Transient-state and steady-state kinetic studies of the mechanism of NADH-dependent aldehyde reduction catalyzed by xylose reductase from the yeast Candida tenuis

Microbial xylose reductase, a representative aldo-keto reductase of primary sugar metabolism, catalyzes the NAD(P)H-dependent reduction of D-xylose with a turnover number approximately 100 times that of human aldose reductase for the same reaction. To determine the mechanistic basis for that physiologically relevant difference and pinpoint features that are unique to the microbial enzyme among other aldo/keto reductases, we carried out stopped-flow studies with wild-type xylose reductase from the yeast Candida tenuis. Analysis of transient kinetic data for binding of NAD(+) and NADH, and reduction of D-xylose and oxidation of xylitol at pH 7.0 and 25 degrees C provided estimates of rate constants for the following mechanism: E + NADH right arrow over left arrow E.NADH right arrow over left arrow E.NADH + D-xylose right arrow over left arrow E.NADH.D-xylose right arrow over left arrow E.NAD(+).xylitol right arrow over left arrow E.NAD(+) right arrow over left arrow E.NAD(+) right arrow over left arrow E + NAD(+). The net rate constant of dissociation of NAD(+) is approximately 90% rate limiting for k(cat) of D-xylose reduction. It is controlled by the conformational change which precedes nucleotide release and whose rate constant of 40 s(-)(1) is 200 times that of completely rate-limiting E.NADP(+) --> E.NADP(+) step in aldehyde reduction catalyzed by human aldose reductase [Grimshaw, C. E., et al. (1995) Biochemistry 34, 14356-14365]. Hydride transfer from NADH occurs with a rate constant of approximately 170 s(-1). In reverse reaction, the E.NADH --> E.NADH step takes place with a rate constant of 15 s(-1), and the rate constant of ternary-complex interconversion (3.8 s(-1)) largely determines xylitol turnover (0.9 s(-1)). The bound-state equilibrium constant for C. tenuis xylose reductase is estimated to be approximately 45 (=170/3.8), thus greatly favoring aldehyde reduction. Formation of productive complexes, E.NAD(+) and E.NADH, leads to a 7- and 9-fold decrease of dissociation constants of initial binary complexes, respectively, demonstrating that 12-fold differential binding of NADH (K(i) = 16 microM) vs NAD(+) (K(i) = 195 microM) chiefly reflects difference in stabilities of E.NADH and E.NAD(+). Primary deuterium isotope effects on k(cat) and k(cat)/K(xylose) were, respectively, 1.55 +/- 0.09 and 2.09 +/- 0.31 in H(2)O, and 1.26 +/- 0.06 and 1.58 +/- 0.17 in D(2)O. No deuterium solvent isotope effect on k(cat)/K(xylose) was observed. When deuteration of coenzyme selectively slowed the hydride transfer step, (D)()2(O)(k(cat)/K(xylose)) was inverse (0.89 +/- 0.14). The isotope effect data suggest a chemical mechanism of carbonyl reduction by xylose reductase in which transfer of hydride ion is a partially rate-limiting step and precedes the proton-transfer step.     

Cl- channels in intact human T lymphocytes.

We recently described a large, multiple-conductance Cl- channel in excised patches from normal T lymphocytes. The properties of this channel in excised patches are similar to maxi-Cl- channels found in a number of cell types. The voltage dependence in excised patches permitted opening only at nonphysiological voltages, and channel activity was rarely seen in cell-attached patches. In the present study, we show that Cl- channels can be activated in intact cells at physiological temperatures and voltages and that channel properties change after patch excision. Maxi-Cl- channels were reversibly activated in 69% of cell-attached patches when the temperature was above 32 degrees C, whereas fewer than 2% of patches showed activity at room temperature. Upon excision, the same patches displayed large, multiple-conductance Cl- channels with characteristics like those we previously reported for excised patches. After patch excision, warm temperatures were not essential to allow channel activity; 37% (114/308) of inside-out patches had active channels at room temperature. The voltage dependence of the channels was markedly different in cell-attached recordings compared with excised patches. In cell-attached patches, Cl- channels could be open at cell resting potentials in the normal range. Channel activation was not related to changes in intracellular Ca2+ since neither ionomycin nor mitogens activated the channels in cell-attached patches, Ca2+ did not rise in response to warming and the Cl- channel was independent of Ca2+ in inside-out patches. Single-channel currents were blocked by internal or external Zn2+ (100-200 microM), 4-acetamido-4' isothiocyanostilbene-2,2'-disulfonate (SITS, 100-500 microM) and 4,4'-diisothiocyanostilbene 2,2'-disulfonate (DIDS, 100 microM). NPPB (5-nitro-2-(3-phenylpropylamino)-benzoate) reversibly blocked the channels in inside-out patches.     

Methylation increases the open probability of the epithelial sodium channel in A6 epithelia

We used single channel methods on A6 renal cells to study the regulation by methylation reactions of epithelial sodium channels. 3-Deazaadenosine (3-DZA), a methyltransferase blocker, produced a 5-fold decrease in sodium transport and a 6-fold decrease in apical sodium channel activity by decreasing channel open probability (P(o)). 3-Deazaadenosine also blocked the increase in channel open probability associated with addition of aldosterone. Sodium channel activity in excised "inside-out" patches usually decreased within 1-2 min; in the presence of S-adenosyl-l-methionine (AdoMet), activity persisted for 5-8 min. Sodium channel mean time open (t(open)) before and after patch excision was higher in the presence of AdoMet than in untreated excised patches but less than t(open) in cell-attached patches. Sodium channel activity in excised patches exposed to both AdoMet and GTP usually remained stable for more than 10 min, and P(o) and the number of active channels per patch were close to values in cell-attached patches from untreated cells. These findings suggest that a methylation reaction contributes to the activity of epithelial sodium channels in A6 cells and is directed to some regulatory element closely connected with the channel, whose activity also depends on the presence of intracellular GTP.     

Light-dependent K(+) channels in the mollusc Onchidium simple photoreceptors are opened by cGMP

Light-dependent K(+) channels underlying a hyperpolarizing response of one extraocular (simple) photoreceptor, Ip-2 cell, in the marine mollusc Onchidium ganglion were examined using cell-attached and inside-out patch-clamp techniques. A previous report (Gotow, T., T. Nishi, and H. Kijima. 1994. Brain Res. 662:268-272) showed that a depolarizing response of the other simple photoreceptor, A-P-1 cell, results from closing of the light-dependent K(+) channels that are activated by cGMP. In the cell-attached patch recordings of Ip-2 cells, external artificial seawater (ASW) was replaced with a modified ASW containing 150 mM K(+) and 200 mM Mg(2+) to suppress any synaptic input and to maintain the membrane potential constant. When Ip-2 cells were equilibrated with this modified ASW, the internal K(+) concentration was estimated to be 260 mM. Light-dependent single-channels in the cell-attached patch on these cells were opened by light but scarcely by voltage. After confirming the light-dependent channel activity in the cell-attached patches, an application of cGMP to the excised inside-out patches newly activated a channel that disappeared on removal of cGMP. Open and closed time distributions of this cGMP-activated channel could be described by the sum of two exponents with time constants tau(o1), tau(o2) and tau(c1), tau(c2), respectively, similar to those of the light-dependent channel. In both the channels, tau(o1) and tau(o2) in ms ranges were similar to each other, although tau(c2) over tens of millisecond ranges was different. tau(o1), tau(o2), and the mean open time tau(o) were both independent of light intensity, cGMP concentration, and voltage. In both channels, the open probability increased as the membrane was depolarized, without changing any of tau(o2) or tau(o). In both, the reversal potentials using 200- and 450-mM K(+)-filled pipettes were close to the K(+) equilibrium potentials, suggesting that both the channels are primarily K(+) selective. Both the mean values of the channel conductance were estimated to be the same at 62 and 91 pS in 200- and 450-mM K(+) pipettes at nearly 0 mV, respectively. Combining these findings with those in the above former report, it is concluded that cGMP is a second messenger which opens the light-dependent K(+) channel of Ip-2 to cause hyperpolarization, and that the channel is the same as that of A-P-1 closed by light.     

Structural and mechanistic insight into the inhibition of aspartic proteases by a slow-tight binding inhibitor from an extremophilic Bacillus sp.: correlation of the kinetic parameters with the inhibitor induced conformational changes

We present here the first report of a hydrophilic peptidic inhibitor, ATBI, from an extremophilic Bacillus sp. exhibiting a two-step inhibition mechanism against the aspartic proteases, pepsin and F-prot from Aspergillus saitoi. Kinetic analysis shows that these proteases are competitively inhibited by ATBI. The progress curves are time-dependent and consistent with slow-tight binding inhibition: E + I right arrow over left arrow (k(3), k(4)) EI right arrow over left arrow (k(5), k(6)) EI. The K(i) values for the first reversible complex (EI) of ATBI with pepsin and F-prot were (17 +/- 0.5) x 10(-9) M and (3.2 +/- 0.6) x 10(-6) M, whereas the overall inhibition constant K(i) values were (55 +/- 0.5) x 10(-12) M and (5.2 +/- 0.6) x 10(-8) M, respectively. The rate constant k(5) revealed a faster isomerization of EI for F-prot [(2.3 +/- 0.4) x 10(-3) s(-1)] than pepsin [(7.7 +/- 0.3) x 10(-4) s(-1)]. However, ATBI dissociated from the tight enzyme-inhibitor complex (EI) of F-prot faster [(3.8 +/- 0.5) x 10(-5) s(-1)] than pepsin [(2.5 +/- 0.4) x 10(-6) s(-1)]. Comparative analysis of the kinetic parameters with pepstatin, the known inhibitor of pepsin, revealed a higher value of k(5)/k(6) for ATBI. The binding of the inhibitor with the aspartic proteases and the subsequent conformational changes induced were monitored by exploiting the intrinsic tryptophanyl fluorescence. The rate constants derived from the fluorescence data were in agreement with those obtained from the kinetic analysis; therefore, the induced conformational changes were correlated to the isomerization of EI to EI. Chemical modification of the Asp or Glu by WRK and Lys residues by TNBS abolished the antiproteolytic activity and revealed the involvement of two carboxyl groups and one amine group of ATBI in the enzymatic inactivation.     

Cyanine dyes as intercalating agents: kinetic and thermodynamic studies on the DNA/Cyan40 and DNA/CCyan2 systems

The interaction of cyanines with nucleic acids is accompanied by intense changes of their optical properties. Consequently these molecules find numerous applications in biology and medicine. Since no detailed information on the binding mechanism of DNA/cyanine systems is available, a T-jump investigation of the kinetics and equilibria of binding of the cyanines Cyan40 [3-methyl-2-(1,2,6-trimethyl-4(1H)pyridinylidenmethyl)-benzothiazolium ion] and CCyan2 [3-methyl-2-[2-methyl-3-(3-methyl-2(3H)-benzothiazolylidene)-1-propenyl]-benzothiazolium ion] with CT-DNA is performed at 25 degrees C, pH 7 and various ionic strengths. Bathochromic shifts of the dye absorption band upon DNA addition, polymer melting point displacement (DeltaT = 8-10 degrees C), site size determination (n = 2), and stepwise kinetics concur in suggesting that the investigated cyanines bind to CT-DNA primary by intercalation. Measurements with poly(dA-dT).poly(dA-dT) and poly(dG-dC).poly(dG-dC) reveal fair selectivity of CCyan2 toward G-C basepairs. T-jump experiments show two kinetic effects for both systems. The binding process is discussed in terms of the sequence D + S left arrow over right arrow D,S left arrow over right arrow DS(I) left arrow over right arrow DS(II), which leads first to fast formation of an external complex D,S and then to a partially intercalated complex DS(I) which, in turn, converts to DS(II), a more stable intercalate. Absorption spectra reveal that both dyes tend to self-aggregate; the kinetics of CCyan2 self-aggregation is studied by T-jump relaxation and the results are interpreted in terms of dimer formation.     

Modal gating behavior of cardiac sodium channels in cell-free membrane patches.

Single voltage-activated Na+ channel currents were obtained from membrane patches of isolated ventricular cells of guinea pig hearts. The currents were compared when measured from cell-attached patches and from the same patch but at least 20 minutes after manual excision. The averaged currents showed a distinctly delayed decay in the excised patches due to the appearance of long lasting openings or bursts of openings. In contrast to control patches, the open time distribution in excised patches requires at least two exponentials. A short mean open time was voltage independent for cell-attached patches (0.38 ms +/- 0.07 ms between -60 and -20 mV, 6 cell-attached patches; and 0.41 +/- 0.1 ms, 7 excised patches). The long mean open time found in excised patches was clearly voltage dependent and increased from 0.48 +/- 0.14 ms (-80 mV) to 2.87 +/- 0.35 ms (-20 mV, regression coefficient +0.88, 7 patches). Sweeps with long openings appeared in clusters. The clustering of records with long openings, short openings, or without openings (nulls) was quantified by a runs analysis which showed a highly significant nonrandom ordering. The results show that in excised patches inactivation is temporally hibernating.     

Interleukin-1 beta folding between pH 5 and 7: experimental evidence for three-state folding behavior and robust transition state positions late in folding

The thermodynamic stability and folding kinetics of the all beta-sheet protein interleukin-1beta were measured between 0 and 4 M GdmCl concentrations and pH 5-7. Native interleukin-1beta undergoes a 3.5 kcal/mol decrease in thermodynamic stability, Delta, as pH is increased from 5 to 7. The native state parameter m(NU), measuring protein destabilization/[GdmCl], remains constant between pH 5 and 7, indicating that the solvent-exposed surface area difference between the native state and unfolded ensemble is unchanged across this pH range. Similarly, pH changes between 5 and 7 decrease only the thermodynamic stability, DeltaG(H)2(O), and not the m-values, of the kinetic intermediate and transition states. This finding is shown to be consistent with transition state configurations which continue to be the high-energy configurations of the transition state in the face of changing stability conditions. A three-state folding mechanism U right arrow over left arrow I right arrow over left arrow N is shown to be sufficient in characterizing IL-1beta folding under all conditions studied. The m-values of refolding transitions are much larger than the m-values of unfolding transitions, indicating that that the fast, T(2) (U right arrow over left arrow I), and slow, T(1) (I right arrow over left arrow N), transition states are highly similar to the intermediate I and native state N, respectively. Many of the folding properties of interleukin-1beta are shared among other members of the beta-trefoil protein family, although clear differences can exist.     

Compact dimension of denatured states of staphylococcal nuclease

Fluorescence and circular dichroism stopped-flow have been widely used to determine the kinetics of protein folding including folding rates and possible folding pathways. Yet, these measurements are not able to provide spatial information of protein folding/unfolding. Especially, conformations of denatured states cannot be elaborated in detail. In this study, we apply the method of fluorescence energy transfer with a stopped-flow technique to study global structural changes of the staphylococcal nuclease (SNase) mutant K45C, where lysine 45 is replaced by cysteine, during folding and unfolding. By labeling the thiol group of cysteine with TNB (5,5'-dithiobis-2-nitrobenzoic acid) as an energy acceptor and the tryptophan at position 140 as a donor, distance changes between the acceptor and the donor during folding and unfolding are measured from the efficiency of energy transfer. Results indicate that the denatured states of SNase are highly compact regardless of how the denatured states (pH-induced or GdmCl-induced) are induced. The range of distance changes between two probes is between 25.6 and 25.4 A while it is 20.4 A for the native state. Furthermore, the folding process consists of three kinetic phases while the unfolding process is a single phase. These observations agree with our previous sequential model: N(0) left arrow over right arrow D(1) left arrow over right arrow D(2) left arrow over right arrow D(3) (Chen et al., J Mol Biol 1991;220:771-778). The efficiency of protein folding may be attributed to initiating the folding process from these compact denatured structures.     

Depolarization increases the single-channel conductance and the open probability of crayfish glutamate channels.

We have studied the voltage sensitivity of glutamate receptors in outside-out patches taken from crayfish muscles. We found that single-channel conductance, measured directly at the single-channel level, increases as depolarization rises. At holding potentials from -90 mV to approximately 20 mV, the conductance is 109 pS. At holding potentials positive to 20 mV, the conductance is 213 pS. This increase in single-channel conductance was also observed in cell-attached patches. In addition, desensitization, rise time, and the dose-response curve were all affected by depolarization. To further clarify these multifaceted effects, we evaluated the kinetic properties of single-channel activity recorded from cell-attached patches in hyperpolarization (membrane potential around -75 mV) and depolarization (membrane potential approximately 105 mV). We found that the glutamate dissociation rate constant (k_) was affected most significantly by membrane potential; it declined 6.5-fold under depolarization. The rate constant of channel closing (k(c)) was also significantly affected; it declined 1.8-fold. The rate constant of channel opening (k(o)) declined only 1.2-fold. The possible physiological significance of the depolarization-mediated changes in the above rate constants is discussed.     

Kinetic properties of single sodium channels in rat heart and rat brain

Single Na channel currents were compared in ventricular myocytes and cortical neurons of neonatal rats using the gigaseal patch-clamp method to determine whether tissue-specific differences in gating can be detected at the single-channel level. Single-channel currents were recorded in cell-attached and excised membrane patches at test potentials of -70 to -20 mV and at 9-11 degrees C. In both cell-attached and excised patches brain Na channel mean open time progressively increased from less than 1 ms at -70 mV to approximately 2 ms at -20 mV. Near threshold, single openings with dispersed latencies were observed. By contrast, in cell-attached patches, heart Na channel mean open time peaked near -50 mV, was three times brain Na channel mean open time, and declined continuously to approximately 2 ms at -20 mV. Near threshold, openings occurred frequently usually as brief bursts lasting several milliseconds and rarely as prolonged bursts lasting tens of milliseconds. Unlike what occurs in brain tissue where excision did not change gating, in excised heart patches both the frequency of prolonged bursting and the mean open time of single units increased markedly. Brain and cardiac Na channels can therefore be distinguished on the basis of their mean open times and bursting characteristics.     

Large-conductance calcium-activated anion channel characteristics in neuroblastoma cells

Large-conductance anion channel characteristics were investigated in neuroblastoma cells (N2A) by using different configurations of the patch-clamp technique. In excised patches, the channel was induced by depolarising potentials in 90% of experiments, had a conductance of 340 pS in symmetrical 135 mmol/l NaCl and exhibited the typical bell-shape activity. Neither the channel induction nor the channel activity was affected by rising the Ca2+ concentration on the cytopasmic side of membranes. In cell-attached configuration the maximal channel activity was shifted towards more positive potentials in comparison to that of excised patches and an increase in intracellular Ca2+, obtained by extracellular application of the Ca2+-ionophore A23187 in the presence of 0.2 micromol/l Ca2+, induced single-channel currents in 80% of patches compared to 31% of cell-attached experiments showing channel activity in normal conditions. In turn, application of 2 micromol/l Ca2+ induced channel activity in 100% of patches. The reversal potential of the channel in cell-attached patches was around -10 mV as the resting potential of cells eliciting channel activity. For cells where channel activity was not detected in cell-attached mode, the resting potential was around -45 mV. Channel activity could be restored in most whole-cell recordings in the presence of 2 micromol/l or more intracellular Ca2+ concentrations. The Ca2+-induction and the relation between channel activity and cell resting potential seem to suggest a role of the large-conductance anion channel in resting potential modulation during some basic functions of the neuroblastoma cell proliferation.     

Barium- and calcium-permeable channels open at negative membrane potentials in rat ventricular myocytes

Summary Ca²⁺- and Ba²⁺-permeable channel activity from adult rat ventricular myocytes, spontaneously appeared in the three single-channel recording configurations: cell-attached, and excised inside-out or outside-out membrane patches. Single-channel activity was recorded at steady-state applied membrane potentials including the entire range of physiologic values, and displayed no rundown in excised patches. This activity occurred in irregular bursts separated by quiescent periods of 5 to 20 min in cell-attached membrane patches, whereas in excised patch experiments, this period was reduced to 2 to 10 min. During activity, a variety of kinetic behaviors could be observed with more or less complex gating patterns. Three conductance levels: 22, 45 and 78 pS were routinely observed in the same excised membrane patch, sometimes combining to give a larger level. These channels were significantly permeable to divalent cations and showed little or no permeability to potassium or sodium ions. The inorganic blockers of voltage-gated Ca channels, cobalt (2mm), cadmium (0.5mm) or nickel (3mm), had no apparent effect on these spontaneous unitary currents carried by barium ions. Under 10^–5 m bay K 8644 or nitrendipine, the activity was clearly increased in about half of the tested excised inside-out membrane patches. Both dihydropyridines enhanced openings of the larger conductance level, which was only very occasionally seen under control conditions. When the single-channel activity became sustained under 5×10^–6 m Bay K 8644, it was possible to calculate the mean unitary current at different membrane potentials and show that the mean current value increased with membrane potential.     

Expression of various scinderin domains in chromaffin cells indicates that this protein acts as a molecular switch in the control of actin filament dynamics and exocytosis

Stimulation-induced chromaffin cell cortical F-actin disassembly allows the movement of vesicles towards exocytotic sites. Scinderin (Sc), a Ca2+-dependent protein, controls actin dynamics. Sc six domains have three actin, two PIP2 and two Ca2+-binding sites. F-actin severing activity of Sc is Ca2+-dependent, whereas Sc-evoked actin nucleation is Ca2+-independent. Sc domain role in secretion was studied by co-transfection of human growth hormone (hGH) reporter gene and green fluorescent protein (GFP)-fusion Sc constructs. Cells over-expressing actin severing Sc1-6 or Sc1-2 (first and second actin binding sites) constructs, increased F-actin disassembly and hGH release upon depolarization. Over-expression of nucleating Sc5-6, Sc5 or ScABP3 (third actin site) constructs decreased F-actin disassembly and hGH release upon stimulation. Over-expression of ScL5-6 or ScL5 (lack of third actin site) produced no changes. During secretion, actin sites 1 and 2 are involved in F-actin severing, whereas site 3 is responsible for nucleation (polymerization). Sc functions as a molecular switch in the control of actin (disassembly left arrow over right arrow assembly) and release (facilitation left arrow over right arrow inhibition). The position of the switch (severing left arrow over right arrow nucleation) may be controlled by [Ca2+]i. Thus, increase in [Ca2+]i produced by stimulation-induced Ca2+ entry would increase Sc-evoked cortical F-actin disassembly. Decrease in [Ca2+]i by either organelle sequestration or cell extrusion would favor Sc-evoked actin nucleation.     

Impaired activation of ATP-sensitive K+ channels in endocardial myocytes from left ventricular hypertrophy

ATP-sensitive K(+) (K(ATP)) channels are essential for maintaining the cellular homeostasis against metabolic stress. Myocardial remodeling in various pathologies may alter this adaptive response to such stress. It was reported that transmural electrophysiological heterogeneity exists in ventricular myocardium. Therefore, we hypothesized that the K(ATP) channel properties might be altered in hypertrophied myocytes from endocardium. To test this hypothesis, we determined the K(ATP) channel currents using the perforated patch-clamp technique, open cell-attached patches, and excised inside-out patches in both endocardial and epicardial myocytes isolated from hypertrophied [spontaneous hypertensive rats (SHR)] vs. normal [Wistar-Kyoto rats (WKY)] left ventricle. In endocardial cells, K(ATP) channel currents (I(K,ATP)), produced by 2 mM CN(-) and no glucose at 0 mV, were significantly smaller (P < 0.01), and time required to reach peak currents after onset of K(ATP) channel opening (Time(onset to peak)) was significantly longer (319 +/- 46 vs. 177 +/- 37 s, P = 0.01) in the SHR group (n = 9) than the WKY group (n = 13). However, in epicardial cells, there were no differences in I(K,ATP) and Time(onset to peak) between the groups (SHR, n = 12; WKY, n = 12). The concentration-open probability-response curves obtained during the exposure of open cells and excised patches to exogenous ATP revealed the impaired K(ATP) channel activation in endocardial myocytes from SHR. In conclusion, K(ATP) channel activation under metabolic stress was impaired in endocardial cells from rat hypertrophied left ventricle. The deficit of endocardial K(ATP) channels to decreased intracellular ATP might contribute to the maladaptive response of hypertrophied hearts to ischemia.     

A comparison of calcium-activated potassium channel currents in cell-attached and excised patches

Single channel currents from Ca-activated K channels were recorded from cell-attached patches, which were then excised from 1321N1 human astrocytoma cells. Cells were depolarized with K (110 mM) so that the membrane potential was known in both patch configurations, and the Ca ionophore A23187 or ionomycin (20-100 microM) was used to equilibrate intracellular and extracellular [Ca] (0.3 or 1 microM). Measurements of intracellular [Ca] with the fluorescent Ca indicator quin2 verified that [Ca] equilibration apparently occurred in our experiments. Under these conditions, where both membrane potential and intracellular [Ca] were known, we found that the dependence of the channel percent open time on membrane potential and [Ca] was similar in both the cell-attached and excised patch configuration for several minutes after excision. Current-voltage relations were also similar, and autocorrelation functions constructed from the single channel currents revealed no obvious change in channel gating upon patch excision. These findings suggest that the results of studies that use excised membrane patches can be extrapolated to the K-depolarized cell-attached configuration, and that the relation between [Ca] and channel activity can be used to obtain a quantitative measure of [Ca] near the membrane intracellular surface.     

Secretory activation of basolateral membrane Cl- channels in guinea pig distal colonic crypts

Cell-attached recordings revealed Cl(-) channel activity in basolateral membrane of guinea pig distal colonic crypts isolated from basement membrane. Outwardly rectified currents ((gp)Cl(or)) were apparent with a single-channel conductance (gamma) of 29 pS at resting membrane electrical potential; another outward rectifier with gamma of 24 pS was also observed ( approximately 25% of (gp)Cl(or)). At a holding potential of -80 mV gamma was 18 pS for both (gp)Cl(or) currents, and at +80 mV gamma was 67 and 40 pS, respectively. Identity as Cl(-) channels was confirmed in excised patches by changing bath ion composition. From reversal potentials, relative permeability of K(+) over Cl(-) (P(K)/P(Cl)) was 0.07 +/- 0.03, with relative permeability of Na(+) over Cl(-) (P(Na)/P(Cl)) = 0.08 +/- 0.04. A second type of Cl(-) channel was seen with linear current-voltage (I-V) relations ((gp)Cl(L)), having subtypes with gamma of 21, 13, and 8 pS. Epinephrine or forskolin increased the number of open (gp)Cl(or) and (gp)Cl(L). Open probabilities (P(o)) of (gp)Cl(or), (gp)Cl(L21), and (gp)Cl(L13) were voltage dependent in cell-attached patches, higher at more positive potentials. Kinetics of (gp)Cl(or) were more rapid with epinephrine activation than with forskolin activation. Epinephrine increased P(o) at the resting membrane potential for (gp)Cl(L13). Secretagogue activation of these Cl(-) channels may contribute to stimulation of electrogenic K(+) secretion across colonic epithelium by increasing basolateral membrane Cl(-) conductance that permits Cl(-) exit after uptake via Na(+)-K(+)-2Cl(-) cotransport.     

Involvement of cysteine residues and domain interactions in the reversible unfolding of lipoxygenase-1

Urea-induced unfolding of lipoxygenase-1 (LOX1) at pH 7.0 was followed by enzyme activity, spectroscopic measurements, and limited proteolysis experiments. Complete unfolding of LOX1 in 9 M urea in the presence of thiol reducing or thiol modifying reagents was observed. The aggregation and oxidative reactions prevented the reversible unfolding of the molecule. The loss of enzyme activity was much earlier than the structural loss of the molecule during the course of unfolding, with the midpoint concentrations being 4.5 and 7.0 M for activity and spectroscopic measurements, respectively. The equilibrium unfolding transition could be adequately fitted to a three-state, two-step model (N left arrow over right arrow I left arrow over right arrow U) and the intermediate fraction was maximally populated at 6.3 M urea. The free energy change (DeltaG(H(2)O)) for the unfolding of native (N) to intermediate (I) was 14.2 +/- 0.28 kcal/mol and for the intermediate to the unfolded state (U) was 11.9 +/- 0.12 kcal/mol. The ANS binding measurements as a function of urea concentration indicated that the maximum binding of ANS was in 6.3 M urea due to the exposure of hydrophobic groups; this intermediate showed significant amount of tertiary structure and retained nearly 60% of secondary structure. The limited proteolysis measurements showed that the initiation of unfolding was from the C-terminal domain. Thus, the stable intermediate observed could be the C-terminal domain unfolded with exposed hydrophobic domain-domain interface. Limited proteolysis experiments during refolding process suggested that the intermediate refolded prior to completely unfolded LOX1. These results confirmed the role of cysteine residues and domain-domain interactions in the reversible unfolding of LOX1. This is the first report of the reversible unfolding of a very large monomeric, multi-domain protein, which also has a prosthetic group.     

A Large-Conductance Chloride Channel in Pigmented Ciliary Epithelial Cells Activated by GTPγS

A large-conductance (or maxi-) chloride channel was identified in bovine pigmented ciliary epithelial (PCE) cells using inside-out excised patch clamp recording. The channel had a mean conductance of 293 pS when excised patches were bathed in symmetrical 130 mm NaCl although the conductance decreased to 209 pS when the solution bathing the cytoplasmic face of the patch contained only 33 mm NaCl. The channel was highly selective for chloride, with a P _Cl/P _Na= 24. A flickery, reversible block was produced by the diuretic stilbene 4-acetamido-4′-isothiocyanatostilbene-2,2′-disulfonic acid (SITS), while 4,4′-diisothiocyanatostilbene-2,2′-disulfonic acid (DIDS) produced a permanent block. The channel was rarely active in cell-attached patches and usually required several minutes of polarization before activity could be detected in excised patches, a process known as metagenesis. Once activated, the channel was voltage-dependent and was mainly open within the voltage range −30 to +30 mV closing when the membrane was polarized to larger values. GTPγS (100 μm) activated the channel with a latency of 170 sec when applied to the cytoplasmic face of patches. This activation was not reversible upon return to control solution within the duration of the experiment. We assess the available evidence and suggest a role for this channel in volume regulation. Received: 24 June 1996/Revised: 18 February 1997     

Immunochemical characterization and taxonomic evaluation of the O polysaccharides of the lipopolysaccharides of Pseudomonas syringae serogroup O1 strains

The O polysaccharide (OPS) of the lipopolysaccharide (LPS) of Pseudomonas syringae pv. atrofaciens IMV 7836 and some other strains that are classified in serogroup O1 was shown to be a novel linear alpha-D-rhamnan with the tetrasaccharide O repeat -->3)-alpha-D-Rhap-(1-->3)-alpha-D-Rhap-(1-->2)-alpha-D-R hap-(1-->2)- alpha-D-Rhap-(1--> (chemotype 1A). The same alpha-D-rhamnan serves as the backbone in branched OPSs with lateral (alpha1-->3)-linked D-Rhap, (beta1-->4)-linked D-GlcpNAc, and (alpha1-->4)-linked D-Fucf residues (chemotypes 1B, 1C, and 1D, respectively). Strains of chemotype 1C demonstrated variations resulting in a decrease of the degree of substitution of the backbone 1A with the lateral D-GlcNAc residue (chemotype 1C-1A), which may be described as branched regular left arrow over right arrow branched irregular --> linear OPS structure alterations (1Cleft arrow over right arrow 1C-1A --> 1A). Based on serological data, chemotype 1D was suggested to undergo a 1D left arrow over right arrow 1D-1A alteration, whereas chemotype 1B showed no alteration. A number of OPS backbone-specific monoclonal antibodies (MAbs), Ps(1-2)a, Ps(1-2)a(1), Ps1a, Ps1a(1), and Ps1a(2), as well as MAbs Ps1b, Ps1c, Ps1c(1), Ps1d, Ps(1-2)d, and Ps(1-2)d(1) specific to epitopes related to the lateral sugar substituents of the OPSs, were produced against P. syringae serogroup O1 strains. By using MAbs, some specific epitopes were inferred, serogroup O1 strains were serotyped in more detail, and thus, the serological classification scheme of P. syringae was improved. Screening with MAbs of about 800 strains representing all 56 known P. syringae pathovars showed that the strains classified in serogroup O1 were found among 15 pathovars and the strains with the linear OPSs of chemotype 1A were found among 9 of the 15 pathovars. A possible role for the LPS of P. syringae and related pseudomonads as a phylogenetic marker is discussed.