Spectroscopic Characterization of Arrestin Interactions with Competitive Ligands: Study of Heparin and Phytic Acid Binding

A combination of intrinsic fluorescence and circular dichroic (CD) spectroscopy has been used to characterize the complexes formed between bovine retinal arrestin and heparin or phytic acid, two ligands that are known to mimic the structural changes in arrestin attending receptor binding. No changes in the CD spectra were observed upon ligand binding, nor did the degree of tryptophan fluorescence quenching change significantly in the complexes. These data argue against any large-scale changes in protein secondary or tertiary structure accompanying ligand binding. The change in tyrosine fluorescence intensity was used to determine the dissociation constants for the heparin and phytic acid complexes of arrestin. The only change observed was a saturable diminution of tyrosine fluorescence signal from the protein. For both ligands, the data suggest two distinct binding interactions with the protein—a high-affinity interaction with K _d between 200 and 300 nM, and a lower affinity interaction with K _d between 2 and 8 M. Study of collisional quenching of tyrosine fluorescence in free arrestin and the ligand-replete complexes indicates that 10 of the 14 tyrosine residues of the protein are solvent-exposed in the free protein; this value drops to between 5 and 6 solvent-exposed residues in the high-affinity complexes of the two ligands. These data suggest that ligand binding leads to direct occlusion of between 4 and 5 tyrosine residues on the solvent-exposed surface of the protein, but not to any large-scale changes in protein structure. The large activation energy previously reported to be associated with arrestin–receptor interactions may therefore reflect localized movements of the N- and C-termini of arrestin, which are proposed to interact in the free protein through electrostatic interactions. Binding of the anionic ligands heparin, phytic acid, or phosphorylated rhodopsin may compete with the C-terminus of arrestin for these electrostatic interactions, thus allowing the C-terminus to swing out of the binding region.     

Heterogeneity of the two tryptophanyl residues in the lac repressor of Escherichia coli.

The wild-type lac repressor of Escherichia coli is a tetrameric protein which contains two tryptophanyl residues per subunit at positions 190 and 209. Solute perturbation studies of the tryptophan fluorescence of the repressor were performed using a polar but uncharged quencher, acrylamide, to prevent possible bias caused by ionic quenchers. The results indicate that the two tryptophan residues have different accessibilities to the quencher. In addition, contrary to a previous report, the accessibility of these tryptophan residues is not altered by isopropyl-β-d-thiogalactoside (IPTG) binding to the repressor. Similar studies with mutant lac repressor containing only a single tryptophan either at positions 190 or 209 suggest that tryptophan 209 is located in a region which is perturbed by inducer binding. That the two tryptophanyl residues have heterogeneous environments was further confirmed by nanosecond fluorescence spectroscopy which showed the wild-type lac repressor exhibiting two excited-state lifetimes, τ₁ = 5.3 ns and τ₂ = 10 ns. In the presence of 10^?3m IPTG, only a single lifetime of 6 ns was observed for the wild-type repressor suggesting that the inducer perturbs the tryptophan residue with the longer lifetime but not the one with the shorter lifetime. This is in accord with the observation that the mutant repressor containing only tryptophan 190 (the Tyr-209 repressor) has a single lifetime of 4.5 ns which is not altered by IPTG binding. The surprising finding that the mutant repressor which contains only tryptophan 209 (the Tyr-190 repressor) shows two excited-state lifetimes has been interpreted to indicate that the repressor either does not exhibit fourfold symmetry in its subunit arrangement or is present in two different conformational states.     

Probing the interactions of hemoglobin with antioxidant flavonoids via fluorescence spectroscopy and molecular modeling studies

Steady state and time resolved fluorescence spectroscopy, combined with molecular modeling computations, have been used to explore the interactions of two therapeutically important flavonoids, fisetin (3,7,3′,4′-OH-flavone) and 3-hydroxyflavone (3-HF), with normal human hemoglobin (HbA). Distinctive ‘two color’ fluorescence signatures and fairly high fluorescence anisotropy (r = 0.12-0.28) of fisetin and 3-HF reveal their specific interactions with HbA. Binding constants estimated from the fluorescence studies were ≈ 4.00 × 10⁴ M^− 1 and 9.83 × 10³ M^− 1 for fisetin and 3-HF respectively. Specific interactions with HbA were further confirmed from flavonoid-induced static quenching of the protein tryptophan fluorescence as indicated by: (a) bimolecular quenching constant K_q ? diffusion controlled limit (b) closely matched values of Stern-Volmer quenching constant and binding constant (c) τ_o/τ ≈ 1 (where τ_o and τ are the unquenched and quenched tryptophan fluorescence lifetimes respectively). Molecular docking and electrostatic surface potential calculations reveal contrasting binding modes of fisetin and 3-HF with HbA.     

Selective Inhibition of Bacterial Tryptophanyl-tRNA Synthetases by Indolmycin Is Mechanism-based

Indolmycin is a natural tryptophan analog that competes with tryptophan for binding to tryptophanyl-tRNA synthetase (TrpRS) enzymes. Bacterial and eukaryotic cytosolic TrpRSs have comparable affinities for tryptophan (K_m ∼ 2 μm), and yet only bacterial TrpRSs are inhibited by indolmycin. Despite the similarity between these ligands, Bacillus stearothermophilus (Bs)TrpRS preferentially binds indolmycin ∼1500-fold more tightly than its tryptophan substrate. Kinetic characterization and crystallographic analysis of BsTrpRS allowed us to probe novel aspects of indolmycin inhibitory action. Previous work had revealed that long range coupling to residues within an allosteric region called the D1 switch of BsTrpRS positions the Mg²⁺ ion in a manner that allows it to assist in transition state stabilization. The Mg²⁺ ion in the inhibited complex forms significantly closer contacts with non-bridging oxygen atoms from each phosphate group of ATP and three water molecules than occur in the (presumably catalytically competent) pre-transition state (preTS) crystal structures. We propose that this altered coordination stabilizes a ground state Mg²⁺·ATP configuration, accounting for the high affinity inhibition of BsTrpRS by indolmycin. Conversely, both the ATP configuration and Mg²⁺ coordination in the human cytosolic (H_c)TrpRS preTS structure differ greatly from the BsTrpRS preTS structure. The effect of these differences is that catalysis occurs via a different transition state stabilization mechanism in H_cTrpRS with a yet-to-be determined role for Mg²⁺. Modeling indolmycin into the tryptophan binding site points to steric hindrance and an inability to retain the interactions used for tryptophan substrate recognition as causes for the 1000-fold weaker indolmycin affinity to H_cTrpRS.     

Isolation and fluorescence studies on a lipophorin from the weevil diaprepes abbreviatus

Lipophorin was isolated from larvae of a root weevil, Diaprepes abbreviatus (Coleoptera: Curculionidae), using density gradient ultracentrifugation. D. abbreviatus lipophorin contained two apoproteins, apolipophorin-I (M_r = 226,000) and apolipophorin-II (M_r = 72,100) and had a density of 1.08. Relative to other larval lipophorins, D. abbreviatus lipophorin contained little cysteine (determined as cysteic acid) and methionine. Fluorescence spectroscopy of intrinsic tyrosine and tryptophan residues excited at 290 nm revealed a single broad emission peak at 330 nm. Upon denaturing and delipidating lipophorin in guanidine HCl, this peak resolved into two peaks with maxima at 305 and 350 nm. Excitation spectra suggested that the two peaks were due to tyrosine and tryptophan, respectively. Fluorescence quenching agents, iodide and acrylamide, were used to determine accessibility of tyrosine and tryptophan residues to the aqueous environment. Iodide, a polar quenching agent, did not quench fluorescent emission from native lipophorin; quenching by iodide increased to moderate levels when lipophorin was denatured in guanidine HCl. Acrylamide quenched the fluorescence of native lipophorin moderately and very efficiently quenched fluorescence of denatured lipophorin. No difference was observed between fluorescence quenching of denatured vs. denatured and delipidated lipophorin by either iodide or acrylamide.     

Effect of ligand binding and conformational changes in proteins on oxygen quenching and fluorescence depolarization of tryptophan residues

The rotational freedom of tryptophan residues in protein-ligand complexes was studied by measuring steady-state fluorescence anisotropies under conditions of oxygen quenching. There was a decrease in the oxygen bimolecular quenching constant upon complexation of trypsin and alpha-chymotrypsin with proteinaceous trypsin inhibitors, of lysozyme with N-acetylglucosamine (NAG) and di(N-acetyl-D-glucosamine) ((NAG)2) and of hexokinase with glucose. Binding of the bisubstrate analogue N-phosphonacetyl-L-aspartate (PALA) to aspartate transcarbamylase (ATCase) and binding of biotin to avidin resulted in increased oxygen quenching constants. The tryptophan of human serum albumin (HSA) in the F state was more accessible to oxygen quenching than that in the N state. With the exception of ATCase, the presence of subnanosecond motions of the tryptophan residues in all the proteins is suggested by the short apparent correlation times for fluorescence depolarization and by the low apparent anisotropies obtained by extrapolation to a lifetime of zero. Complex formation evidently resulted in more rigid structures in the case of trypsin, alpha-chymotrypsin and lysozyme. The effects of glucose binding on hexokinase were not significant. Binding of biotin to avidin resulted in a shorter correlation time for the tryptophan residues. The N --> F transition in HSA resulted in a more rigid environment for the tryptophan residue. Overall, these changes in the dynamics of the protein matrix and motional freedom of tryptophan residues due to complex formation and subsequent conformational changes are in the same direction as those observed by other techniques, especially hydrogen exchange. Significantly, the effects of complex formation on protein dynamics are variable. Among the limited number of cases we examined, the effects of complex formation were to increase, decrease or leave unchanged the apparent dynamics of the protein matrix.     

A phosphorylation-dependent gating mechanism controls the SH2 domain interactions of the Shc adaptor protein

The Shc (Src homology collagen-like) adaptor protein plays a crucial role in linking stimulated receptors to mitogen-activated protein kinase activation through the formation of dynamic signalling complexes. Shc comprises an N-terminal phosphotyrosine binding (PTB) domain, a C-terminal Src homology 2 (SH2) domain and a central proline-rich collagen homology 1 domain. The latter domain contains three tyrosine residues that are known to become phosphorylated. We have expressed and purified the human p52Shc isoform and characterised its binding to different ligands. CD spectra revealed that some parts of the Shc protein are not fully folded, remaining largely unaffected by the binding of ligands. The PTB domain binds peptide and Ins-1,4,5-P₃ (but not Ins-1,3,5-P₃) independently, suggesting two distinct sites of interaction. In the unphosphorylated Shc, the SH2 domain is non-functional. Ligand binding to the PTB domain does not affect this. However, phosphorylation of the three tyrosine residues promotes binding to the SH2 domain. Thus, Shc has an intrinsic phosphorylation-dependent gating mechanism where the SH2 domain adopts an open conformation only when tyrosine phosphorylation has occurred.     

Nitric Oxide Activation of Guanylate Cyclase Pushes the α1 Signaling Helix and the β1 Heme-binding Domain Closer to the Substrate-binding Site

The complete structure of the assembled domains of nitric oxide-sensitive guanylate cyclase (NOsGC) remains to be determined. It is also unknown how binding of NO to heme in guanylate cyclase is communicated to the catalytic domain. In the current study the conformational change of guanylate cyclase on activation by NO was studied using FRET. Endogenous tryptophan residues were used as donors, the substrate analog 2′-Mant-3′-dGTP as acceptor. The enzyme contains five tryptophan residues distributed evenly over all four functional domains. This provides a unique opportunity to detect the movement of the functional domains relative to the substrate-binding catalytic region. FRET measurements indicate that NO brings tryptophan 22 in the αB helix of the β₁ heme NO binding domain and tryptophan 466 in the second short helix of the α₁ coiled-coil domain closer to the catalytic domain. We propose that the respective domains act as a pair of tongs forcing the catalytic domain into the nitric oxide-activated conformation.     

An NMR-based docking model for the physiological transient complex between cytochrome f and cytochrome c6

The physiological transient complex between cytochrome f (Cf) and cytochrome c₆ (Cc₆) from the cyanobacterium Nostoc sp. PCC 7119 has been analysed by NMR spectroscopy. The binding constant at low ionic strength is 8 ± 2 mM⁻¹, and the binding site of Cc₆ for Cf is localized around its exposed haem edge. On the basis of the experimental data, the resulting docking simulations suggest that Cc₆ binds to Cf in a fashion that is analogous to that of plastocyanin but differs between prokaryotes and eukaryotes.     

Role of the O-phosphoserine clusters in the interaction of the bovine milk αs1-, β-, κ-caseins and the PP3 component with immobilized iron (III) ions

α_s1- and β-Caseins have a sequence cluster -Ser(P)-Ser(P)-Ser(P)-Glu-Glu- which is not present in κ-casein and the whey PP3 component. The affinity of these phosphoproteins for the iron(III)-iminodiacetic acid (IDA) complex immobilized on Sepharose was studied a a function of pH, urea concetnration, calcium ion concentration, enzymatic dephosphorylation and temperature. The affinity of the three polyphosphorylated proteins (α_s1- and β-caseins, PP3) was similar. The sequence cluster was not a specific recognition pattern for the iron(III) ion. These three proteins presented a site of high affinity and a site of weak affinity. κ-Casein, which had only one Ser(P) residue, presented only the site of weak affinity. Their primary site which was absent after dephosphorylation or calcium ion addition required the presence of at least two Ser(P) residues close in space. Their secondary site was sensitive to the presence of urea. It was sensitive to pH variation for PP3 and κ-casein. The study of the affinity of a few free amino acids towards iron(III)-IDA showed that the secondary site involved tryptophan and tyrosine residues for α_s1- and β-caseins, histidine residues for PP3 and cysteine residues for κ-casein.     

Stabilization of the human beta2-adrenergic receptor TM4-TM3-TM5 helix interface by mutagenesis of Glu122(3.41), a critical residue in GPCR structure

G protein-coupled receptor (GPCR) instability represents one of the most profound obstacles in the structural study of GPCRs that bind diffusible ligands. The introduction of targeted mutations at nonconserved residues that lie proximal to helix interfaces has the potential to enhance the fold stability of the receptor helix bundle while maintaining wild-type receptor function. To test this hypothesis, we studied the effect of amino acid substitutions at Glu122^3.41 in the well-studied β₂-adrenergic receptor (β₂AR), which was predicted from sequence conservation to lie at a position equivalent to a tryptophan residue in rhodopsin at the 3,4,5 helix interface among transmembrane (TM) domains 3, 4, and 5. Replacement of Glu122^3.41 with bulky hydrophobic residues, such as tryptophan, tyrosine, and phenylalanine, increases the yield of functionally folded β₂AR by as much as 5-fold. Receptor stability in detergent solution was studied by isothermal denaturation, and it was found that the E122W and E122Y mutations enhanced the β₂AR thermal half-life by 9.3- and 6.7-fold, respectively, at 37 °C. The β₁AR was also stabilized by the introduction of tryptophan at Glu147^3.41, and the effect on protein behavior was similar to the rescue of the unstable wild-type receptor by the antagonist propranolol. Molecular modeling of the E122W and E122Y mutants revealed that the tryptophan ring edge and tyrosine hydroxyl are positioned proximal to the helical break in TM5 introduced by the conserved Pro211^5.50 and may stabilize the helix by interacting favorably with the unpaired carbonyl oxygen of Val206^5.45. Conformational flexibility of TM5 is likely to be a general property of class A GPCRs; therefore, engineering of the TM4-TM3-TM5 interface at the 3.41 position may provide a general strategy for the stabilization of other receptors.     

The structure and orientation of class-A amphipathic peptides on a phospholipid bilayer surface

The amphipathic α-helix is a recognised structural motif that is shared by membrane-associating proteins and peptides of diverse function. The aim of this paper is to determine the orientation of an α-helical amphipathic peptide on the bilayer surface. We use five amphipathic 18-residue peptide analogues of a class A amphipathic peptide that is known to associate with a bilayer surface. Tyrosine and tryptophan are used as spectroscopic probes to sense local environments in the peptide in solution and when bound to the surface of unilamellar phosphatidylcholine vesicles. In a series of peptides, tryptophan is moved progressively along the sequence from the nonpolar face (positions 3, 7, 4) to the polar face of the peptide (positions 2, 12). The local environment of the tryptophan residue at each position is determined using fluorescence spectroscopy employing quantum yield, and the wavelength of the emission maximum as indicators of micropolarity. The exposure of the tryptophan residues at each site is assessed by acrylamide quenching. On association with vesicles, the tryptophan residues at positions 3, 7 and 14 are in nonpolar water-shielded environments, and the tryptophan at position 12 is in an exposed polar environment. The tryptophan at position 2, which is located near the bilayer-water interface, exhibits intermediate behaviour. Analysis of the second-derivative absorption spectrum confirmed that the tyrosine residue at position 7 is in a nonpolar water-shielded environment in the peptide-lipid complex. We conclude that these class A amphipathic peptides lie parallel to the lipid surface and penetrate no deeper than the ester linkages of the phospholipids. Received: 8 April 1998 / Revised version: 6 July 1998 / Accepted: 7 August 1998     

Structure and properties of a bovine liver UGA suppressor serine tRNA with a tryptophan anticodon

A bovine liver serine tRNA with a variety of unusual features has been sequenced and characterized. This tRNA is aminoacylated with serine, although it has a tryptophan anticodon CmCA. In ribosome binding assays, this tRNA (tRNA_CmCA^Ser) binds to the termination codon UGA and shows little or no binding in response to a variety of other codons including those for tryptophan and serine. The unusual codon recognition properties of this molecule were confirmed in an in vitro assay where this tRNA suppressed UGA termination. This is the first naturally occurring eucaryotic suppressor tRNA to be so characterized. Other unusual features, possibly related to the ability of this tRNA to read UGA, are the presence of two extra nucleotides, compared to all other tRNAs, between the universal residues U at position 8 and A at position 14 and the presence of an extra unpaired nucleotide within the double-stranded loop IV stem. This tRNA is also the largest eucaryotic tRNA sequenced to date (90 nucleotides). Despite its size, however, it contains only six modified residues. tRNA_CmCA^Ser shows extremely low homology to other mammalian serine (47–52% homology) or tryptophan (49% homology) tRNAs.     

Chemical modification studies onCocos nucifera pollen allergens

Two highly active allergens C_n II (M _r 158,000) and C_nVII (M _r 2900) isolated fromCocos nucifera pollen extract were treated with various protein modifying reagents in order to ascertain the amino acid residues responsible for their allergenicity, In C_n II modification of carboxy group and tryptophan residue led to 30 and ≽ 75% loss in allergenicity and those of lysine and tyrosine reduced 62 and 38% activity, Lysine, tyrosine, tryptophan and carboxy group of C_nVII were also modified causing 81, 17, ≽ 70 and 26% loss of allergenicity respectively, Allergenicity of both was highly affected by pronase and moderately affected by heat, Periodate destroyed about 50% of their allergenicity and other chemical reagents except urea had no remarkable effect     

Dopamine release and molecular modeling study of some coumarin derivatives

4-aryl-2-amino-6-(4-hydroxy-2-oxo-2H-chromen-3-yl)-pyridin-3-carbonitrile (1), 4-aryl-2-oxo-6-(4-hydroxy-2-oxo-2H-chromen-3-yl)-pyridin-3-carbonitriles (2a-2c), 3-(6-aryl-1,2,5,6- tetrahydro-2-thioxopyrimidin-4-yl)-4-hydroxy-2H-chromen-2-one (3a, 3b) and pyrazol-3-yl-4-hydroxycoumarin derivatives (4a-4c, 5, 6a, 6b, 7a, 7b, and 8a-8c) were prepared in order to measure their % change dopamine release in comparison to amphetamine as reference, using PC-12 cells in different concentrations. In addition, the molecular modeling study of the compounds into 3BHH receptor was also demonstrated. The calculated inhibition constant (k_i) implemented in the AutoDock program revealed identical correlation with the experimental results to that obtained binding free energy (ΔG_b) as both parameters revealed reasonable correlation coefficients (R²) being 0.51 involving 10 compounds; (1, 2b, 2c, 3a, 3b, 4a, 4b, 6a, and 8c).     

Chemical and Photochemical Modification of Colicin E1 and Gramicidin A in Bilayer Lipid Membranes

Chemical modification and photodynamic treatment of the colicin E1 channel-forming domain (P178) in vesicular and planar bilayer lipid membranes (BLMs) was used to elucidate the role of tryptophan residues in colicin E1 channel activity. Modification of colicin tryptophan residues by N-bromosuccinimide (NBS), as judged by the loss of tryptophan fluorescence, resulted in complete suppression of wild-type P178 channel activity in BLMs formed from fully saturated (diphytanoyl) phospholipids, both at the macroscopic-current and single-channel levels. The similar effect on both the tryptophan fluorescence and the electric current across BLM was observed also after NBS treatment of gramicidin channels. Of the single-tryptophan P178 mutants studied, W460 showed the highest sensitivity to NBS treatment, pointing to the importance of the water-exposed Trp460 in colicin channel activity. In line with previous work, the photodynamic treatment (illumination with visible light in the presence of a photosensitizer) led to suppression of P178 channel activity in diphytanoyl-phospholipid membranes concomitant with the damage to tryptophan residues detected here by a decrease in tryptophan fluorescence. The present work revealed novel effects: activation of P178 channels as a result of both NBS and photodynamic treatments was observed with BLMs formed from unsaturated (dioleoyl) phospholipids. These phenomena are ascribed to the effect of oxidative modification of double-bond-containing lipids on P178 channel formation. The pronounced stimulation of the colicin-mediated ionic current observed after both pretreatment with NBS and sensitized photomodification of the BLMs support the idea that distortion of membrane structure can facilitate channel formation.Abbreviations: AlP_cS₃, almininum trisulfophthalocyanine; BLM, bilayer lipid membrane; DOPC, dioleoylphosphatidylcholine; DOPG, dioleoylphosphatidyl-glycerol; DPhPG, diphytanoylphos-phatidylglycerol; DPhPg, diphytanoylphosphatidylcholine; gA, gramicidin A; NBS, N-bromosuccinimideThis revised version was published online in August 2005 with a corrected cover date.     

Design and synthesis of (5-amino-1, 2, 4-triazin-6-yl)(2-(benzo[d] isoxazol-3-yl) pyrrolidin-1-yl)methanone derivatives as sodium channel blocker and anticonvulsant agents

Abstract

A series of novel (5-amino-3-substituted-1, 2, 4-triazin-6-yl) (2-(6-halo-substituted benzo[d]isoxazol-3-yl) pyrrolidin-1-yl) methanone 5a–5r was synthesized. Their anticonvulsant activities were evaluated by the maximal electroshock (MES) test and neurotoxicity was evaluated by the rotorod test. The MES test showed that (5-amino-3-phenyl-1, 2, 4-triazin-6-yl)(2-(6-fluorobenzo[d]isoxazol-3-yl) pyrrolidin-1-yl) methanone 5c was found to be the most potent compound with ED₅₀ value of 6.20?mg/kg (oral/rat) and a protective index (PI?=?ED₅₀/TD₅₀) value of >48.38, which was much higher than the PI of the reference drug phenytoin. To explain the possible mechanism of action of selected derivatives 5b, 5c, 5i and 5o, their influence on sodium channel was evaluated in vitro.     

Effects of Nucleotides on [3H]Bradykinin Binding in Guinea Pig: Further Evidence for Multiple B2 Receptor Subtypes

Abstract: We have suggested recently the existence of three subtypes of B₂ bradykinin receptors in tissues of guinea pigs. We have classified these B₂ bradykinin receptors into B_2a, B_2b, and B_2c subtypes depending on their affinity for various bradykinin antagonists. Because the actions of bradykinin in different cell systems appear to be both dependent on and independent of G proteins, we sought to determine whether the binding of [³H]bradykinin to the B₂ subtypes is sensitive to guanine nucleotides and, therefore, possibly coupled to G proteins. In the ileum, where we have demonstrated B_2a and B_2b subtypes, specific [³H]bradykinin binding was reduced with GDP (100 μM) and the nonmetabolized analogue of GTP, guanyl-5′-yl-imidodiphosphate (GppNHp; 100 μM). Competition studies with bradykinin and with [Hyp³]-bradykinin, which shows approximately 20-fold greater selectivity for the B_2a subtype than bradykinin, were performed in the presence or absence of GppNHp (100 μM). The competition experiments demonstrated that binding to the B_2a subtype, which has higher affinity for [Hyp³]-bradykinin and bradykinin than the B_2b subtype, was lost in the presence of GppNHp, whereas binding to the B_2b subtype was unaffected. In contrast, GppNHp (100 μM) and GDP (100 μM) failed to alter specific [³H]bradykinin binding to B_2b and B_2c subtypes in lung. [³H]Bradykinin binding was unaffected by AMP, ADP, ATP, and GMP (100 μM each). Based on this evidence, we suggest that the B_2a bradykinin subtype is coupled to G proteins. The B_2b and B_2c subtypes are either not coupled to G proteins, or may be coupled to the G^o-type GTP binding proteins, which have been suggested to be less sensitive to guanine nucleotides. These data provide further evidence for three subtypes of B₂-type bradykinin receptors in guinea pig.     

Adsorption studies for the separation ofl-tryptophan froml-serine and indole in a bioconversion medium

l-tryptophan was produced froml-serine and indole by immobilized Escherichia coli cells in organic-aqueous systems. Selective adsorption was the method chosen to enable both product separation andl-serine reutilization. Amongst various adsorbents tested activated carbons and neutral polymeric resins (XAD-4 and XAD-7) showed good performance. The neutral resins could selectively concentrate thel-tryptophan from dilute aqueous solutions and adsorbed only 5% of the unconvertedl-serine. High separation factors (l-tryptophan/l-serine and indole/l-tryptophan) were obtained with these adsorbents. Despite a lower capacity, the XAD-7 resin had the advantage of desorbingl-tryptophan with basic or acidic solutions, while organic solvents were required to desorb, at the same concentration levels, this compound from XAD-4.In a packed bed column filled with XAD-4 resin or activated carbon, totall-tryptophan adsorption and recovery were achieved at linear velocities up to 5.0 cm/min and 3.2 cm/min respectively. Successive sorbent reutilization, following continuous sorption and elution steps, was carried out in packed bed columns with the neutral resins and activated carbon.Thel-form of tryptophan, after crystallization, was identified by HPTLC.List of Symbols HPLC High Performance Liquid Chromatography - HPTLC High Performance Thin Layer Chromatography - Trp tryptophan - Ser Serine - A amount of sorbent(g) - c equilibrium solute concentration in the aqueous phase (g/dm³) - c _i initial (before adding the sorbent) liquid phase concentration (g/dm³) - C _T tryptophan concentration in the inlet solution (g/dm³) - C _To tryptophan concentration in the outlet solution (g/dm³) - E _z axial dispersion coefficient (m²/s) - k experimental constant (Eq. 1, 2 and 3) - K ₁ rate constant of adsorption (min^–1) - L column length(m) - n experimental constant (eq. 1, 2 and 3) - q equilibrium solid phase concentration (g solute/g sorbent) - q _max maximum capacity of sorbent (g solute/g sorbent) - t time(s) - v liquid velocity (m/s) - V volume of liquid phase(dm³) - V _e eluted volume(dm³) - V _r volume needed to saturate the column (dm³)