or, in rare cases, both cell poles, peaks at square pulse durations,t _E, of 70 to 120 ms. 3) The same frequency response appears if the pulse number is kept constant at 8s^–1 by variation of the interval between the pulses,t _o. Only fort _oo > 200 ms,i.e. a pulse number of 3s^–1 the response declines markedly. The data support our hypothesis that imposed electric fields induce cell polarityvia differential shift of the membrane potential rather than transcellular current flow. Furthermore, the given dose-response curves strikingly resemble those due to the other morphogenetically active signals: percent response consistently approximates the per cent signal intensity gradient which evokes it.     

Maturation of Receptor Proteins in Eukaryotic Expression Systems

Abstract

Transient and stable expression in eukaryotic cells is commonly used to examine receptor function. Characterization of the V2 vasopressin receptor synthesized in transiently transfected cells revealed the presence of large quantities of immature protein and a small fraction of fully mature protein. The immature protein was characterized by its sensitivity to endoglycosidase H treatment, abnormal migration in SDS PAGE, and a tendency to form aggregates. Prevention of protein glycosylation by mutagenesis increased the fraction of mature protein produced, but did not eliminate the need for the maturation step. On the other hand, stably transfected cells produce almost exclusively mature receptor protein with a t_1/2 of 6 h, while the immature form has a t_1/2 of 20 min. In the absence of N-linked glycosylation the t_1/2 of the mature V2 receptor in stably transfected cells was reduced to 4.5 h. In transient expression experiments the immature receptor proteins exhibited a prolonged t_1/2 of about 8 h. Comparison of the half life of the immature form of the wild type and the R137H mutant V2 receptor did not reveal differences despite the lower amounts of mutant mature receptor detected by binding.