Direct Comparison of NPPB and DPC as Probes of CFTR Expressed in Xenopus Oocytes

Blockers of CFTR with well-characterized kinetics and mechanism of action will be useful as probes of pore structure. We have studied the mechanism of block of CFTR by the arylaminobenzoates NPPB and DPC. Block of macroscopic currents by NPPB and DPC exhibited similar voltage-dependence, suggestive of an overlapping binding region. Kinetic analysis of single-channel currents in the presence of NPPB indicate drug-induced closed time constants averaging 2.2 msec at −100 mV. The affinity for NPPB calculated from single-channel block, K _D = 35 μm, exceeds that for other arylaminobenzoates studied thus far. These drugs do not affect the rate of activation of wild-type (WT) channels expressed in oocytes, consistent with a simple mechanism of block by pore occlusion, and appear to have a single binding site in the pore. Block by NPPB and DPC were affected by pore-domain mutations in different ways. In contrast to its effects on block by DPC, mutation T1134F-CFTR decreased the affinity and reduced the voltage-dependence for block by NPPB. We also show that the alteration of macroscopic block by NPPB and DPC upon changes in bath pH is due to both direct effects (i.e., alteration of voltage-dependence) and indirect effects (alteration of cytoplasmic drug loading). These results indicate that both NPPB and DPC block CFTR by entering the pore from the cytoplasmic side and that the structural requirements for binding are not the same, although the binding regions within the pore are similar. The two drugs may be useful as probes for overlapping regions in the pore. Received: 14 October 1999/Revised: 18 January 2000     

Probing an open CFTR pore with organic anion blockers

The cystic fibrosis transmembrane conductance regulator (CFTR) is an ion channel that conducts Cl- current. We explored the CFTR pore by studying voltage-dependent blockade of the channel by two organic anions: glibenclamide and isethionate. To simplify the kinetic analysis, a CFTR mutant, K1250A-CFTR, was used because this mutant channel, once opened, can remain open for minutes. Dose-response relationships of both blockers follow a simple Michaelis-Menten function with K(d) values that differ by three orders of magnitude. Glibenclamide blocks CFTR from the intracellular side of the membrane with slow kinetics. Both the on and off rates of glibenclamide block are voltage dependent. Removing external Cl- increases affinity of glibenclamide due to a decrease of the off rate and an increase of the on rate, suggesting the presence of a Cl- binding site external to the glibenclamide binding site. Isethionate blocks the channel from the cytoplasmic side with fast kinetics, but has no measurable effect when applied extracellularly. Increasing the internal Cl- concentration reduces isethionate block without affecting its voltage dependence, suggesting that Cl- and isethionate compete for a binding site in the pore. The voltage dependence and external Cl- concentration dependence of isethionate block are nearly identical to those of glibenclamide block, suggesting that these two blockers may bind to a common binding site, an idea further supported by kinetic studies of blocking with glibenclamide/isethionate mixtures. By comparing the physical and chemical natures of these two blockers, we propose that CFTR channel has an asymmetric pore with a wide internal entrance and a deeply embedded blocker binding site where local charges as well as hydrophobic components determine the affinity of the blockers.     

Kinetic Analysis of the Inhibitory Effect of Glibenclamide on KATP Channels of Mammalian Skeletal Muscle

We investigated the block of K_ATP channels by glibenclamide in inside-out membrane patches of rat flexor digitorum brevis muscle. (1) We found that glibenclamide inhibited K_ATP channels with an apparent K _i of 63 nm and a Hill coefficient of 0.85. The inhibition of K_ATP channels by glibenclamide was unaffected by internal Mg²⁺. (2) Glibenclamide altered all kinetic parameters measured; mean open time and burst length were reduced, whereas mean closed time was increased. (3) By making the assumption that binding of glibenclamide to the sulphonylurea receptor (SUR) leads to channel closure, we have used the relation between mean open time, glibenclamide concentration and K _D to estimate binding and unbinding rate constants. We found an apparent rate constant for glibenclamide binding of 9.9 × 10⁷ m ⁻¹ sec⁻¹ and an unbinding rate of 6.26 sec⁻¹. (4) Glibenclamide is a lipophilic molecule and is likely to act on sulfonylurea receptors from within the hydrophobic phase of the cell membrane. The glibenclamide concentration within this phase will be greater than that in the aqueous solution and we have taken this into account to estimate a true binding rate constant of 1.66 × 10⁶ m ⁻¹ sec⁻¹. Received: 7 July 1996/Revised: 4 October 1996     

Measurement of cystic fibrosis transmembrane conductance regulator activity using fluorescence spectrophotometry

Cystic fibrosis (CF) is a frequent autosomal recessive disease caused by mutations that impair the CF transmembrane conductance regulator (CFTR) protein function. CFTR is a chloride channel activated by cyclic AMP (cAMP) via protein kinase A (PKA) and ATP hydrolysis. We describe here a method to measure CFTR activity in a monolayer of cultured cells using a fluorescence spectrophotometer and the chloride-sensitive probe 6-methoxy-N-(3-sulfopropyl)quinolinium (SPQ). Modifying a slice holder, the spectrophotometer quartz cuvette was converted in a perfusion chamber, allowing measurement of CFTR activity in real time, in a monolayer of T84 colon carcinoma cells. The SPQ Stern–Volmer constant (K_Cl^-) for chloride in water solution was 115.0 ± 2.8 M⁻¹, whereas the intracellular K_Cl^- was 17.8 ± 0.8 M⁻¹, for T84 cells. A functional analysis was performed by measuring CFTR activity in T84 cells. The CFTR transport inhibitors CFTR(inh)-172 (5 μM) and glibenclamide (100 μM) showed a significant reduction (P < 0.05) in CFTR activity. This simple method allows measuring CFTR activity in a very simple, reproducible, and sensitive way.     

Catalyst-like modulation of transition states for CFTR channel opening and closing: New stimulation strategy exploits nonequilibrium gating

Cystic fibrosis transmembrane conductance regulator (CFTR) is the chloride ion channel mutated in cystic fibrosis (CF) patients. It is an ATP-binding cassette protein, and its resulting cyclic nonequilibrium gating mechanism sets it apart from most other ion channels. The most common CF mutation (ΔF508) impairs folding of CFTR but also channel gating, reducing open probability (P_o). This gating defect must be addressed to effectively treat CF. Combining single-channel and macroscopic current measurements in inside-out patches, we show here that the two effects of 5-nitro-2-(3-phenylpropylamino)benzoate (NPPB) on CFTR, pore block and gating stimulation, are independent, suggesting action at distinct sites. Furthermore, detailed kinetic analysis revealed that NPPB potently increases P_o, also of ΔF508 CFTR, by affecting the stability of gating transition states. This finding is unexpected, because for most ion channels, which gate at equilibrium, altering transition-state stabilities has no effect on P_o; rather, agonists usually stimulate by stabilizing open states. Our results highlight how for CFTR, because of its unique cyclic mechanism, gating transition states determine P_o and offer strategic targets for potentiator compounds to achieve maximal efficacy.     

Steady-State Interactions of Glibenclamide with CFTR: Evidence for Multiple Sites in the Pore

The objective of the present study was to clarify the mechanism by which the sulfonylurea drug, glibenclamide, inhibits single CFTR channels in excised patches from Xenopus oocytes. Glibenclamide blocks the open pore of the channel via binding at multiple sites with varying kinetics. In the absence of glibenclamide, open-channel bursts exhibited a flickery intraburst closed state (C1); this is due to block of the pore by the pH buffer, TES. Application of 25 microM glibenclamide to the cytoplasmic solution resulted in the appearance of two drug-induced intraburst closed states (C2, C3) of widely different duration, which differed in pH-dependence. The kinetics of interaction with the C3 state, but not the C2 state, were strongly voltage-dependent. The durations of both the C2 and C3 states were concentration-dependent, indicating a non-linear reaction scheme. Application of drug also increased the burst duration, which is consistent with an open-channel blocking mechanism. A kinetic model is proposed. These results indicate that glibenclamide interacts with open CFTR channels in a complex manner, involving interactions with multiple binding sites in the channel pore.     

Kinetics of reversible N-ethylmaleimide alkylation of metallothionein and the subsequent metal release

 The model alkylating agent N-ethylmaleimide (NEM) reacts reversibly at the metal-bound thiolates of Zn₇MT and Cd₇MT. An unprecedented feature of this reaction is that it approaches equilibrium and requires a large excess of NEM (>1 mM for 3 μM protein) to drive it to completion. The complex kinetics of the reaction can be followed by monitoring the release of bound metal ions using the metallochromic dyes Zincon (ZI) for Zn₇MT and pyridylazoresorcinol for Cd₇MT. An initial lag phase is followed by more rapid release of zinc ions. The observed pseudo-first-order rate constants for the two phases are independent of the ZI and Zn₇MT concentrations. The complex NEM concentration dependence of each phase, k _f, obs=k ^f ₁+k ^f ₂ [NEM] and k _s, obs=k ^s ₁+k ^s ₂ [NEM], demonstrates that the forward reactions are second order and the reverse reactions are first order. The alkylation can be reversed using 2-mercaptoethanol to compete for the protein-bound NEM and regenerate the Zn-binding capability of alkylated MT. An explanation of these observations, based on the reversibility of cysteine alkylation by NEM, was developed and tested. The reactions of Cd₇MT are less complete than those of Zn₇MT and occur more slowly. ¹¹¹Cd-NMR studies of the partially alkylated ¹¹¹Cd₇MT reveal that reaction with only four equivalents of NEM completely alters the cluster structure and eliminates the spectral signatures of the α and β clusters, although very little cadmium has been removed from the protein. This finding substantiates the proposed kinetic intermediate, a partially alkylated MT with complete or nearly complete retention of the metal ions, and rules out the possibility of cooperative reactions at either cluster. Received: 5 August 1996 / Accepted: 24 October 1996     

Inhibitory effect of memantine, an NMDA-receptor antagonist, on electoporation-induced inward currents in pituitary GH3 cells

The membrane electroporation-induced inward current (I_MEP) in pituitary tumor (GH₃) cells was characterized. This current emerges irregularly when membrane hyperpolarizations to −200 mV with a holding potential of −80 mV were elicited. Neither E-4031 (10 μM), glibenclamide (30 μM), nor ZD7288 (30 μM) caused any effects on I_MEP. The single-channel conductance and pore radius were estimated to be around 1.12 nS and 1.7 nm, respectively. LaCl₃- and memantidine (MEM)-induced block of this current was also examined. The IC₅₀ value for LaCl₃- and MEM-induced inhibition of I_MEP was 35 and 75 μM, respectively. However, unlike LaCl₃, MEM (300 μM) did not exert any effect on voltage-gated Ca²⁺ current. In inside-out configuration, MEM applied to either external or internal surface of the excised patch did not suppress the activity of ATP-sensitive K⁺ channels expressed in GH₃ cells, although glibenclamide significantly suppressed channel activity. This study provides the first evidence to show that MEM, a non-competitive antagonist of N-methyl D-aspartate receptors, directly inhibits the amplitude of I_MEP in pituitary GH₃ cells. MEM-mediated block of I_MEP in these cells is unlinked to its inhibition of glutamate-induced currents or ATP-sensitive K⁺ currents. The channel-suppressing properties of MEM might contribute to the underlying mechanisms by which it and its structurally related compounds affect neuronal or neuroendocrine function.     

Cardiac ion channel current modulation by the CFTR inhibitor GlyH-101

The role in the heart of the cardiac isoform of the cystic fibrosis transmembrane conductance regulator (CFTR), which underlies a protein kinase A-dependent Cl⁻ current (I_Cl.PKA) in cardiomyocytes, remains unclear. The identification of a CFTR-selective inhibitor would provide an important tool for the investigation of the contribution of CFTR to cardiac electrophysiology. GlyH-101 is a glycine hydrazide that has recently been shown to block CFTR channels but its effects on cardiomyocytes are unknown. Here the action of GlyH-101 on cardiac I_Cl.PKA and on other ion currents has been established. Whole-cell patch-clamp recordings were made from rabbit isolated ventricular myocytes. GlyH-101 blocked I_Cl.PKA in a concentration- and voltage-dependent fashion (IC₅₀ at +100 mV = 0.3 ± 1.5 μM and at −100 mV = 5.1 ± 1.3 μM). Woodhull analysis suggested that GlyH-101 blocks the open pore of cardiac CFTR channels at an electrical distance of 0.15 ± 0.03 from the external membrane surface. A concentration of GlyH-101 maximally effective against I_Cl.PKA (30 μM) was tested on other cardiac ion currents. Inward current at −120 mV, comprised predominantly of the inward-rectifier background K⁺ current, I_K1, was reduced by ∼43% (n = 5). Under selective recording conditions, the Na⁺ current (I_Na) was markedly inhibited by GlyH-101 over the entire voltage range (with a fractional block at −40 mV of ∼82%; n = 8). GlyH-101 also produced a voltage-dependent inhibition of L-type Ca²⁺ channel current (I_Ca,L); fractional block at +10 mV of ∼49% and of ∼28% at −10 mV; n = 11, with a ∼−3 mV shift in the voltage-dependence of I_Ca,L activation. Thus, this study demonstrates for the first time that GlyH-101 blocks cardiac I_Cl.PKA channels in a similar fashion to that reported for recombinant CFTR. However, inhibition of other cardiac conductances may limit its use as a CFTR-selective blocker in the heart.     

Cystic Fibrosis Transmembrane Conductance Regulator (CFTR) Confers Glibenclamide Sensitivity to Outwardly Rectifying Chloride Channel (ORCC) in Hi-5 Insect Cells

Increasing evidence is now accumulating for the involvement of the cystic fibrosis transmembrane conductance regulator (CFTR) in the control of the outwardly rectifying chloride channel (ORCC). We have examined the sensitivity of ORCC to the sulfonylurea drug glibenclamide in Hi-5 (Trichoplusia ni) insect cells infected with recombinant baculovirus expressing either wild-type CFTR, ΔF508-CFTR or E. coliβ galactosidase cDNA and in control cells either infected with virus alone or uninfected. Iodide efflux and single channel patch-clamp experiments confirmed that forskolin and 1-methyl-3-isobutyl xanthine (IBMX) or 7-methyl-1,3 dipropyl xanthine (DPMX) activate CFTR channels (unitary conductance: 9.1 ± 1.6 pS) only in cells expressing CFTR. In contrast, we identified 4-acetamido-4′-isothiocyanatostilbene-2,2′-disulfonic acid (SITS)-sensitive ORCC in excised membrane patches in any of the cells studied, with similar conductance (22 ± 2.5 pS at −80 mV; 55 ± 4.1 pS at +80 mV) and properties. In the presence of 500 μm SITS, channel open probability (P _o ) of ORCC was reversibly reduced to 0.05 ± 0.01 in CFTR-cells, to 0.07 ± 0.02 in non-CFTR expressing cells and to 0.05 ± 0.02 in ΔF508-cells. In Hi-5 cells that did not express CFTR, glibenclamide failed to inhibit ORCC activity even at high concentrations (100 μm), whereas 500 μm SITS reversibly inhibited ORCC. In contrast in cells expressing CFTR or ΔF508, glibenclamide dose dependently (IC₅₀= 17 μm, Hill coefficient 1.2) and reversibly inhibited ORCC. Cytoplasmic application of 100 μm glibenclamide reversibly reduced P _o from 0.88 ± 0.03 to 0.09 ± 0.02 (wash: P _o = 0.85 ± 0.1) in CFTR cells and from 0.89 ± 0.05 to 0.08 ± 0.05 (wash: P _o = 0.87 ± 0.1) in ΔF508 cells. In non-CFTR expressing cells, glibenclamide (100 μm) was without effect on P _o (control: P _o = 0.89 ± 0.09, glib.: P _o = 0.86 ± 0.02; wash: P _o = 0.87 ± 0.05). These data strongly suggest that the expression of CFTR confers glibenclamide sensitivity to the ORCC in Hi-5 cells. Received: 23 October 1998/Revised: 29 December 1998     

CFTR Regulation of Intracellular Calcium in Normal and Cystic Fibrosis Human Airway Epithelia

In cystic fibrosis airway epithelia, mutation of the CFTR protein causes a reduced response of Cl⁻ secretion to secretagogues acting via cAMP. Using a Ca²⁺ imaging system, the hypothesis that CFTR activation may permit ATP release and regulate [Ca²⁺] _i via a receptor-mediated mechanism, is tested in this study. Application of external nucleotides produced a significant increase in [Ca²⁺] _i in normal (16HBE14o⁻ cell line and primary lung culture) and in cystic fibrosis (CFTE29o⁻ cell line) human airway epithelia. The potency order of nucleotides on [Ca²⁺] _i variation was UTP ≫ ATP > UDP > ADP > AMP > adenosine in both cell types. The nucleotide [Ca²⁺] _i response could be mimicked by activation of CFTR with forskolin (20 μm) in a temperature-dependent manner. In 16HBE14o⁻ cells, the forskolin-induced [Ca²⁺] _i response increased with increasing temperature. In CFTE29o⁻ cells, forskolin had no effect on [Ca²⁺] _i at body temperature-forskolin-induced [Ca²⁺] _i response in CF cells could only be observed at low experimental temperature (14°C) or when cells were cultured at 26°C instead of 37°C. Pretreatment with CFTR channel blockers glibenclamide (100 μm) and DPC (100 μm), with hexokinase (0.5 U/mg), and with the purinoceptor antagonist suramin (100 μm), inhibited the forskolin [Ca²⁺] _i response. Together, these results demonstrate that once activated, CFTR regulates [Ca²⁺] _i by mediating nucleotide release and activating cell surface purinoceptors in normal and CF human airway epithelia. Received: 3 April 2000/Revised: 30 June 2000     

L'alcool deshydrogénase de tomate (Lycopersicon esculentum): Étude cinétique et mécanisme d'action

In order to investigate the enzymatic mechanism of tomato alcohol dehydrogenase, kinetic studies were carried out at pH 5.8 and 9.4 for the forward and reverse reactions, respectively. Primary double reciprocal plots for several fixed concentrations of the associated substrate in all cases intersect, suggesting a sequential mechanism. Exploitation of secondary plots (slope-intercept values on the primary plots versus the reciprocals of the non-varied substrates) gives the following values: K_ms 500 μM for MeCHO, 30 μM for NADH, 2700 μM for EtOH, 12 μM for NAD⁺; K_is 40 μM for MeCHO, 3 μM for NADH, 10⁴ μM for EtOH and 45 μM for NAD⁺. The results obtained in product inhibition studies agree with an ordered bi-bi mechanism for both forward and reverse reactions. Application of Cleland's rules shows that the coenzyme was the first substrate to complex with the enzyme in both cases.     

Ca2+-activated K+ channels in the apical membrane ofNecturus choroid plexus

Summary The properties of Ca²⁺-activated K⁺ channels in the apical membrane of theNecturus choroid plexus were studied using single-channel recording techniques in the cell-attached and excised-patch configurations. Channels with large unitary conductances clustered around 150 and 220 pS were most commonly observed. These channels exhibited a high selectivity for K⁺ over Na⁺ and K⁺ over Cs⁺. They were blocked by high cytoplasmic Na⁺ concentrations (110mm). Channel activity increased with depolarizing membrane potentials, and with increasing cytoplasmic Ca²⁺ concentrations. Increasing Ca²⁺ from 5 to 500nm, increased open probability by an order of magnitude, without changing single-channel conductance. Open probability increased up to 10-fold with a 20-mV depolarization when Ca²⁺ was 500nm. Lowering intracellular pH one unit, decreased open probability by more than two orders of magnitude, but pH did not affect single-channel conductance. Cytoplasmic Ba²⁺ reduced both channel-open probability and conductance. The sites for the action of Ba²⁺ are located at a distance more than halfway through the applied electric field from the inside of the membrane. Values of 0.013 and 117mm were calculated as the apparent Ba²⁺ dissociation constants (K _d (0 mV) for the effects on probability and conductance, respectively. TEA⁺ (tetraethylammonium) reduced single-channel current. Applied to the cytoplasmic side, it acted on a site 20% of the distance through the membrane, with aK _d (0 mV)=5.6mm. A second site, with a higher affinity,K _d (0 mV)=0.23mm, may account for the near total block of chanel conductance by 2mm TEA⁺ applied to the outside of the membrane. It is concluded that the channels inNecturus choroid plexus exhibit many of the properties of maxi Ca²⁺-activated K⁺ channels found in other tissues.     

Kinetic study on unzippering of polynucleotides and order-order transition in polypeptides

Solutions are presented for N + 1 sequential and reversible first-order reactions for which the magnitude of the reverse rate constant, k_b, for all steps except the last is identical. Also the magnitude of the forward rate constant, k_f, for all steps except the first and last is identical. The initial and final steps are nucleation reactions; therefore, the initial and final k_f are modified by the factors σ′ and γ respectively. The final k_b is modified by the factor γ σ. The ratio k_b/k_f is defined as s, which has the same meaning as s in the Zimm-Bragg theory. The mathematical model is intended to apply to polymeric molecules of N segments and allows the calculation of the mole fraction of molecules in state i at any time t, C_i(t). A molecule in state i has i unreacted segments and N – i reacted ones. Because the reactions are sequential, all reacted segments are contiguous. Our numerical results show that when σ′ is much less than unity and the forward reaction is favored, the relaxation curve is sigmoidal. If, however, the forward and reverse reactions are equally favored (i.e., s ? 1) the relaxation curve is a straight line. When s and σ′ are near unity, the curve is exponential for a considerably large fraction of the reaction. Further, in the exponential for a considerably large fraction of the reaction. Further, in the exponential phase of the reaction, the relaxation time is proportional to N² for highly cooperative systems (i.e., Nσ ? 1). As found by Pipkin and Gibbs, if N is sufficiently large and s is less than unity (e.g., N ? 50 and s ?0.9) the relaxation curve is largely linear with a slope inversely proportional to N. Applications are given for the unwinding of double-helical poly(A·U) and the order–order transition in poly-L -proline.     

Homoserine dehydrogenase in Pisum sativum and Ricinus communis

Homoserine dehydrogenase was extracted from Ricinus communis and Pisum sativum. The kinetic parameters of the forward and reverse reactions were determined. In the forward reaction only the enzyme from Ricinus is inhibited by threonine. The response to K⁺ is different for the enzyme from the two sources.     

Kinetics and Mechanism of the Peptide Synthesis in Solution

The kinetics of the reaction of Boc-Xaa fluorophenyl esters (where Xaa = Ala, Val, Phe, Ser, Leu, Gly, Met, Pro, or Ile) with leucinamide was studied in order to measure changes in fluorescence emission at 375 nm of the fluorophenyl chromophore accompanying the reaction. It was found that the experimental kinetic data could not be described by a simple scheme of the second order reaction. Measurements of the kinetic parameters of the reaction at various initial concentrations of reagents indicated that the reaction rate can be expressed as: = kC _N ^a C _AE ^b , where k is the reaction rate constant, C _N is the concentration of leucinamide, and C _AE is the concentration of fluorophenyl ester. The a and b reaction orders were close to 1/2 and 3/2 for Xaa = Ala, Val, Phe, Ser, or Leu, 1/2 and 1 for Gly, Met, or Pro, and 1 and 2 for Ile. The experimental equations for the reaction rate can theoretically be derived from a single scheme of chain reactions with various deactivation ways for active intermediates.     

Adenylosuccinate synthetase from Azotobacter vinelandii: purification, properties and steady-state kinetics.

Adenylosuccinate synthetase has been purified to homogeneity from Azotobacter, vinelandii. The purification method involves affinity chromatography on blue dextran-Sepharose, and hydrophobic chromatography, in addition to heat treatment, ammonium sulfate fractionation, and ion-exchange chromatography. The purified enzyme displays a single protein band after electrophoresis in the presence or absence of sodium dodecyl sulfate (SDS). Molecular weights of 110,000 and 54,000 are estimated by gel filtration and SDS gel electrophoresis, respectively.Steady-state kinetic measurements of the forward and reverse reactions and of the reaction in which arsenate replaces phosphate reveal a sequential mechanism with a fully random order of substrate addition in all cases. The maximal velocities of the reverse reaction and arsenolysis are virtually identical, and are approximately 10% of the maximal velocity for the forward reaction. In common with this enzyme from other sources, hadacidin is a potent competitive inhibitor with respect to aspartate (K_i = 0.3 μm). Specific anions, e.g. nitrate and thiocyanate, are competitive inhibitors with respect to GTP; their effectiveness follows the Hofmeister series. Anion inhibition is synergized by GDP, but binding is exclusive with respect to guanylylimidodiphosphate, suggesting binding of the anions at the site normally occupied by the transferable phosphoryl group of GTP.     

ATP binding to noncatalytic sites of chloroplast coupling factor CF1

A kinetic analysis of ATP binding to noncatalytic sites of chloroplast coupling factor CF₁ was made. The ATP binding proved to be unaffected by reduction of the disulfide bridge of the CF₁ -subunit. The first-order equation describing nucleotide binding to noncatalytic sites allowed for two vacant nucleotide binding sites different in their kinetics. As suggested by nucleotide concentration dependence of the rate of nucleotide binding, the tight binding was preceded by rapid reversible binding of nucleotides. Preincubation of CF₁ with Mg²⁺ resulted in a decreased rate of ATP binding. ATP dissociation from noncatalytic sites was described by the first order equation for similar sites with a dissociation rate constant k _d (ATP) 10^–3 min^–1. Noncatalytic sites of CF₁ were shown to be not homogeneous. One of them retained the major part of endogenous ADP after precipitation of CF₁ with ammonium sulfate. Its two other sites differed in kinetic parameters and affinity for ATP. Anions of phosphate, sulfite, and especially, pyrophosphate inhibited the interaction between ATP and the noncatalytic sites.     

Stereoselective sulfate conjugation of isoproterenol in humans: Comparison of hepatic,intestinal, and platelet activity

The stereochemistry of sulfate conjugation of isoproterenol (ISO) was examined with human liver, intestine, and platelets as the phenolsulfotransferase (PST) enzyme source and PAP³⁵S as the cosubstrate. With the hepatic cytosol, two distinct sulfation reactions were identified, a high affinity reaction (K_m 5 to 50 μM) and a low affinity reaction (K_m 360 to 2,900 μM). The efficiency of sulfation (V_max/K_m) for both reactions was 5-fold higher for (+)- than for (?)-ISO. When the hepatic PSTs were resolved by ionexchange chromatography, it could be shown that the high affinity reaction was catalyzed by the monoamine (M) form and the low affinity reaction by the phenol (P) form of PST. Only the high affinity (M form) sulfation was detected in the jejunal cytosol with a V_max/K_m value 6.1-fold higher for (+)- than for (?)-ISO. Finally the platelet, as a potentially useful model tissue, also demonstrated only the high affinity M form reaction with a V_max/K_m value 5.7-fold higher for (+)- than for (?)-ISO. In summary, this study has shown that sulfation of ISO by PSTs in various human tissues is stereoselective and favors the inactive (+)-enantiomer over the active (?)-enantiomer by about 5-fold, a finding which should be considered in the therapeutic use of chiral drugs cleared by sulfate conjugation. © 1993 Wiley-Liss, Inc.     

Sulfhydryl groups regulate thyroid hormone binding at nuclear receptor sites: further evidence for a separate binding site for reverse T3

Pig and rat liver nuclei possess specific, high affinity, low capacity binding sites for 3,3′,5′-triiodothyronine (reverse T₃) distinct from known 3,5,3′-triiodothyronine (T₃) binding sites. Sulfhydryl (SH) stabilishing and oxidising agents have profound and opposite, but not equal, effects upon in vitro binding of reverse T₃ and T₃. In the absence of SH stabilising agents T₃ and reverse T₃ bind with similar affinity (Ka 0.83 × 10⁹ v.s. 0.57 × 10⁹ M^?1). SH stabilising agents produce a small increase in the binding affinity of T₃ and a profound decrease in the binding affinity of reverse T₃. Chromatography of nuclear protein preincubated with both radioligands revealed two separate peaks of protein bound radioactivity consistent with two nuclear binding sites. These data suggest that SH groups may regulate binding of T₃ and reverse T₃ to nuclear receptors, and provide a mechanism for biological action of reverse T₃.