scholarly journals Diethyl pyrocarbonate modification of the ryanodine receptor/Ca2+ channel from skeletal muscle

1994 ◽  
Vol 299 (1) ◽  
pp. 177-181 ◽  
Author(s):  
V Shoshan-Barmatz ◽  
S Weil
Keyword(s):  
Binding Site ◽  
Ryanodine Receptor ◽  
Time Course ◽  
Ph Dependence ◽  
Histidine Residue ◽  
Binding Affinities ◽  
Single Class ◽  
Diethyl Pyrocarbonate ◽  
Dependent Inactivation ◽  
Specific Reagent

Exposure of junctional sarcoplasmic reticulum (SR) membranes or purified ryanodine receptor to the histidine-specific reagent diethyl pyrocarbonate (DEPC) led to concentration- and time-dependent inactivation of ryanodine binding. The pH-dependence of the inactivation of ryanodine binding by DEPC and the reversal of this inactivation by hydroxylamine suggests the modification of histidine residue(s) by the reagent. Kinetic analysis of the time course of inactivation of ryanodine binding by DEPC suggests that the inactivation resulted from modification of a single class of histidine residue per ryanodine-binding site. The degree of inactivation of ryanodine binding by DEPC was decreased when high NaCl concentrations were present in the modification medium. The binding affinities for ryanodine and Ca2+ were not altered by DEPC modification. This modification decreased the total ryanodine-binding sites. DEPC modification increased the Ca(2+)-permeability of the SR vesicles. A variety of bivalent cations prevented the DEPC inactivation of ryanodine binding in a series of decreasing efficiency: Mn2+ > Ba2+ > Mg2+ > Ca2+, similar to their effectiveness in inhibiting ryanodine binding. It is suggested that a histidine residue(s) in the ryanodine receptor is involved, either in the binding of Ca2+, or in a conformational change that may be required for Ca2+ binding to its binding site(s). This modification of the ryanodine receptor resulted in inactivation of ryanodine binding and activation of Ca2+ release.

scholarly journals Interactions between inhibitors of dihydrofolate reductase

1989 ◽  
Vol 258 (2) ◽  
pp. 335-342 ◽  
Author(s):  
K Bowden ◽  
A D Hall ◽  
B Birdsall ◽  
J Feeney ◽  
G C K Roberts
Keyword(s):  
Steady State ◽  
Binding Site ◽  
Dihydrofolate Reductase ◽  
Binding Sites ◽  
High Field ◽  
Ph Dependence ◽  
Histidine Residue ◽  
Adenine Ring ◽  
Steady State Kinetics ◽  
Pka Value

The binding of substrates and inhibitors to dihydrofolate reductase was studied by steady-state kinetics and high-field 1H-n.m.r. spectroscopy. A series of 5-substituted 2,4-diaminopyrimidines were examined and were found to be ‘tightly binding’ inhibitors of the enzyme (Ki less than 10(-9) M). Studies on the binding of 4-substituted benzenesulphonamides and benzenesulphonic acids also established the existence of a ‘sulphonamide-binding site’ on the enzyme. Subsequent n.m.r. experiments showed that there are two binding sites for the sulphonamides on the enzyme, one of which overlaps the coenzyme (NADPH) adenine-ring-binding site. An examination of the pH-dependence of the binding of sulphonamides to the enzyme indicated the influence of an ionizable group on the enzyme that was not directly involved in the sulphonamide binding. The change in pKa value from 6.7 to 7.2 observed on sulphonamide binding suggests the involvement of a histidine residue, which could be histidine-28.

scholarly journals pH-dependence of warfarin binding to α1-acid glycoprotein (orosomucoid)

1993 ◽  
Vol 289 (3) ◽  
pp. 767-770 ◽  
Author(s):  
S Urien ◽  
F Brée ◽  
B Testa ◽  
J P Tillement
Keyword(s):  
Hydrogen Bond ◽  
Binding Site ◽  
Ph Dependence ◽  
Protonated Form ◽  
Association Constants ◽  
Histidine Residue ◽  
Difference Spectra ◽  
Acid Glycoprotein ◽  
Binding Data ◽  
Decreasing Ph

The binding of warfarin to alpha 1-acid glycoprotein (AAG) was found to increase with decreasing pH. The u.v.-visible difference spectra generated upon binding to AAG at pH 5.0 or 7.4 showed warfarin to bind as the anion. Warfarin-binding data were satisfactorily fitted to a model that incorporates the effect of pH and discriminates the association constants of the non-protonated and protonated binding site of the protein. It was shown that AAG-binding site in the protonated form had a markedly higher affinity for warfarin than the non-protonated form, with a pK value of 7.7 +/- 0.1, which is likely to be a histidine residue. Among other possible interactions, it is suggested that ligand binding to AAG involves a reinforced hydrogen bond.

Glu106 in the Orai1 pore contributes to fast Ca2+-dependent inactivation and pH dependence of Ca2+ release-activated Ca2+ (CRAC) current

2011 ◽  
Vol 441 (2) ◽  
pp. 743-753 ◽  
Author(s):  
Nathan R. Scrimgeour ◽  
David P. Wilson ◽  
Grigori Y. Rychkov
Keyword(s):  
Binding Site ◽  
Voltage Dependence ◽  
Ph Dependence ◽  
Low Ph ◽  
Wild Type ◽  
Channel Pore ◽  
Crac Channels ◽  
Dependent Inactivation ◽  
Crac Channel ◽  
Ca2 Channels

FCDI (fast Ca2+-dependent inactivation) is a mechanism that limits Ca2+ entry through Ca2+ channels, including CRAC (Ca2+ release-activated Ca2+) channels. This phenomenon occurs when the Ca2+ concentration rises beyond a certain level in the vicinity of the intracellular mouth of the channel pore. In CRAC channels, several regions of the pore-forming protein Orai1, and STIM1 (stromal interaction molecule 1), the sarcoplasmic/endoplasmic reticulum Ca2+ sensor that communicates the Ca2+ load of the intracellular stores to Orai1, have been shown to regulate fast Ca2+-dependent inactivation. Although significant advances in unravelling the mechanisms of CRAC channel gating have occurred, the mechanisms regulating fast Ca2+-dependent inactivation in this channel are not well understood. We have identified that a pore mutation, E106D Orai1, changes the kinetics and voltage dependence of the ICRAC (CRAC current), and the selectivity of the Ca2+-binding site that regulates fast Ca2+-dependent inactivation, whereas the V102I and E190Q mutants when expressed at appropriate ratios with STIM1 have fast Ca2+-dependent inactivation similar to that of WT (wild-type) Orai1. Unexpectedly, the E106D mutation also changes the pH dependence of ICRAC. Unlike WT ICRAC, E106D-mediated current is not inhibited at low pH, but instead the block of Na+ permeation through the E106D Orai1 pore by Ca2+ is diminished. These results suggest that Glu106 inside the CRAC channel pore is involved in co-ordinating the Ca2+-binding site that mediates fast Ca2+-dependent inactivation.

scholarly journals Evidence for a histidine and a cysteine residue in the substrate-binding site of yeast alcohol dehydrogenase

1975 ◽  
Vol 145 (3) ◽  
pp. 581-590 ◽  
Author(s):  
V Leskovac ◽  
D Pavkov-Peričin
Keyword(s):  
Alcohol Dehydrogenase ◽  
Binding Site ◽  
Ternary Complex ◽  
Substrate Binding ◽  
Cysteine Residue ◽  
Histidine Residue ◽  
Thiol Groups ◽  
Substrate Binding Site ◽  
Yeast Alcohol Dehydrogenase ◽  
Diethyl Pyrocarbonate

1. Yeast alcohol dehydrogenase (EC 1.1.1.1) is inhibited by stoicheiometric concentrations of diethyl pyrocarbonate. The inhibition is due to the acylation of a single histidine residue/monomer (mol.wt. 36000). 2. Alcohol dehydrogenase is also inhibited by stoicheiometric amounts of 5,5′-dithiobis-(2-nitrobenzoate), owing to the modification of a single cysteine residue/monomer. 3. Native alcohol dehydrogenase binds two molecules of reduced coenzyme/molecule of enzyme (mol.wt. 144000). 4. Modification of a single histidine residue/monomer by treatment with diethyl pyrocarbonate prevents the binding of acetamide in the ternary complex, enzyme-NADH-acetamede, but does not prevent the binding of NADH to the enzyme. 5. Modification of a single cysteine residue/monomer does not prevent the binding of acetamide to the ternary complex. After the modification of two thiol groups/monomer by treatment with 5,5′-dithiobis-(2-nitrobenzoate), the capacity of enzyme to bind coenzyme in the ternary complex was virtually abolished. 6. From the results presented in this paper we conclude that at least one histidine and one cysteine residue are closely associated in the substrate-binding site of alcohol dehydrogenase.

scholarly journals New Insights into the Pleiotropic Drug Resistance Network from Genome-Wide Characterization of the YRR1 Transcription Factor Regulation System

2002 ◽  
Vol 22 (8) ◽  
pp. 2642-2649 ◽  
Author(s):  
Stéphane Le Crom ◽  
Frédéric Devaux ◽  
Philippe Marc ◽  
Xiaoting Zhang ◽  
W. Scott Moye-Rowley ◽  
...  
Keyword(s):  
Drug Resistance ◽  
Transcription Factor ◽  
Binding Site ◽  
Time Course ◽  
Target Genes ◽  
Regulation System ◽  
Dependent Manner ◽  
Pleiotropic Drug Resistance ◽  
Genome Wide

ABSTRACT Yrr1p is a recently described Zn2Cys6 transcription factor involved in the pleiotropic drug resistance (PDR) phenomenon. It is controlled in a Pdr1p-dependent manner and is autoregulated. We describe here a new genome-wide approach to characterization of the set of genes directly regulated by Yrr1p. We found that the time-course production of an artificial chimera protein containing the DNA-binding domain of Yrr1p activated the 15 genes that are also up-regulated by a gain-of-function mutant of Yrr1p. Gel mobility shift assays showed that the promoters of the genes AZR1, FLR1, SNG1, YLL056C, YLR346C, and YPL088W interacted with Yrr1p. The putative consensus Yrr1p binding site deduced from these experiments, (T/A)CCG(C/T)(G/T)(G/T)(A/T)(A/T), is strikingly similar to the PDR element binding site sequence recognized by Pdr1p and Pdr3p. The minor differences between these sequences are consistent with Yrr1p and Pdr1p and Pdr3p having different sets of target genes. According to these data, some target genes are directly regulated by Pdr1p and Pdr3p or by Yrr1p, whereas some genes are indirectly regulated by the activation of Yrr1p. Some genes, such as YOR1, SNQ2, and FLR1, are clearly directly controlled by both classes of transcription factor, suggesting an important role for the corresponding membrane proteins.

scholarly journals The pKa of the catalytic histidine residue of chloramphenicol acetyltransferase

1993 ◽  
Vol 290 (1) ◽  
pp. 15-19 ◽  
Author(s):  
A Lewendon ◽  
W V Shaw
Keyword(s):  
Chemical Modification ◽  
Ph Dependence ◽  
Thiol Group ◽  
Chloramphenicol Acetyltransferase ◽  
Histidine Residue ◽  
Pka Values ◽  
Primary Hydroxy Group ◽  
Pka Value ◽  
Catalytic Role ◽  
Kinetics Of

A catalytically essential histidine residue (His-195) of chloramphenicol acetyltransferase (CAT) acts as a general base in catalysis, abstracting a proton from the primary hydroxy group of chloramphenicol. The pKa of His-195 has been determined from the pH-dependence of chemical modification. Both methyl 4-nitrobenzenesulphonate and iodoacetamide inactivate CAT by irreversible modification of His-195. The kinetics of inactivation by methyl 4-nitrobenzenesulphonate are pseudo-first-order, and the pH-dependence of inactivation yields a pKa value of 6.60. Iodoacetamide inactivation proceeds with second-order kinetics and a pKa value of 6.80. An alternative site of modification at the active site of CAT is the thiol group of Cys-31, a residue which has no catalytic role. On replacement of Cys-31 with alanine (Ala-31 CAT), the pH-dependence of iodoacetamide inactivation gives a pKa value of 6.66. The pKa values derived from chemical-modification experiments directed at His-195 are in agreement with the pKa values of 6.62 and 6.61 determined for wild-type and Ala-31 CAT respectively from the pH-dependence of kcat/Km.

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