scholarly journals Role of the N-terminal region of phospholipase A2 subunit of β1-bungarotoxin in the toxin-Ca2+ complex-formation

1991 ◽  
Vol 278 (2) ◽  
pp. 481-486 ◽  
Author(s):  
S T Chu ◽  
Y H Chen
Keyword(s):  
Phospholipase A2 ◽  
Metal Ions ◽  
Binding Sites ◽  
Association Constant ◽  
Enzymic Activity ◽  
Association Constants ◽  
High Affinity ◽  
Toxin Molecule ◽  
High Affinity Site

beta 1-Bungarotoxin consists of a phospholipase A2 subunit and a non-phospholipase A2 subunit. Modification of beta 1-bungarotoxin with CNBr resulted in cleavage at Met-6 and Met-8 of its phospholipase A2 subunit. Analysis of the fluorescence data of both the toxin-Ca2+ complex at 300-350 nm and the toxin-Tb3+ complex at 450-650 nm showed the existence of two binding sites for both metal ions on the different domains of the toxin molecule. At pH 7.6 the association constants for the high-affinity and low-affinity sites of the toxin-Ca2+ complex were determined to be 2.79 x 10(3) +/- 0.21 x 10(3) M-1 and 0.47 x 10(3) +/- 0.06 x 10(3) M-1 respectively. For the toxin-Tb3+ complex the association constant for the high-affinity site was 2.95 x 10(3) +/- 0.43 x 10(3) M-1 and that for the low-affinity site was 0.11 x 10(3) +/- 0.03 x 10(3) M-1. Removal of the N-terminal octapeptide of the phospholipase A2 subunit from the toxin molecule caused disintegration of the low-affinity site but did not disrupt the high-affinity site. This might accompany a change in the configuration around His-48 of the phospholipase A2 subunit. Between pH 6 and 8 the binding of metal ions to the high-affinity site increased but that to the low-affinity site did not change with increasing pH. The neurotoxicity and enzymic activity of the toxin were lost on removal of the low-affinity site.

scholarly journals Roles of Two ATPase-Motif-containing Domains in Cyanobacterial Circadian Clock Protein KaiC

2004 ◽  
Vol 279 (50) ◽  
pp. 52331-52337 ◽  
Author(s):  
Fumio Hayashi ◽  
Noriyo Itoh ◽  
Tatsuya Uzumaki ◽  
Ryo Iwase ◽  
Yuka Tsuchiya ◽  
...  
Keyword(s):  
Reaction Mechanism ◽  
Circadian Clock ◽  
Binding Sites ◽  
Wild Type ◽  
High Affinity ◽  
High Affinity Site ◽  
Clock Protein

Cyanobacterial clock protein KaiC has a hexagonal, pot-shaped structure composed of six identical dumbbell-shaped subunits. Each subunit has duplicated domains, and each domain has a set of ATPase motifs. The two spherical regions of the dumbbell are likely to correspond to two domains. We examined the role of the two sets of ATPase motifs by analyzing thein vitroactivity of ATPγS binding, AMPPNP-induced hexamerization, thermostability, and phosphorylation of KaiC and byin vivorhythm assays both in wild type KaiC (KaiCWT) and KaiCs carrying mutations in either Walker motif A or deduced catalytic Glu residues. We demonstrated that 1) the KaiC subunit had two types of ATP-binding sites, a high affinity site in N-terminal ATPase motifs and a low affinity site in C-terminal ATPase motifs, 2) the N-terminal motifs were responsible for hexamerization, and 3) the C-terminal motifs were responsible for both stabilization and phosphorylation of the KaiC hexamer. We proposed the following reaction mechanism. ATP preferentially binds to the N-terminal high affinity site, inducing the hexamerization of KaiC. Additional ATP then binds to the C-terminal low affinity site, stabilizing and phosphorylating the hexamer. We discussed the effect of these KaiC mutations on circadian bioluminescence rhythm in cells of cyanobacteria.

scholarly journals Novel Three-Finger Neurotoxins from Naja melanoleuca Cobra Venom Interact with GABAA and Nicotinic Acetylcholine Receptors

Toxins ◽  
2021 ◽  
Vol 13 (2) ◽  
pp. 164
Author(s):  
Lina Son ◽  
Elena Kryukova ◽  
Rustam Ziganshin ◽  
Tatyana Andreeva ◽  
Denis Kudryavtsev ◽  
...  
Keyword(s):  
Nicotinic Acetylcholine Receptors ◽  
Gabaa Receptors ◽  
Binding Sites ◽  
Acetylcholine Receptors ◽  
Nicotinic Acetylcholine ◽  
High Affinity ◽  
Central Loop ◽  
Acetylcholine Binding Proteins ◽  
Α7 Nachrs

Cobra venoms contain three-finger toxins (TFT) including α-neurotoxins efficiently binding nicotinic acetylcholine receptors (nAChRs). As shown recently, several TFTs block GABAA receptors (GABAARs) with different efficacy, an important role of the TFTs central loop in binding to these receptors being demonstrated. We supposed that the positive charge (Arg36) in this loop of α-cobratoxin may explain its high affinity to GABAAR and here studied α-neurotoxins from African cobra N. melanoleuca venom for their ability to interact with GABAARs and nAChRs. Three α-neurotoxins, close homologues of the known N. melanoleuca long neurotoxins 1 and 2, were isolated and sequenced. Their analysis on Torpedocalifornica and α7 nAChRs, as well as on acetylcholine binding proteins and on several subtypes of GABAARs, showed that all toxins interacted with the GABAAR much weaker than with the nAChR: one neurotoxin was almost as active as α-cobratoxin, while others manifested lower activity. The earlier hypothesis about the essential role of Arg36 as the determinant of high affinity to GABAAR was not confirmed, but the results obtained suggest that the toxin loop III may contribute to the efficient interaction of some long-chain neurotoxins with GABAAR. One of isolated toxins manifested different affinity to two binding sites on Torpedo nAChR.

scholarly journals Functional characterization of the dimerization domain of the ferric uptake regulator (Fur) of Pseudomonas aeruginosa

2006 ◽  
Vol 400 (3) ◽  
pp. 385-392 ◽  
Author(s):  
Erdeni Bai ◽  
Federico I. Rosell ◽  
Bao Lige ◽  
Marcia R. Mauk ◽  
Barbara Lelj-Garolla ◽  
...  
Keyword(s):  
Pseudomonas Aeruginosa ◽  
Metal Ions ◽  
Binding Site ◽  
Metal Ion ◽  
Association Constants ◽  
Ion Binding ◽  
Ferric Uptake Regulator ◽  
Metal Ion Binding ◽  
Dimerization Domain ◽  
High Affinity

The functional properties of the recombinant C-terminal dimerization domain of the Pseudomonas aeruginosa Fur (ferric uptake regulator) protein expressed in and purified from Escherichia coli have been evaluated. Sedimentation velocity measurements demonstrate that this domain is dimeric, and the UV CD spectrum is consistent with a secondary structure similar to that observed for the corresponding region of the crystallographically characterized wild-type protein. The thermal stability of the domain as determined by CD spectroscopy decreases significantly as pH is increased and increases significantly as metal ions are added. Potentiometric titrations (pH 6.5) establish that the domain possesses a high-affinity and a low-affinity binding site for metal ions. The high-affinity (sensory) binding site demonstrates association constants (KA) of 10(±7)×106, 5.7(±3)×106, 2.0(±2)×106 and 2.0(±3)×104 M−1 for Ni2+, Zn2+, Co2+ and Mn2+ respectively, while the low-affinity (structural) site exhibits association constants of 1.3(±2)×106, 3.2(±2)×104, 1.76(±1)×105 and 1.5(±2)×103 M−1 respectively for the same metal ions (pH 6.5, 300 mM NaCl, 25 °C). The stability of metal ion binding to the sensory site follows the Irving–Williams order, while metal ion binding to the partial sensory site present in the domain does not. Fluorescence experiments indicate that the quenching resulting from binding of Co2+ is reversed by subsequent titration with Zn2+. We conclude that the domain is a reasonable model for many properties of the full-length protein and is amenable to some analyses that the limited solubility of the full-length protein prevents.

Temperature-dependence of the DnaA–DNA interaction and its effect on the autoregulation of dnaA expression

2012 ◽  
Vol 449 (2) ◽  
pp. 333-341 ◽  
Author(s):  
Chiara Saggioro ◽  
Anne Olliver ◽  
Bianca Sclavi
Keyword(s):  
Temperature Dependence ◽  
Binding Sites ◽  
Dna Interaction ◽  
Bacterial Dna ◽  
Dnaa Protein ◽  
High Affinity ◽  
Key Factor

The DnaA protein is a key factor for the regulation of the timing and synchrony of initiation of bacterial DNA replication. The transcription of the dnaA gene in Escherichia coli is regulated by two promoters, dnaAP1 and dnaAP2. The region between these two promoters contains several DnaA-binding sites that have been shown to play an important role in the negative auto-regulation of dnaA expression. The results obtained in the present study using an in vitro and in vivo quantitative analysis of the effect of mutations to the high-affinity DnaA sites reveal an additional effect of positive autoregulation. We investigated the role of transcription autoregulation in the change of dnaA expression as a function of temperature. While negative auto-regulation is lost at dnaAP1, the effects of both positive and negative autoregulation are maintained at the dnaAP2 promoter upon lowering the growth temperature. These observations can be explained by the results obtained in vitro showing a difference in the temperature-dependence of DnaA–ATP binding to its high- and low-affinity sites, resulting in a decrease in DnaA–ATP oligomerization at lower temperatures. The results of the present study underline the importance of the role for autoregulation of gene expression in the cellular adaptation to different growth temperatures.

Role of external Na+ and cytosolic pH in agonist-evoked cytosolic Ca2+ response in human platelets

1994 ◽  
Vol 267 (6) ◽  
pp. C1543-C1552 ◽  
Author(s):  
M. Kimura ◽  
K. Nakamura ◽  
J. W. Fenton ◽  
T. T. Andersen ◽  
J. P. Reeves ◽  
...  
Keyword(s):  
Binding Sites ◽  
Human Platelets ◽  
Thrombin Receptor ◽  
Free Medium ◽  
Amidolytic Activity ◽  
Cytosolic Ph ◽  
Amino Acid Peptide ◽  
High Affinity ◽  
Inositol 1,4,5 Trisphosphate

The role of external Na+ in agonist-evoked platelet Ca2+ response is poorly understood. This was explored in this study. Removal of external Na+ decreased both cytosolic Ca2+ mobilization and external Ca2+ entry, induced by thrombin but not by ADP or vasopressin. That external Na+ regulates thrombin activities was demonstrated by 1) Na+ dependency of the amidolytic activity of thrombin, 2) inhibition of thrombin binding to the high-affinity binding sites in Na(+)-free medium, and 3) attenuation of thrombin-induced inositol 1,4,5-trisphosphate production in Na(+)-free medium. Moreover, Ca2+ response to the thrombin receptor 6-amino acid peptide was independent of external Na+. The role of external Na+ in modifying agonist-evoked Ca2+ response through activation of Na+/H+ antiport and cytosolic alkalinization was then explored. Cytosolic alkalinization by monensin or NH4Cl enhanced thrombin, ADP, and thimerosal-induced external Ca2+ entry. Thimerosal-induced acceleration of external Ca2+ entry was diminished by the inhibition of Na+/H+ antiport. Thus external Na+ enhances thrombin activities, and cytosolic pH mediates store-regulated external Ca2+ entry. However, Na+/H+ antiport activation is not essential for agonist-evoked Ca2+ mobilization and external Ca2+ entry.

scholarly journals Oxidation of human haemoglobin by copper. Mechanism and suggested role of the thiol group of residue β-93

1977 ◽  
Vol 165 (1) ◽  
pp. 141-148 ◽  
Author(s):  
C C Winterbourn ◽  
R W Carrell
Keyword(s):  
Electron Transfer ◽  
Binding Sites ◽  
Equilibrium Dialysis ◽  
Thiol Group ◽  
Thiol Groups ◽  
Human Haemoglobin ◽  
High Affinity ◽  
Rate Determining Step ◽  
Haemoglobin Molecule

Addition of Cu(II) ions to human oxyhaemoglobin caused the rapid oxidation of the haem groups of the beta-chain. Oxidation required binding of Cu(II) to sites involving the thiol group of beta-93 residues and was prevented when these groups were blocked with iodoacetamide or N-ethylmaleimide. Equilibrium-dialysis studies showed three pairs of binding sites, two pairs with high affinity for Cu(II) and one pair with lower affinity. It was the second pair of high-affinity sites that were blocked with iodoacetamide and were involved in haem oxidation. Cu(II) oxidized deoxyhaemoglobin at least ten times as fast as oxyhaemoglobin, and analysis of rates suggested that binding rather than electron transfer was the rate-determining step. No thiol-group oxidation to disulphides occurred during the period of haem oxidation, although it did occur subsequently in the presence of oxygen, or when Cu(II) was added to methaemoglobin. It is proposed that thiol oxidation did not occur because there exists a pathway of electron transfer between the haem group and copper bound to the beta-93 thiol groups. The route for this electron transfer is discussed, as well as the implications as to the function of the beta-93 cysteine in the haemoglobin molecule.

scholarly journals Binding of Adenosine Diphosphate (ADP) to Isolated Human Platelet Membranes

1977 ◽  
Author(s):  
J. Lips ◽  
J. J. Sixma
Keyword(s):  
Binding Sites ◽  
Association Constant ◽  
Plasma Membranes ◽  
Human Platelet ◽  
Membrane Vesicles ◽  
Adenosine Diphosphate ◽  
Ph Optimum ◽  
High Affinity ◽  
Absolute Requirement ◽  
Millipore Filtration

Human platelet plasma membranes were isolated according to the glycerol loading technique of Barber and Jamieson. The binding of 14C ADP was studied with Millipore filtration in a Ca2+ and Mg2+ containing buffer at pH 7,4. At least two types of binding sites were found: A high affinity system with a maximum binding of 160 pMoles/mg protein and an association constant of 1,1 χ 106 M-1; and alow affinity system with a maximum binding of about 4500 pMoles/mg protein and an association constant of 0,6 χ 1θ4 M-1. The binding according to the high affinity system showed little temperature dependency (Q10 = 1,10). The pH optimum was at 7,3. Ca2+ ions were an absolute requirement for binding.Nucleoside diphosphokinase (NDPK) was found in the membrane vesicles. Evidence that this enzyme was not responsible for ADP binding was obtained. The enzyme is Mg2+ dependent and is inhibited by AMP, in contrast to ADP binding. The Q10 was 1,44.ADP binding was inhibited by ATP, IDP and β/γ-imidoadenosine triphosphate.

Bradykinin receptors localized by quantitative autoradiography in kidney, ureter, and bladder

1989 ◽  
Vol 256 (5) ◽  
pp. F909-F915 ◽  
Author(s):  
D. C. Manning ◽  
S. H. Snyder
Keyword(s):  
Guinea Pig ◽  
Binding Sites ◽  
Basal Membrane ◽  
Epithelial Layer ◽  
Bradykinin Receptors ◽  
High Affinity ◽  
Collecting Tubule ◽  
Tubule Cells

We have localized high affinity [3H]bradykinin receptor binding sites by in vitro autoradiography in kidney, ureter, and bladder of the guinea pig. The peptide pharmacology of the binding sites corresponds to that of high affinity physiological bradykinin receptors previously described (Manning, D. C., R. Vavrek, J. M. Stewart, and S. H. Snyder. J. Pharmacol. Exp. Ther. 237:504-512, 1986). In the kidney, receptors are concentrated in the medulla with negligible binding in the cortex. Medullary receptors are localized to the interstitium just beneath the basal membrane of collecting tubule cells and between tubules. In the ureter and bladder, receptors are confined to the lamina propria just beneath the epithelial layer. Localizations in the kidney may relate to the diuretic and natriuretic actions of bradykinin. Ureteral and bladder receptors may be associated with a role of bradykinin in pain and inflammation.

scholarly journals Effect of Phospholipase A2 on Temperature-Induced High-Affinity (3H)Tryptamine Binding Sites in Rat Brain.

1991 ◽  
Vol 56 (4) ◽  
pp. 413-419 ◽  
Author(s):  
Michiko Saito ◽  
Shigefumi Serikyaku ◽  
Ryoichi Ishitani
Keyword(s):  
Phospholipase A2 ◽  
Rat Brain ◽  
Binding Sites ◽  
High Affinity
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