Shallow Dependence Research Articles

Retardation of acetal hydrolysis by cyclodextrins and its use in probing cyclodextrin–guest binding

Hydrolysis of benzaldehyde dimethyl acetal 1 in aqueous acid is slowed down greatly by cyclodextrins (CDs): α-CD, β-CD, hp-β-CD (hydroxypropyl-β-cyclodextrin) and γ-CD. The variations of the observed first-order rate constants (kobs) with [CD] exhibit saturation behaviour, consistent with 1∶1 binding between 1 and the CDs. In the case of β-CD and hp-β-CD, the binding is relatively strong and the CD-bound acetal is unreactive. In contrast, binding of the acetal by α-CD and γ-CD is much weaker, but only with α-CD does the CD-bound form show significant reactivity. The four CD-mediated reactions have been evaluated as probe reactions for determining dissociation constants of {CD– ‘guest’} complexes. In this approach, added guests attenuate the retarding effect of CD–substrate binding and cause an increase in the rate of acetal hydrolysis. The method works well for aliphatic alcohols and ketones binding to β-CD and hp-β-CD, but it is less successful with α-CD because of the shallow dependence of kobs on [α-CD] in the probe reaction. With γ-CD, the approach is not applicable at all, because added guests cause a further reduction in the rate of acetal hydrolysis, not an increase. Various implications of these findings are discussed.

An unusual pH-independent and metal-ion-independent mechanism for hairpin ribozyme catalysis.

Hairpin ribozymes (RNA enzymes) catalyze the same chemical reaction as ribonuclease A and yet RNAs do not usually have functional groups analogous to the catalytically essential histidine and lysine sidechains of protein ribonucleases. Some RNA enzymes appear to recruit metal ions to act as Lewis acids in charge stabilization and metal-bound hydroxide for general base catalysis, but it has been reported that the hairpin ribozyme functions in the presence of metal ion chelators. This led us to investigate whether the hairpin ribozyme exploits a metal-ion-independent catalytic strategy. Substitution of sulfur for nonbridging oxygens of the reactive phosphate of the hairpin ribozyme has small, stereospecific and metal-ion-independent effects on cleavage and ligation mediated by this ribozyme. Cobalt hexammine, an exchange-inert metal complex, supports full hairpin ribozyme activity, and the ribozyme's catalytic rate constants display only a shallow dependence on pH. Direct metal ion coordination to phosphate oxygens is not essential for hairpin ribozyme catalysis and metal-bound hydroxide does not serve as the general base in this catalysis. Several models might account for the unusual pH and metal ion independence: hairpin cleavage and ligation might be limited by a slow conformational change; a pH-independent or metal-cation-independent chemical step, such as breaking the 5' oxygen-phosphorus bond, might be rate determining; or finally, functional groups within the ribozyme might participate directly in catalytic chemistry. Whichever the case, the hairpin ribozyme appears to employ a unique strategy for RNA catalysis.

Identification and analysis of long-range electrostatic effects in proteins by computer modeling:aspartate transcarbamylase.

While ion pairs are readily identified in crystal structures, longer range electrostatic interactions cannot be identified from the three-dimensional structure alone. These interactions are likely to be important in large, multisubunit proteins that are regulated by allosteric interactions. In this paper, we show that these interactions are readily detected by electrostatic modeling, using, as an example, unliganded Escherichia coli aspartate transcarbamylase, a widely studied allosteric enzyme with 12 subunits and a molecular weight of 310 kD. The Born, dipolar, and site-site interaction terms of the free energy of protonation of the 810 titratable sites in the holoenzyme were calculated using the multigrid solution of the nonlinear Poisson-Boltzmann equation. Calculated titration curves are in good agreement with experimental titration curves, and the structural asymmetry observed in the crystal structure is readily apparent in the calculated free energies and pK1/2 values. Most of the residues with pK1/2 values that differ substantially from those of model compounds are buried in the low dielectric medium of the protein, particularly at the intersubunit interfaces. The dependence of the site-site interaction free energies on distance is complex, with a steep dependence at distances less than 5 A and a more shallow dependence at longer distances. Interactions over distances of 6 to 15 A require a bridging residue and are often not apparent in the structure. The network of interactions between ionizable groups extends across and between subunits and provides a potential mechanism for transmitting long-range structural effects and allosteric signals.

Imaging the HL Tauri Disk at λ = 2.7 Millimeters with the BIMA Array

We have obtained a subarcsecond image of the disk associated with the T Tauri star HL Tau at a wavelength of 2.7 mm using the new high-resolution capability of the BIMA Array. The disk is elongated with a deconvolved Gaussian source size of 10 ± 02 × 05 ± 02, implying a semimajor axis of 70 ± 15 AU for a distance of 140 pc; the minor axis may be unresolved. The position angle of the major axis (125° ± 10°) is orthogonal to the axis of the optical jet. The disk centroid is coincident with the VLA λ = 3.6 cm source position and nearly coincident with recent measurements of the near-infrared emission peak. The λ = 2.7 mm images, along with previous interferometric measurements at λ = 0.87 mm and flux measurements from 10 μm to 1.3 cm, are well fitted by a simple power-law disk model with a shallow radial dependence to the surface density [Σ(r) ∝ r0 to r-1], an outer radius between 90 and 160 AU, and a dust opacity law proportional to ν1.

Stereoselective block of a human cardiac potassium channel (Kv1.5) by bupivacaine enantiomers

Stereoselective drug-channel interactions may help to elucidate the molecular basis of voltage-gated potassium channel block by local anesthetic drugs. We studied the effects of the enantiomers of bupivacaine on a cloned human cardiac potassium channel (hKv1.5). This channel was stably expressed in a mouse Ltk- cell line and studied using the whole-cell configuration of the patch-clamp technique. Both enantiomers modified the time course of this delayed rectifier current. Exposure to 20 microM of either S(-)-bupivacaine or R(+)-bupivacaine did not modify the activation time constant of the current, but reduced the peak outward current and induced a subsequent exponential decline of current with time constants of 18.7 +/- 1.1 and 10.0 +/- 0.9 ms, respectively. Steady-state levels of block (assessed with 250-ms depolarizing pulses to +60 mV) averaged 30.8 +/- 2.5% (n = 6) and 79.5 +/- 3.2% (n = 6) (p < 0.001), for S(-)- and R(+)-bupivacaine, respectively. The concentration dependence of hKv1.5 inhibition revealed apparent KD values of 27.3 +/- 2.8 and 4.1 +/- 0.7 microM for S(-)-bupivacaine and R(+)-bupivacaine, respectively, with Hill coefficients close to unity, suggesting that binding of one enantiomer molecule per channel was sufficient to block potassium permeation. Analysis of the rate constants of association (k) and dissociation (l) yielded similar values for l (24.9 s-1 vs. 23.6 s-1 for S(-)- and R(+)-bupivacaine, respectively) but different association rate constants (1.0 x 10(6) vs. 4.7 x 10(6) M-1 s-1 for S(-)- and R(+)-bupivacaine, respectively). Block induced by either enantiomer displayed a shallow voltage dependence in the voltage range positive to 0 mV, i.e., where the channel is fully open, consistent with an equivalent electrical distance delta of 0.16 +/- 0.01. This suggested that at the binding site, both enantiomers of bupivacaine experienced 16% of the applied transmembrane electrical field, referenced to the inner surface. Both bupivacaine enantiomers reduced the tail current amplitude recorded on return to -40 mV and slowed their time course relative to control, resulting in a "crossover" phenomenon. These data indicate 1) the charged form of both bupivacaine enantiomers block the hKv1.5 channel after it opens, 2) binding occurs within the transmembrane electrical field, 3) unbinding is required before the channel can close, 4) block of hKv1.5 channels by bupivacaine is markedly stereoselective, with the R(+)-enantiomer being the more potent one, 5) this stereoselective block was associated with a 1.11 -kcal/mol difference in binding energy between both enantiomers, and 6) the stereoselectivity derives mainly from a difference in the association rate constants, suggesting that the S(-)-enantiomer is less likely to access the binding site in an optimal configuration.

Inwardly rectifying currents of saccular hair cells from the leopard frog

1. Inwardly rectifying currents were characterized in sensory hair cells isolated from the saccules of leopard frogs, using the whole cell configuration of the patch-clamp technique in voltage-clamp mode. 2. Two types of inwardly rectifying currents were distinguishable based on their ionic selectivity, activation and deactivation kinetics, voltage dependence, dependence on external K+ and sensitivity to divalent cations. 3. One inwardly rectifying current displayed K+ selectivity, rapid monoexponential activation (tau approximately 1 ms at -120 mV), steep voltage dependence, dependence of the activation voltage range on external K+ and block by external Ba2+. We refer to this current as IK1, consistent with the terminology used for a similar current in cardiac cells. In 5 mM external K+, IK1 activated negative to -60 mV, was half-activated at -86 mV and fully activated by -110 mV. 4. The other inwardly rectifying current was a mixed K+/Na+ current with slow sigmoidal activation (slow tau approximately 100 ms at -120 mV) and deactivation, shallow voltage dependence and no dependence of the activation curve on external K+ and which was blocked by external Cd2+. This current was called Ih because of its similarities to Ih of photoreceptors. Ih activated negative to -50 mV, was half-activated at -90 mV and was fully activated at -130 mV. 5. A correlation between cell shape and the type of inwardly rectifying current was noted; the more spherical cells had Ih alone and the more cylindrically shaped cells had Ih and IK1. 6. The mean resting potential of 115 cells with IK1 and Ih was -68 +/- 0.5 mV (mean +/- SE) and that of 53 cells with Ih alone was -50 +/- 0.5 mV. This suggests that IK1 contributes to the more negative resting potential of the cylindrical cells. 7. In current-clamp mode, the voltage responses to current steps of the two cell populations differed. Small negative current steps evoked faster, smaller responses in cells with IK1 and Ih than in cells with Ih alone. In cells with Ih alone, long (> 100 ms) negative current steps evoked a hyperpolarization that partly repolarized as Ih activated. Cells with Ih alone showed electrical resonance at rest whereas cells with IK1 resonated only in response to positive current steps. 8. A model developed to explain electrical resonance in bullfrog saccular hair cells was adapted to include Ih or IK1 and Ih.(ABSTRACT TRUNCATED AT 400 WORDS)

Voltage dependence of Na-Ca exchanger conformational currents

Properties of a transient current (Icont) believed to reflect a conformational change of the Na-Ca exchanger molecules after Ca2+ binding were investigated. Intracellular Ca2+ concentration jumps in isolated cardiac myocytes were generated with flash photolysis of caged Ca2+ dimethoxynitrophenamine, and membrane currents were simultaneously measured using the whole-cell variant of the patch-clamp technique. A previously unresolved shallow voltage dependence of Icont was revealed after developing an experimental protocol designed to compensate for the photoconsumption of the caged compound. This voltage dependence can be interpreted to reflect the distribution of Na-Ca exchanger conformational states with the Ca2+ binding site exposed to the inside of the cell immediately before the flash. Analysis performed by fitting a Boltzmann distribution to the observed data suggests that under control conditions most exchanger molecules reside in states with the Ca2+ binding site facing the outside of the cell. Dialysis of the cytosol with 3',4'-dichlorobenzamil, an organic inhibitor of the Na-Ca exchange, increased the magnitude of Icont and changed the voltage dependence, consistent with a parallel shift of the charge/voltage curve. This shift may result from intracellular DCB interfering with an Na(+)-binding or Na(+)-translocating step. These observations are consistent with Icont arising from a charge movement mediated by the Na-Ca exchanger molecules after binding of Ca2+.

Free‐Exciton Recombination in Stoichiometry‐Controlled GaP

Shallow donor concentration dependence of the free‐exciton recombination in epitaxial layers grown by the temperature‐difference method under controlled vapor pressure (TDM‐CVP) has been investigated. The intensity of the free‐exciton recombination linearly increases with shallow donor concentration as long as , which means that the recombination is enhanced by the presence of shallow impurities. The facet regions have a difference in the shallow donor concentration dependence, which can be attributed to lower deep level concentrations there than in the non‐facet regions.

Activation of Na-Ca exchange current by photolysis of "caged calcium"

Intracellular photorelease of Ca2+ from "caged calcium" (DM-nitrophen) was used to investigate the Ca(2+)-activated currents in ventricular myocytes isolated from guinea pig hearts. The patch-clamp technique was applied in the whole-cell configuration to measure membrane current and to dialyze the cytosol with a pipette solution containing the caged compound. In the presence of inhibitors for Ca2+, K+, and Na+ channels, concentration jumps of [Ca2+]i induced a rapidly activating inward Na-Ca exchange current which then decayed slowly (tau approximately 500 ms). The initial peak of the inward current and the time-course of current decay were voltage-dependent, and no reversal of the current direction was found between -100 and +100 mV. The observed shallow voltage dependence can be described in terms of the movement of an apparently fractional elementary charge (+0.44e-) across an energy barrier located symmetrically in the electrical field of the membrane. The currents were dependent on extracellular Na+ with a half-maximal activation at 73 mM and a Hill coefficient of 2.8. No change of membrane conductance was activated by the Ca2+ concentration jump when extracellular Na+ was completely replaced by Li+ or N-methyl-D-glucamine (NMG) or when the Na-Ca exchange was inhibited by extracellular Ni2+, La3+, or dichlorobenzamil (DCB). The velocity of relengthening after a twitch induced by photorelease of Ca2+ was only reduced drastically when both the sarcoplasmic reticulum and the Na-Ca exchange were inhibited suggesting that all other Ca2+ removing mechanisms have a low transport capacity under these conditions. In conclusion, we have used a novel approach to study Na-Ca exchange activity with photolysis of "caged" calcium. We found that in guinea pig heart muscle cells the Na-Ca exchange is a potent mechanism for Ca2+ extrusion, is weakly voltage-dependent (118 mV for e-fold change) and can be studied without contamination with other Ca(2+)-activated currents.

Alkaloid-modified sodium channels from lobster walking leg nerves in planar lipid bilayers.

Alkaloid-modified, voltage-dependent sodium channels from lobster walking leg nerves were studied in planar neutral lipid bilayers. In symmetrical 0.5 M NaCl the single channel conductance of veratridine (VTD) (10 pS) was less than that of batrachotoxin (BTX) (16 pS) modified channels. At positive potentials, VTD- but not BTX-modified channels remained open at a flickery substate. VTD-modified channels underwent closures on the order of milliseconds (fast process), seconds (slow process), and minutes. The channel fractional open time (f(o)) due to the fast process, the slow process, and all channel closures (overall f(o)) increased with depolarization. The fast process had a midpoint potential (V(a)) of -122 mV and an apparent gating charge (z(a)) of 2.9, and the slow process had a V(a) of -95 mV and a z(a) of 1.6. The overall f(o) was predominantly determined by closures on the order of minutes, and had a V(a) of about -24 mV and a shallow voltage dependence (z(a) approximately 0.7). Augmenting the VTD concentration increased the overall f(o) without changing the number of detectable channels. However, the occurrence of closures on the order of minutes persisted even at super-saturating concentrations of VTD. The occurrence of these long closures was nonrandom and the level of nonrandomness was usually unaffected by the number of channels, suggesting that channel behavior was nonindependent. BTX-modified channels also underwent closures on the order of milliseconds, seconds, and minutes. Their characterization, however, was complicated by the apparent low BTX binding affinity and by an apparent high binding reversibility (channel disappearance) of BTX to these channels. VTD- but not BTX-modified channels inactivated slowly at high positive potentials (greater than +30 mV). Single channel conductance versus NaCl concentrations saturated at high NaCl concentrations and was non-Langmuirian at low NaCl concentrations. At all NaCl concentrations the conductance of VTD-modified channels was lower than that of BTX-modified channels. However, this difference in conductance decreased as NaCl concentrations neared zero, approaching the same limiting value. The permeability ratio of sodium over potassium obtained under mixed ionic conditions was similar for VTD (2.46)- and BTX (2.48)-modified channels, whereas that obtained under bi-ionic conditions was lower for VTD (1.83)- than for BTX (2.70)-modified channels. Tetrodotoxin blocked these alkaloid-modified channels with an apparent binding affinity in the nanomolar range.

Heterogeneity of V 2O 5/Al 2O 3 as studied by water vapour adsorption microcalorimetry

The adsorption of water vapour on 773 K-outgassed γ-Al 2O 3, V 2O 5 and alumina-supported V 2O 5 has been studied calorimetrically at 303 K. Measurements have been made incrementally in the region of sub-monolayer H 2O coverage in order to map the hydrophilicity of the surface. For γ-Al 2O 3 the heat of adsorption is initially very high (155 kJ mol −1) and about 30% of the surface chemisorbs water with a heat of adsorption greater than 120 kJ mol −1. The heat then falls rapidly with increasing coverage, reaching 55–60 kJ mol −1 at the monolayer value. V 2O 5, by contrast, is much less hydrophilic, with the heat of adsorption initially 90 kJ mol −1 and a more shallow dependence of the heat on coverage to give a final value of 55 kJ mol −1. This distinction in hydrophilicity between the two oxides has been exploited to investigate V 2O 5 supported on γ-Al 2O 3 in the region of loading up to an amount capable of giving monolayer coverage of vanadia. Oxides containing 10, 15 and 20 wt.% V 2O 5 have been compared. 10 wt.% loading, capable of ∼40% coverage if spread as a monolayer, leads to a marked change in the hydrophilic profile of alumina. The high energy sites are eliminated preferentially. Increasing the loading to 15 and 20 wt.% produces further changes, but they are relatively much smaller. On the basis of the measured water vapour isotherms an estimate is made of the vanadia coverage. The conclusion is that up to 10 wt.% V 2O 5 there is monolayering of the alumina by vanadia, but that further loading leads to much less effective spreading. At 20 wt.% loading there is still much alumina surface which is not covered by vanadia.

A Critical Review of the Ferric Chloride Leaching of Galena

Abstract Ferric chloride leaching is a potentially attractive method of processing lead concentrates, and many studies have been carried out on the kinetics of dissolution of galena (PbS). In this paper, the extensive literature on galena dissolution is critically reviewed and the various results are compared on a consistent basis. There is a consensus that the leaching reaction is rapid and that elemental sulphur is the dominant reaction product. Although quantitative agreement among the various studies is lacking because of differences in galena purity, experimental procedures and methods, and data interpretation, the overall trends indicate a shallow temperature dependence, a direct total chloride concentration effect and a modest influence of solution agitation. Furthermore, there is an abrupt change in the reaction mechanism at FeCl3 concentrations less than ∼0.1 M. The experimental observations are consistent with rate control either by the outward diffusion of the PbCl2 reaction product through the...

Veratridine modification of the purified sodium channel alpha-polypeptide from eel electroplax.

In the interest of continuing structure-function studies, highly purified sodium channel preparations from the eel electroplax were incorporated into planar lipid bilayers in the presence of veratridine. This lipoglycoprotein originates from muscle-derived tissue and consists of a single polypeptide. In this study it is shown to have properties analogous to sodium channels from another muscle tissue (Garber, S. S., and C. Miller. 1987. Journal of General Physiology. 89:459-480), which have an additional protein subunit. However, significant qualitative and quantitative differences were noted. Comparison of veratridine-modified with batrachotoxin-modified eel sodium channels revealed common properties. Tetrodotoxin blocked the channels in a voltage-dependent manner indistinguishable from that found for batrachotoxin-modified channels. Veratridine-modified channels exhibited a range of single-channel conductance and subconductance states. The selectivity of the veratridine-modified sodium channels for sodium vs. potassium ranged from 6-8 in reversal potential measurements, while conductance ratios ranged from 12-15. This is similar to BTX-modified eel channels, though the latter show a predominant single-channel conductance twice as large. In contrast to batrachotoxin-modified channels, the fractional open times of these channels had a shallow voltage dependence which, however, was similar to that of the slow interaction between veratridine and sodium channels in voltage-clamped biological membranes. Implications for sodium channel structure are discussed.

Influence of lipid headgroup on the specificity and exchange dynamics in lipid-protein interactions. A spin-label study of myelin proteolipid apoprotein-phospholipid complexes.

The pH and salt dependences of the interaction of phosphatidic acid, phosphatidylserine, and stearic acid with myelin proteolipid apoprotein (PLP) in dimyristoylphosphatidylcholine (DMPC) recombinants have been studied by electron spin resonance spectroscopy, using spin-labeled lipids. The two-component spin-label spectra have been analyzed both by spectral subtraction and by simulation using the exchange-coupled Bloch equations to give the fraction of lipids motionally restricted by the protein and the rate of lipid exchange between the fluid and motionally restricted lipid populations. For stearic acid, phosphatidic acid, and phosphatidylserine, the fraction of motionally restricted spin-label increases with increasing pH, with pKa's of 7.7, 7.6, and ca. 9.4, respectively. The corresponding pKa's for the bulk lipid regions of the bilayer are estimated, from changes in the ESR spectra, to be 6.7, 7.4, and 11, respectively. In the dissociated state at pH 9.0, the fraction of motionally restricted component decreases with increasing salt concentration, reaching an approximately constant value at [NaCl] = 0.5-1.0 M for all three negatively charged lipids. The net decreases for stearic acid and phosphatidic acid are considerably smaller (by ca. 30%) than those obtained on protonating the two lipids, whereas for phosphatidylserine the fraction of motionally restricted lipid in high salt is reduced to that corresponding to phosphatidylcholine. For a fixed lipid/protein ratio, the on-rate for exchange at the lipid-protein interface is independent of the degree of selectivity and has a shallow temperature dependence, as expected for a diffusion-controlled process.(ABSTRACT TRUNCATED AT 250 WORDS)

Dose‐response of acetylcholine receptor channels opened by a flash‐activated agonist in voltage‐clamped rat myoballs.

Whole-cell or single-channel currents through acetylcholine (ACh) receptor channels were studied in voltage-clamped rat myoballs or in excised membrane patches from myoballs. The recording pipette contained CsCl to suppress outward currents, and tetrodotoxin was used to help suppress Na+ currents. To minimize problems associated with bath applied agonists, myoballs were bathed in a solution containing the inactive (cis) isomer of the photo-isomerizable azobenzene derivative, Bis-Q. Calibrated light flashes of varying intensity were presented to produce concentration jumps of agonist, trans-Bis-Q. The resulting whole-cell current relaxations through ACh channels approach a steady state along an exponential time course, then decline as the newly created agonist diffuses away over the next few seconds. The dose-response relationship was inferred from Hill (double-log) plots for myoballs bathed in 500 nM-cis-Bis-Q at three membrane potentials. At low agonist concentrations (less than 300 nM-trans-Bis-Q), the slope of the Hill plot averaged 1.62 at -150 mV, 1.89 at -100 mV, and 2.05 at +80 mV. These results are consistent with an apparent agonist affinity constant that decreases with membrane depolarization and shifts the responses further down on the dose-response curve. When the myoballs were bathed in higher concentrations of cis-Bis-Q (1.5-20 microM), the slope of the Hill plot was reduced at all membrane potentials, although it was still closer to two at positive potentials. This is expected from the known sigmoid shape of the dose-response relation. The shallow dependence of the Hill slope on agonist concentration suggests the presence of negative cooperativity in the over-all binding of agonist molecules. Following treatment of the membrane with dithiothreitol to reduce disulphide groups, the Hill slope for the reversibly bound agonist, trans-Bis-Q, remained near two. The kinetics of currents at hyperpolarized membrane potentials became complicated at higher agonist concentrations in a manner that was consistent with open-channel block by trans-Bis-Q; the currents showed a slow secondary increase in conductance. Averaged single-channel recordings at higher agonist concentrations resemble macroscopic relaxations under comparable conditions. Furthermore, those recordings also suggested that open channels are blocked by trans-Bis-Q at concentrations greater than 2 microM; the block depends strongly on membrane potential and increases with hyperpolarization. Currents at positive membrane potentials showed no evidence of open-channel block.(ABSTRACT TRUNCATED AT 400 WORDS)

Inverse Relations between 0.25 keV counts and local interstellar dust

A preliminary comparison of color excesses and HEA0-1, A2 0.25 keV counts have been performed on two angular levels in order to search for indications of local photoelectric absorption.a. Color excesses and background counts are smoothed to a common 1.5 1.5 sq.deg. beam. Although distance information on the color excesses are available it is not used in this first attempt. The lines of sight to which the excesses pertain have however a length comparable to the expected viewing distance at these energies in the local medium. Figure 1 displays the resulting average counts/pixel versus average excess/pixel for the five areas investigated. A general decrease of the counts with excess is apparent. The wide scatter in the individual regions is probably due to excess variations on scales smaller than the collimated beam. The minimum counts on the diagram come from a region where an almost coherent sheet of dust is present within a distance ~200 pc. The constancy of the minimum counts in this particular direction indicates that the soft X-ray emission probably originate exclusively in front of the matter observed to be present. Note that the SGP counts show a shallow inverse dependence on excess. The latitude distribution of the five areas assures that the general inverse relation of the 0.25 keV counts on color excesses is not a latitude effect.

Properties of single-step mutants of Syrian hamster cell lines resistant to N-(phosphonacetyl)-L-aspartate.

Eleven independent lines of Syrian hamster cells were selected by using very low levels of N-(phosphonacetyl)-L-aspartate (PALA), an inhibitor of aspartate transcarbamylase. The protocol employed insured that each resistant cell arose during one of the last divisions before selection was applied. Cells of each mutant line contained an amplification of the structural gene for CAD, a trifunctional protein which includes aspartate transcarbamylase and two other enzymes of UMP biosynthesis. Strikingly, despite the minimal selection employed, the degree of amplification of the CAD gene was 6 to 10 times the normal diploid number in all 11 cases. In situ hybridization indicated that the amplified CAD genes were almost always present at a single chromosomal site in each line. Therefore, one of the two alleles was amplified 11- to 19-fold. The rates at which cells became resistant to PALA, determined by fluctuation analysis, were 100 times less dependent on drug concentration than were the frequencies of resistant cells in steady-state populations. The relatively shallow dependence of this rate upon PALA concentration is consistent with our independent observation that most events gave rise to a similar degree of amplification. In six of six cell lines examined, the levels of CAD mRNA and aspartate transcarbamylase activity were elevated two- to fourfold. These lines were resistant to PALA concentrations 20- to 80-fold higher than the ones used for selection. The organization of amplified DNA was examined by hybridizing Southern blots with cloned DNA fragments containing amplified sequences, previously isolated from two cell lines resistant to high levels of PALA. A contiguous region of DNA approximately 44 kilobases long which included the CAD gene was amplified in five of five single-step mutants examined. Outside this region, these mutants shared amplified sequences with only one of the two highly resistant lines.

A transient calcium‐dependent chloride current in the immature Xenopus oocyte.

Ionic currents were studied in immature full-grown Xenopus oocytes using the two-micro-electrode voltage-clamp technique. Recordings of total membrane current showed a transient outward peak during depolarizations from the approximate resting voltage (-70 or -80 mV) to voltages more positive than -20 mV. The current-voltage relation for peak outward current was U-shaped, with a maximum at about 0 mV. Replacement of external Cl with methanesulphonate reversed this transient outward current to a transient inward current. Current relaxations recorded after the membrane potential was stepped to different voltages at the time of the peak showed a component that inverted at about -25 to -30 mV. This value was close to ECl as determined by measurement of the intracellular Cl ion concentration. The reversal potential for these current relaxations changed with the external Cl concentration as predicted by the Nernst relation. Replacement of external Ca with Mg, Sr or Ba, or addition of low concentrations of Ni in the presence of Ca, eliminated the transient outward current. Increasing the external Ca concentration increased the amplitude of the transient outward current without affecting the amplitude of the steady-state current. It was concluded that the outward peak in records of total membrane current represented the contribution of a transient outward current carried by Cl ions which was dependent on the entry of external Ca. It will be noted as ICl(Ca). Decay of ICl(Ca) could be described at the normal Ca concentration by a single exponential function whose time constant showed a shallow U-shaped voltage dependence. ICl(Ca) was maximally activatable by depolarizations from a holding potential of about -100 mV, but could not be activated by depolarizations from -40 mV. The amplitude of ICl(Ca) showed a large temperature dependence as compared to the steady-state current, suggesting complex control of its activation.

Kinetics of amiloride action in the hen coprodaeum in vitro

The kinetics of amiloride action on the isolated epithelium of the hen coprodaeum are reported. Tissues were taken from birds fed on low salt diets for 9-10 days, conditions which induce a high resting short circuit current due to sodium and sensitive to amiloride. The relation between the inhibition of amiloride sensitive short circuit current and blocker concentration obeyed simple mass laws with an apparent stoichiometry of 1:1 between amiloride and the sodium entry sites. The concentration of amiloride producing its half maximal effect (Ki) was 1.77 +/- 0.20 microM at a sodium concentration of 130 mM. There was a shallow dependence of Ki on sodium concentration, the value of Ki falling to 0.78 +/- 0.1 microM at 1.3 mM Na. The relation of Ki to Na concentration was linear indicating competitive antagonism. The sodium concentration which half saturates the amiloride site (KNa) was 80 mM. This value is very different from the concentration of sodium which half saturates SCC (Kscc = 5-7 mM) suggesting there are at least two sites at which sodium can modify the transporting characteristics. These data are compared to those for other epithelia where Kscc and KNa are rather similar. The benzyl derivative of amiloride (benzamil) was found to be 11.6 times more potent than amiloride on this tissue. The potency ration is similar to that for other sodium transporting epithelia suggesting that the structure of the ion translocation mechanism is partly conserved between species although the Ki for amiloride may vary by an order of magnitude.

The tension-depolarization relationship of frog atrial trabeculae as determined by potassium contractures.

1. In the presence of extracellular Na ions K contractures evoked from isolated frog atrial trabeculae show an initial phasic and a subsequent tonic contractile response. 2. The phasic response shows a steep dependence on membrane potential, persists in Na-free fluid, but is blocked by Mn ions, D600 and tetracaine. It has an indirect dependence on the [Ca]0 and would seem to be associated with both the secondary inward current and the release of Ca2+ from intracellular stores. 3. The tonic component of the K contracture is unaffected by D600 or tetracaine, shows a shallow dependence on membrane potential but is absent in Na-free fluid. Its tension-depolarization curve is immediately affected by alteration of either the [Ca]0 or the [Na]0. The form of the tension-depolarization relationship and the effects of [Ca]0 and [Na]0 are consistent with the strength of the tonic tension being determined by a 3Na+ for 1Ca2+ exchange across the cell membrane. 4. The results agree well with those obtained previously with voltage-clamp experiments on the same tissue, and may also help with the interpretation of Ca-flux experiments.