Use of 3-D computer modelling and kinetic studies to analyse grapefruit pyrophosphate-dependent phosphofructokinase

The glycolytic reaction of grapefruit PPi-dependent phosphofructokinase (PFP) depends on the presence of Fru-2,6-P₂ (K_a=6.7 nM). This molecule was further demonstrated in grapefruit juice sac cells. Citrate, -ketoglutarate and isocitrate competitively inhibited the binding of Fru-2,6-P₂ to PFP. The affinity for Fru-6-P (K_m=159 μM) and PPi (K_m=33 μM) were not affected by the addition of these molecules. In the gluconeogenic reaction, the presence of Fru-2,6-P₂ did not affect the K_m of Fru-1,6-P₂ (61 μM) in contrast to orange fruit PFP. These results led to the building of a computer model of PFP, based on the known structure of Bacillus stearothermophilus ATP-dependent phosphofructokinase (ATP-PFK). The results show that catalysis of Fru-6-P in the chain is most unlikely, due to amino-acid substitutions and that Fru-2,6-P₂ can bind between the and β subunits.     

Acetylcholine receptor binding site contains a disulfide cross-link between adjacent half-cystinyl residues

A conserved feature of all nicotinic receptors is the presence of a readily reducible disulfide bond adjacent to the acetylcholine binding site. Previously we showed that in intact receptor from Torpedo californica electric tissue reduction of this disulfide followed by affinity alkylation with 4-(N-maleimido)benzyltri[3H] methylammonium iodide specifically and uniquely labels the alpha subunit residues Cys-192 and Cys-193. To identify all of the half-cystinyl residues contributing to the binding site disulfide(s), we have now reduced receptor under mild conditions and alkylated with a mixture of 4-(N-maleimido)benzyltri[3H]methylammonium iodide and N-[1-14C]ethylmaleimide and find that Cys-192 and Cys-193 are labeled exclusively. Furthermore, from unreduced receptor we have isolated two cyanogen bromide peptides of alpha, one containing Cys-192 and Cys-193, and the other containing Cys-128 and Cys-142 (which are the other potential contributors to the binding site disulfide(s]. These isolated peptides incorporate iodo[1-14C]acetamide only following reduction by dithiothreitol. Our results demonstrate that: 1) the binding site disulfide is between Cys-192 and Cys-193; 2) Cys-128 is disulfide-cross-linked to Cys-142; and 3) under conditions that reduce Cys-192 and Cys-193 completely, Cys-128 and Cys-142 remain cross-linked. At the acetylcholine binding site, agonists induce a local conformational change that stabilizes the binding site disulfide against reduction. We suggest that a transition between two stable conformations of the vicinal disulfide, both involving a nonplanar cis peptide bond between Cys-192 and Cys-193, is associated with receptor activation by agonists.     

Redox potential of human thioredoxin 1 and identification of a second dithiol/disulfide motif

Thioredoxin (Trx1) is a redox-active protein containing two active site cysteines (Cys-32 and Cys-35) that cycle between the dithiol and disulfide forms as Trx1 reduces target proteins. Examination of the redox characteristics of this active site dithiol/disulfide couple is complicated by the presence of three additional non-active site cysteines. Using the redox Western blot technique and matrix assisted laser desorption ionization time-of-flight mass spectrometry mass spectrometry, we determined the midpoint potential (E0) of the Trx1 active site (-230 mV) and identified a second redox-active dithiol/disulfide (Cys-62 and Cys-69) in an alpha helix proximal to the active site, which formed under oxidizing conditions. This non-active site disulfide was not a substrate for reduction by thioredoxin reductase and delayed the reduction of the active site disulfide by thioredoxin reductase. Within actively growing THP1 cells, most of the active site of Trx1 was in the dithiol form, whereas the non-active site was totally in the dithiol form. The addition of increasing concentrations of diamide to these cells resulted in oxidation of the active site at fairly low concentrations and oxidation of the non-active site at higher concentrations. Taken together these results suggest that the Cys-62-Cys-69 disulfide could provide a means to transiently inhibit Trx1 activity under conditions of redox signaling or oxidative stress, allowing more time for the sensing and transmission of oxidative signals.     

Expression and mutagenesis of the novel serpin endopin 2 demonstrates a requirement for cysteine-374 for dithiothreitol-sensitive inhibition of elastase

The primary sequence of the serpin endopin 2 predicts a reactive site loop (RSL) region that possesses high homology to bovine elastase inhibitor, suggesting inhibition of elastase. Moreover, endopin 2 possesses two cysteine residues that implicate roles for reduced Cys residue(s) for inhibitory activity. To test these predicted properties, mutagenesis and chemical modification of recombinant endopin 2 were performed to examine the influence of dithiothreitol (DTT), a reducing agent, on endopin 2 activity. Endopin 2 inhibited elastase in a DTT-dependent manner, with enhanced inhibition in the presence of DTT. The stoichiometry of inhibition in the presence of DTT occurred at a molar ratio of endopin 2 to elastase of 8/1, resulting in complete inhibition of elastase. However, a higher molar ratio (25/1) was required in the absence of DTT. DTT enhanced the formation of SDS-stable complexes of endopin 2 and elastase, a characteristic property of serpins. Site-directed mutagenesis of endopin 2, with substitution of Ala for Cys-232 or Cys-374, demonstrated that Cys-374 (but not Cys-232) was required for the DTT-sensitive nature of endopin 2. Chemical modification of Cys-374 by bis(maleimido)ethane also reduced inhibitory activity. Modified electrophoretic mobilities of mutant endopin 2 suggested the presence of intramolecular disulfide bonds; in addition, chemical modification suggested that Cys-374 influences the electrophoretic and conformational properties of endopin 2. Moreover, the reducing agent glutathione enhanced endopin 2 activity, suggesting that glutathione can function as an endogenous reducing agent for endopin 2 in vivo. These findings demonstrate the importance of Cys-374 for DTT-sensitive inhibition of elastase by endopin 2.     

Effects of retinoic acid on rat forebrain cells derived from embryonic and perinatal rats

All-trans retinoic acid (RA), a potent inducer of neural development in non-committed neuroectodermal precursors and also, a teratogenic agent for early prosencephalic development is reported to promote the survival and differentiation of embryonic forebrain neurons, in vitro. In cultures of embryonic (E13, E15) rat forebrain cells, long-term (2–5 days) treatment with RA increased the number of neurons and the overall neurofilament immunoreactivity. Treatment with RA for periods longer than 1 h resulted in enhanced binding of the non-competitive NMDA-receptor antagonist, TCP, by embryonic and fetal (E17, E18) cells, but not by cells derived from perinatal (E19, P0) forebrains. As TCP binding-sites are localised within the channel-complex, treatment with RA was thought to result in an opening of the NMDA receptor channel. In direct binding assays, however, RA had no detectable effect, while conditioned media taken from RA-treated embryonic or fetal cells increased the TCP-binding, immediately. Analyses on conditioned media taken from control cultures of cells with various in vivo or in vitro ages revealed a stable extracellular glutamate level ([Glu]_e) of 1–3 μM. This basal [Glu]_e was restored within 24 h after addition of 100 μM exogenous glutamate. In the presence of RA, however, [Glu]_e was stabilised at an approximately three-fold higher (4–10 μM) level by cells derived from embryonic and fetal brains. RA-treatment did not influence the [Glu]_e in cultures of perinatal cells. The RA-induced rise in the neurofilament-immunoreactivity of embryonic brain cell cultures was prevented by simultaneous treatment with APV, a competitive antagonist of NMDA-receptors. The data suggest that a RA-induced shift in the set-point of extracellular glutamate-balance plays an important role in the promotion of survival and maturation of developing neurons, in culture.     

Sulfhydryl Modification of Two Nicotinic Binding Sites in Mouse Brain

Two distinct binding sites with properties corresponding to those expected for nicotinic cholinergic receptors can be identified in brain by the specific binding of nicotine (or acetylcholine) and alpha-bungarotoxin. The effects of modification of these binding sites by treatment with the disulfide-reducing agent dithiothreitol were examined in tissue prepared from DBA mouse brains. Treatment with dithiothreitol reduced the binding measured with either ligand, and reoxidization of the disulfides fully restored binding. The effects of dithiothreitol treatment appeared to be due to a reduction in the maximal binding of nicotine and to a decrease in the binding affinity for alpha-bungarotoxin. Agonist affinity for the alpha-bungarotoxin binding site was reduced by treatment with low concentrations of dithiothreitol. The nicotine binding sites remaining after disulfide treatment displayed rates of ligand association and dissociation similar to those of unmodified tissue, but treatment of previously unmodified tissue with dithiothreitol accelerated the rate of nicotine dissociation. After reduction, both binding sites could be selectively alkylated with bromoacetylcholine. The results suggest that both putative nicotinic receptors in brain respond similarly to disulfide reduction and that their responses resemble those known for the nicotinic receptor of electric tissue.     

Arachidonic acid production by Mortierella alpina with growth-coupled lipid synthesis

The fungus Mortierella alpina LPM 301, a producer of arachidonic acid (ARA), was found to possess a unique property of a growth-coupled lipid synthesis. An increase in specific growth rate (μ) from 0.03 to 0.05 h⁻¹ resulted in a two-fold increase in the specific rate of lipid synthesis (milligram lipid (gram per lipid-free biomass) per hour). Under batch cultivation in glucose-containing media with urea or potassium nitrate as nitrogen sources, the ARA content was 46.0 and 60.4% of lipid; 16.4 and 18.8% of dry biomass; and 4.2 and 4.5 g l⁻¹, respectively. Under continuous cultivation of the strain, the productivity of ARA synthesis was 16.2 and 19.2 mg l⁻¹ h⁻¹ at μ=0.05 and 0.03 h⁻¹, respectively.     

Interaction of the nicotinic agonist (R,S)-3-pyridyl-1-methyl-2-(3-pyridyl)-azetidine (MPA) with nicotinic acetylcholine receptor subtypes expressed in cell lines and rat cortex

The interaction of the nicotinic agonist (R,S)-3-pyridyl-1-methyl-2-(3-pyridyl)-azetidine (MPA) with different nicotinic acetylcholine receptor (nAChR) subtypes was studied in cell lines and rat cortex. MPA showed an affinity (Ki = 1.21 nM) which was higher than anatoxin-a > (−)-nicotine > (+)-[R]nornicotine > (−)-[S]nornicotine > and (+)-nicotine, but lower than cytisine (Ki = 0.46 nM) in competing for (−)-[³H]nicotine binding in M10 cells, which stably express the recombinant ₄β₂ nAChR subtype. A one-binding site model was observed in all competing experiments between (−)-[³H]nicotine binding and each of the agonists studied in M10 cells. MPA showed a 13-fold higher affinity for (−)-[³H]nicotine binding sites compared to the [³H]epibatidine binding sites in rat cortical membranes. In human neuroblastoma SH-SY5Y cells, which predominantly express the ₃ nAChR subunit mRNA, MPA displaced [³H]epibatidine binding from a single population of the binding sites with an affinity in the same nM range as that observed MPA in displacing [³H]epibatidine binding in rat cortical membranes. Chronic treatment of M10 cells with MPA significantly up-regulated the number of (−)-[³H]nicotine binding sites in a concentration dependent manner. Thus MPA appears to have higher affinity to 4-subunit containing receptor subtype than ₃-subunit containing receptor subtype of nAChRs. Furthermore MPA binds to ₄β₂ receptor subtype with higher affinity than (−)-nicotine and behaves, opposite to cytisine, as a full agonist in up-regulating the number of nAChRs. © 1998 Elsevier Science Ltd. All rights reserved.     

In vitro culture of the seagrass Halophila decipiens

The seagrass Halophila decipiens Ostenfeld was grown axenically in a culture medium consisting of 20% artificial seawater, f/4 nutrients (except that glutamic acid was the nitrogen source), and 1% sucrose (w:v). The culture medium was adjusted to pH 5.0. A root–rhizome layer was created by solidifying a portion of the medium with 0.9% agar (w:v) and 1% activated charcoal (w:v). The rhizome layer also contained the following vitamins: 0.5 mg l⁻¹ nicotinic acid, 0.5 mg l⁻¹ pyridoxine, 0.5 mg l⁻¹ biotin, 0.5 mg l⁻¹ cyanocobalamin and 0.1 mg l⁻¹ of thiamine HCl. The liquid overlay (without vitamins or charcoal) was poured onto the agar-solidified root–rhizome layer. Growth of H. decipiens was not improved by the addition of the auxins indoleacetic acid (IAA), indolebutyric acid (IBA) or naphthaleneacetic acid (NAA) at either of the tested concentrations (10 and 50 μM). At a concentration of 10 μM, the cytokinins 6-(γ,γ-dimethylallylamino) purine (2iP) and benzylaminopurine (BA) stimulated shoot and branch production compared to controls with no cytokinins. Among the tested nitrogen sources, growth was best on 1.7 mM glutamic acid. Cultures grown on 1.7 mM NH₄Cl showed the same growth rates as those grown on glutamic acid, but the leaves were smaller and curled, suggesting incipient ammonium toxicity. Use of nitrate or urea led to mortality of the cultures. Long term axenic culture of H. decipiens appears to require the added vitamins. Hence, H. decipiens is the first seagrass known to need exogenous vitamins. Cultures of H. decipiens died when grown suspended in liquid cultures or in a biphasic medium system without activated charcoal in the root–rhizome layer. The use of more highly charged κ-carrageenan could not replace the use of activated charcoal and agar in the root–rhizome layer.     

Effects of thio-group modifications of Torpedo californica acetylcholine receptor on ion flux activation and inactivation kinetics

The effects of thio-group modifications on the ion permeability control and ligand binding properties of the acetylcholine receptor were measured in reconstituted membranes prepared from purified Torpedo californica acetylcholine receptor and soybean lipids (asolectin). A quench flow device was used to obtain subsecond time resolution for agonist-stimulated cation influx using carbamylcholine chloride (Carb) as the ligand and 86Rb+ as the cation. The effects of disulfide reduction with dithiothreitol (DTT), affinity alkylation with [4-(N-maleimido)benzyl]trimethylammonium ion and bromoacetylcholine, and nonspecific alkylation with N-ethylmaleimide and N-benzylmaleimide were examined. Activation, fast inactivation, and slow inactivation rates were measured on the chemically modified membranes. The flux results were compared with similar measurements on native membranes, and the role of vesicle size, heterogeneity, and influx time on ion flux results was analyzed. Major conclusions are that the binding sites that react with affinity labels are the same sites that mediate ligand-activated ion flux and that blockade of one of the two ligand binding sites is sufficient to block about 95% of the ion flux response. The main effect of DTT reduction is to shift the EC50 values for activation and slow inactivation to higher Carb concentrations, consistent with a decrease in binding affinity for Carb. The EC50 value for fast inactivation was not affected by DTT. However, the maximum rate of ion flux activation and the maximum rate of fast inactivation were decreased 2-fold after DTT treatment.     

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

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

Identification of a Disulfide Bridge Important for Transport Function of SNAT4 Neutral Amino Acid Transporter

SNAT4 is a member of system N/A amino acid transport family that primarily expresses in liver and muscles and mediates the transport of L-alanine. However, little is known about the structure and function of the SNAT family of transporters. In this study, we showed a dose-dependent inhibition in transporter activity of SNAT4 with the treatment of reducing agents, dithiothreitol (DTT) and Tris(2-carboxyethyl)phosphine (TCEP), indicating the possible involvement of disulfide bridge(s). Mutation of residue Cys-232, and the two highly conserved residues Cys-249 and Cys-321, compromised the transport function of SNAT4. However, this reduction was not caused by the decrease of SNAT4 on the cell surface since the cysteine-null mutant generated by replacing all five cysteines with alanine was equally capable of being expressed on the cell surface as wild-type SNAT4. Interestingly, by retaining two cysteine residues, 249 and 321, a significant level of L-alanine uptake was restored, indicating the possible formation of disulfide bond between these two conserved residues. Biotinylation crosslinking of free thiol groups with MTSEA-biotin provided direct evidence for the existence of a disulfide bridge between Cys-249 and Cys-321. Moreover, in the presence of DTT or TCEP, transport activity of the mutant retaining Cys-249 and Cys-321 was reduced in a dose-dependent manner and this reduction is gradually recovered with increased concentration of H₂O₂. Disruption of the disulfide bridge also decreased the transport of L-arginine, but to a lesser degree than that of L-alanine. Together, these results suggest that cysteine residues 249 and 321 form a disulfide bridge, which plays an important role in substrate transport but has no effect on trafficking of SNAT4 to the cell surface.     

Investigations of the catalytic mechanism of thioredoxin glutathione reductase from Schistosoma mansoni

Thioredoxin glutathione reductase from Schistosoma mansoni (SmTGR) catalyzes the reduction of both thioredoxin and glutathione disulfides (GSSG), thus playing a crucial role in maintaining redox homeostasis in the parasite. In line with this role, previous studies have demonstrated that SmTGR is a promising drug target for schistosomiasis. To aid in the development of efficacious drugs that target SmTGR, it is essential to understand the catalytic mechanism of SmTGR. SmTGR is a dimeric flavoprotein in the glutathione reductase family and has a head-to-tail arrangement of its monomers; each subunit has the components of both a thioredoxin reductase (TrxR) domain and a glutaredoxin (Grx) domain. However, the active site of the TrxR domain is composed of residues from both subunits: FAD and a redox-active Cys-154/Cys-159 pair from one subunit and a redox-active Cys-596'/Sec-597' pair from the other; the active site of the Grx domain contains a redox-active Cys-28/Cys-31 pair. Via its Cys-28/Cys-31 dithiol and/or its Cys-596'/Sec-597' thiol-selenolate, SmTGR can catalyze the reduction of a variety of substrates by NADPH. It is presumed that SmTGR catalyzes deglutathionylation reactions via the Cys-28/Cys-31 dithiol. Our anaerobic titration data suggest that reducing equivalents from NADPH can indeed reach the Cys-28/Cys-31 disulfide in the Grx domain to facilitate reductions effected by this cysteine pair. To clarify the specific chemical roles of each redox-active residue with respect to its various reactivities, we generated variants of SmTGR. Cys-28 variants had no Grx deglutathionylation activity, whereas Cys-31 variants retained partial Grx deglutathionylation activity, indicating that the Cys-28 thiolate is the nucleophile initiating deglutathionylation. Lags in the steady-state kinetics, found when wild-type SmTGR was incubated at high concentrations of GSSG, were not present in Grx variants, indicating that this cysteine pair is in some way responsible for the lags. A Sec-597 variant was still able to reduce a variety of substrates, albeit slowly, showing that selenocysteine is important but is not the sole determinant for the broad substrate tolerance of the enzyme. Our data show that Cys-520 and Cys-574 are not likely to be involved in the catalytic mechanism.     

Egg white sulfhydryl oxidase: kinetic mechanism of the catalysis of disulfide bond formation

The flavin-dependent sulfhydryl oxidase from chicken egg white catalyzes the oxidation of sulfhydryl groups to disulfides with reduction of oxygen to hydrogen peroxide. The oxidase contains FAD and a redox-active cystine bridge and accepts a total of 4 electrons per active site. Dithiothreitol (DTT; the best low molecular weight substrate known) reduces the enzyme disulfide bridge with a limiting rate of 502/s at 4 degrees C, pH 7.5, yielding a thiolate-to-flavin charge-transfer complex. Further reduction to EH4 is limited by the slow internal transfer of reducing equivalents from enzyme dithiol to oxidized flavin (3.3/s). In the oxidative half of catalysis, oxygen rapidly converts EH4 to EH2, but Eox appearance is limited by the slow internal redox equilibration. During overall turnover with DTT, the thiolate-to-flavin charge-transfer complex accumulates with an apparent extinction coefficient of 4.9 mM-1 cm-1 at 560 nm. In contrast, glutathione (GSH) is a much slower reductant of the oxidase to the EH2 level and shows a kcat/Km 100-fold smaller than DTT. Full reduction of EH2 by GSH shows a limiting rate of 3.6/s at 4 degrees C comparable to that seen with DTT. Reduced RNase is an excellent substrate of the enzyme, with kcat/Km per thiol some 1000- and 10-fold better than GSH and DTT, respectively. Enzyme-monitored steady-state turnover shows that RNase is a facile reductant of the oxidase to the EH2 state. This work demonstrates the basic similarity in the mechanism of turnover between all of these three substrates. A physiological role for sulfhydryl oxidase in the formation of disulfide bonds in secreted proteins is discussed.     

Separate sites of low and high affinity for agonists on Torpedo californica acetylcholine receptor

We have studied alkylation of the membrane-bound acetylcholine receptor (AcChR) from Torpedo californica electric organ by the cholinergic agonist bromo-acetylcholine (BrAcCh). Following reduction of the AcChR with dithiothreitol (DTT) under strictly controlled conditions, a single class of binding sites was covalently labeled by BrAcCh. The extent of alkylation was dependent on the concentration of both DTT and BrAcCh and reached a maximum when a number of sites equivalent to the number of alpha-bungarotoxin (alpha-BTx) binding sites were labeled. The reaction with BrAcCh was completely inhibited by saturating concentrations of alpha-BTx. On the contrary, complete alkylation of the AcChR with [3H]BrAcCh consistently inhibited only approximately 50% of alpha-BTx binding. The effects of DTT reduction and subsequent BrAcCh alkylation on the cation-gating properties of the AcChR were investigated in rapid kinetic experiments. DTT reduction resulted in a slight decrease in the maximum cation flux and a small shift in the effective dissociation constant to higher acetylcholine (AcCh) concentration. The flux response was completely inhibited by maximal alkylation of the membrane vesicles by BrAcCh. A low-affinity binding site for AcCh, which is likely to be important in AcChR activation, has been revealed for T. californica AcChR by studying the effects of cholinergic ligands on the fluorescence of a probe, 4-[(iodoacetoxy)ethylmethylamino]-7-nitro-2,1,3-benzoxadiazole (IANBD), covalently bound to the AcChR protein. Maximal labeling by BrAcCh did not affect the binding of AcCh to the low-affinity binding site, as monitored by changes in the fluorescence of this probe. This low-affinity binding site must therefore be distinct from the site labeled by BrAcCh. The results strongly support the notion that the nicotinic AcChR contains multiple binding sites for cholinergic ligands.     

Involvement of a Disulfide Bond in the Binding of Flunitrazepam to the Benzodiazepine Receptor from Bovine Cerebral Cortex

Abstract: The effects of chemical modification of a disulfide bond(s) (-SS-) or sulfhydryl group(s) (-SH) on the [³H]-flunitrazepam ([³H]FNZ) binding to membrane-bound or immunoprecipitated benzodiazepine (BZD) receptors (BZD-R) from bovine cerebral cortex were examined. Reduction of -SS- with dithiothreitol (DTT) brought about a reversible, time- and dose-dependent inhibition of [³H]FNZ binding to the membrane-bound BZD-R. Alkylation of the membranes with the -SH-modifying reagent iodoacetamide (IAA) or 5,5'-dithiobis-(2-nitrobenzoic acid) (DTNB) produced a slight inhibition of [³H]FNZ binding in a dose-dependent manner. Scatchard analysis of saturation curves of [³H]FNZ binding in the presence and absence of 5 m M DTT revealed changes in affinity without modification in the maximal binding capacity, thus indicating a competitive mode of interaction. DTT pretreatment of both the membrane-bound and the immunoprecipitated BZD-R led to [³H]FNZ binding inhibition. Consistent with the modification of a binding site is the observation that reduction of -SS- does not bear on the binding affinity, but rather reduces the number of sites. Complete protection from DTT inhibition of [³H]FNZ binding by FNZ (an agonist) or by Ro 15–1788 (an antagonist) suggests the presence of -SS- at, or very close to, the BZD recognition binding site. No protection against IAA or DTNB inhibition was provided by FNZ. Photoaffinity labeling experiments with [³H]FNZ revealed a clear-cut band of 50 kDa in native and alkylated membranes but an extremely weak label in 5 m M DTT/IAA-treated membranes. The present results provide evidence for the participation of a disulfide bond in the recognition binding site of the bovine cerebral cortex BZD-R.     

Role of extracellular disulfide-bonded cysteines in the ligand binding function of the beta 2-adrenergic receptor

Evidence is presented for a role of disulfide bridging in forming the ligand binding site of the beta 2-adrenergic receptor (beta AR). The presence of disulfide bonds at the ligand binding site is indicated by "competitive" inhibition by dithiothreitol (DTT) in radioligand binding assays, by specific protection by beta-adrenergic ligands of these effects, and by the requirement of disulfide reduction for limit proteolysis of affinity ligand labeled receptor. The kinetics of binding inhibition by DTT suggest at least two pairs of disulfide-bonded cysteines essential for normal binding. Through site-directed mutagenesis, we indeed were able to identify four cysteines which are critical for normal ligand binding affinities and for the proper expression of functional beta AR at the cell surface. Unexpectedly, the four cysteines required for normal ligand binding are not those located within the hydrophobic transmembrane domains of the receptor (where ligand binding is presumed to occur) but lie in the extracellular hydrophilic loops connecting these transmembrane segments. These findings indicate that, in addition to the well-documented involvement of the membrane-spanning domains of the receptor in ligand binding, there is an important and previously unsuspected role of the hydrophilic extracellular domains in forming the ligand binding site.     

Reaction of quinacrine mustard with the acetylcholine receptor from Torpedo californica

Amines with local anesthetic activity are typically also noncompetitive inhibitors of the agonist-induced increase in cation permeability mediated by the nicotinic acetylcholine receptor. Quinacrine is such an agent, and we have synthesized tritiated quinacrine mustard, a derivative capable of reacting with nucleophiles. Quinacrine mustard was reacted with receptor-rich membrane from torpedo electric tissue, excess reagent was removed by partition into liposomes, and the modified receptor was extracted and reconstituted with exogenous phospholipid. After reaction of the native membrane with 10 microM quinacrine mustard for 5 min, binding of cobratoxin to the acetylcholine binding sites is inhibited 15%; in contrast, receptor-mediated 86Rb uptake in the reconstituted vesicles is inhibited 70%. When the reaction with quinacrine mustard is carried out in the presence of 10 microM carbamylcholine or 10 microM d-tubocurarine, there is no block of the acetylcholine binding sites; nevertheless, the inhibition of Rb uptake is greater than that resulting from reaction in the absence of acetylcholine binding site ligands. Conversely, when the reaction is carried out in the presence of either 100 microM quinacrine or 100 microM proadifen (also a potent noncompetitive inhibitor), either with or without carbamylcholine or d-tubocurarine, the inhibition of 86Rb uptake is about 70% smaller. Under the same conditions that we used in the functional studies, quinacrine mustard reacts with the four types of chains that constitute the receptor complex, alpha 2 beta gamma delta. The presence of the acetylcholine binding site ligands, however, results in increased reaction with the alpha and beta chains, while the presence of the noncompetitive inhibitors, with or without the acetylcholine binding site ligands, results in decreased reaction with the alpha and beta chains. We conclude that the alpha and beta chains contribute to one or more functionally significant binding sites for noncompetitively inhibiting amines.     

The effect of amantadine on nicotinic acetylcholine receptor (nAchR) in reconstituted membranes

The antiviral drug amantadine is also a potent neuromuscular blocking agent. When the nicotinic receptor from a Torpedinidae species is reconstituted into soybean liposomes, the binding of α-bungarotoxin is not altered although the carbamylcholine induced radioactive cation influx is blocked.By studying cation fluxes in amantadine preincubated membranes previously exposed to different concentrations of carbamylcholine for different periods of time, we have shown that the drug accelerates the conversion of the nicotinic acetylcholine receptor from a state of low affinity to a state of high affinity for carbamyalcholine, a phenomenon correlated with receptor desensitization. The drug did not induce such a shift by itself.The present data and those by Earnest et al. (Biochemistry22, 5523–5535, 1984) show that the nicotinic acetylcholine receptor reconstituted into liposomes is a good model for studying the effects of noncompetitive blockers of nicotinic acetylcholine receptor function.