scholarly journals Palytoxin-induced Effects on Partial Reactions of the Na,K-ATPase

2006 ◽  
Vol 128 (1) ◽  
pp. 103-118 ◽  
Author(s):  
Nadine Harmel ◽  
Hans-Jürgen Apell
Keyword(s):  
Rate Constant ◽  
Reaction Pathway ◽  
Molecular Model ◽  
Forward Rate ◽  
Channel State ◽  
Toxin Binding ◽  
Styryl Dye ◽  
Forward Rate Constant ◽  
Experimental Findings ◽  
Rate Limiting

The interaction of palytoxin with the Na,K-ATPase was studied by the electrochromic styryl dye RH421, which monitors the amount of ions in the membrane domain of the pump. The toxin affected the pump function in the state P-E2, independently of the type of phosphorylation (ATP or inorganic phosphate). The palytoxin-induced modification of the protein consisted of two steps: toxin binding and a subsequent conformational change into a transmembrane ion channel. At 20°C, the rate-limiting reaction had a forward rate constant of 105 M−1s−1 and a backward rate constant of about 10−3 s−1. In the palytoxin-modified state, the binding affinity for Na+ and H+ was increased and reached values between those obtained in the E1 and P-E2 conformation under physiological conditions. Even under saturating palytoxin concentrations, the ATPase activity was not completely inhibited. In the Na/K mode, ∼50% of the enzyme remained active in the average, and in the Na-only mode 25%. The experimental findings indicate that an additional exit from the inhibited state exists. An obvious reaction pathway is a slow dephosphorylation of the palytoxin-inhibited state with a time constant of ∼100 s. Analysis of the effect of blockers of the extracellular and cytoplasmic access channels, TPA+ and Br2-Titu3+, respectively, showed that both access channels are part of the ion pathway in the palytoxin-modified protein. All experiments can be explained by an extension of the Post-Albers cycle, in which three additional states were added that branch off in the P-E2 state and lead to states in which the open-channel conformation is introduced and returns into the pump cycle in the occluded E2 state. The previously suggested molecular model for the channel state of the Na,K-ATPase as a conformation in which both gates between binding sites and aqueous phases are simultaneously in their open state is supported by this study.

scholarly journals P3331Obesity is associated with an increase in the forward rate constant of the creatine kinase reaction

2017 ◽  
Vol 38 (suppl_1) ◽  
Author(s):  
J. Rayner ◽  
W. Clarke ◽  
M.A. Peterzan ◽  
C.T. Rodgers ◽  
S. Neubauer ◽  
...  
Keyword(s):  
Creatine Kinase ◽  
Rate Constant ◽  
Forward Rate ◽  
Kinase Reaction ◽  
Forward Rate Constant ◽  
Creatine Kinase Reaction

scholarly journals Volume-Sensitive K+/Cl− Cotransport in Rabbit Erythrocytes

1999 ◽  
Vol 114 (6) ◽  
pp. 743-758 ◽  
Author(s):  
Michael L. Jennings
Keyword(s):  
Cell Volume ◽  
Time Course ◽  
Red Cells ◽  
Cell Swelling ◽  
Forward Rate ◽  
Inactivation Kinetics ◽  
Forward Rate Constant ◽  
The Individual ◽  
Rate Limiting ◽  
Individual Rate

The kinetics of activation and inactivation of K+/Cl− cotransport (KCC) have been measured in rabbit red blood cells for the purpose of determining the individual rate constants for the rate-limiting activation and inactivation events. Four different interventions (cell swelling, N-ethylmaleimide [NEM], low intracellular pH, and low intracellular Mg2+) all activate KCC with a single exponential time course; the kinetics are consistent with the idea that there is a single rate-limiting event in the activation of transport by all four interventions. In contrast to LK sheep red cells, the KCC flux in Mg2+-depleted rabbit red cells is not affected by cell volume. KCC activation kinetics were examined in cells pretreated with NEM at 0°C, washed, and then incubated at higher temperatures. The forward rate constant for activation has a very high temperature dependence (Ea ∼ 32 kCal/mol), but is not affected measurably by cell volume. Inactivation kinetics were examined by swelling cells at 37°C to activate KCC, and then resuspending at various osmolalities and temperatures to inactivate most of the transporters. The rate of transport inactivation increases steeply as cell volume decreases, even in a range of volumes where nearly all the transporters are inactive in the steady state. This finding indicates that the rate-limiting inactivation event is strongly affected by cell volume over the entire range of cell volumes studied, including normal cell volume. The rate-limiting inactivation event may be mediated by a protein kinase that is inhibited, either directly or indirectly, by cell swelling, low Mg2+, acid pH, and NEM.

scholarly journals 31P-saturation-transfer nuclear-magnetic-resonance measurements of phosphocreatine turnover in guinea-pig brain slices

1985 ◽  
Vol 227 (3) ◽  
pp. 777-782 ◽  
Author(s):  
P G Morris ◽  
J Feeney ◽  
D W Cox ◽  
H S Bachelard
Keyword(s):  
Guinea Pig ◽  
Rate Constant ◽  
Brain Slices ◽  
Forward Rate ◽  
Saturation Transfer ◽  
Reverse Rate Constant ◽  
Forward Rate Constant ◽  
Guinea Pig Brain

The technique of 31P saturation-transfer n.m.r. was used to determine the forward and the reverse rate constants of creatine phosphotransferase in superfused guinea-pig cerebral tissues in vitro. The calculated forward rate constant of 0.22 +/- 0.03s-1 compared well with a previously reported value for rat brain in vivo [Shoubridge, Briggs & Radda (1982) FEBS Lett. 140, 288-292]. The reverse rate constant was found to be 0.55 +/- 0.10s-1. 3. By using concentrations of ATP and phosphocreatine estimated previously for this superfused preparation [Cox, Morris, Feeney & Bachelard (1983) Biochem. J. 212, 365-370], forward and reverse flux rates were calculated to be 0.68 and 0.72 mumol X s-1 X g-1 respectively. The concordance of forward and reverse fluxes contrasts with the situation observed in vitro in other tissues, and suggests that the creatine phosphotransferase reaction is at equilibrium under the conditions used here. 4. Lowering the concentration of glucose in the superfusing medium from 10mM to 0.5mM had no significant effect on phosphocreatine concentration or on the forward (ATP-generating) flux through creatine phosphotransferase. The results indicate that a normal phosphocreatine content in the presence of lowered glucose availability is reflected by an unchanged turnover rate.

The Effect of p-Toluidine on the Two-Step Electroreduction of Zn(II) in Water-Methanol and Water-Dimethylformamide

1999 ◽  
Vol 64 (4) ◽  
pp. 585-594 ◽  
Author(s):  
Barbara Marczewska
Keyword(s):  
Electron Transfer ◽  
Binary Mixtures ◽  
Rate Constant ◽  
Rate Constants ◽  
Electrode Surface ◽  
Mercury Electrode ◽  
Forward Rate ◽  
Solvent Molecules ◽  
Activated Complex

The acceleration effect of p-toluidine on the electroreduction of Zn(II) on the mercury electrode surface in binary mixtures water-methanol and water-dimethylformamide is discussed. The obtained apparent and true forward rate constants of Zn(II) reduction indicate that the rate constant of the first electron transfer increases in the presence of p-toluidine. The acceleration effect may probably be accounted for by the concept of the formation on the mercury electrode an activated complex, presumably composed of p-toluidine and solvent molecules.

Kinetic and Equilibrium Studies of the Reactions of 2,4,6-Trinitrotoluene and 2,4,6-Trinitrotoluene-d3 with Sodium and Potassium t-Butoxide in t-Butanol

1974 ◽  
Vol 52 (12) ◽  
pp. 2306-2315 ◽  
Author(s):  
Erwin Buncel ◽  
Albert Richard Norris ◽  
Kenneth Edwin Russell ◽  
Harold Wilson
Keyword(s):  
Initial Rate ◽  
Forward Rate ◽  
Specific Rate ◽  
Equilibrium Studies ◽  
Proton Abstraction ◽  
Kinetic Isotope ◽  
Forward Rate Constant ◽  
Hydrogen Deuterium ◽  
Reaction In Solution ◽  
Sodium And Potassium

The reactions of 2,4,6-trinitrotoluene (TNT) and 2,4,6-trinitrotoluene fully deuterated at the methyl position (TNT-d3) with sodium and potassium t-butoxide in t-butanol have been studied. With TNT as the substrate, proton abstraction by ion-paired sodium or potassium t-butoxide appears to be the predominant reaction in solution. With sodium t-butoxide as base, the forward rate constant for proton abstraction at 30.0 °C (Kf,ip) is 6000 ± 400 M−1 s−1 while ΔH≠ and ΔS≠ for the reaction are 4.2 ± 0.3 kcal mol−1 and −27 ± 2 cal deg−1 mol−1, respectively. With TNT-d3 as the substrate, formation of a TNT-d3-t-butoxide ion σ-complex occurs simultaneously with deuteron abstraction. Specific rate constants for the two processes have been determined at 30.0 °C. Initial rate studies establish a hydrogen-deuterium kinetic isotope effect of 8 ± 1 for the formation of the anion in t-butanol.

scholarly journals Hydrogen sulfide bypasses the rate-limiting oxygen activation of heme oxygenase

2018 ◽  
Vol 293 (43) ◽  
pp. 16931-16939 ◽  
Author(s):  
Toshitaka Matsui ◽  
Ryota Sugiyama ◽  
Kenta Sakanashi ◽  
Yoko Tamura ◽  
Masaki Iida ◽  
...  
Keyword(s):  
Heme Oxygenase ◽  
Mammalian Cells ◽  
Culture Media ◽  
Reaction Pathway ◽  
Hypoxic Conditions ◽  
Research Areas ◽  
Generating Capacity ◽  
Rate Limiting ◽  
Sulfur Containing ◽  
Induced Reaction

Discovery of unidentified protein functions is of biological importance because it often provides new paradigms for many research areas. Mammalian heme oxygenase (HO) enzyme catalyzes the O2-dependent degradation of heme into carbon monoxide (CO), iron, and biliverdin through numerous reaction intermediates. Here, we report that H2S, a gaseous signaling molecule, is part of a novel reaction pathway that drastically alters HO's products, reaction mechanism, and catalytic properties. Our prediction of this interplay is based on the unique reactivity of H2S with one of the HO intermediates. We found that in the presence of H2S, HO produces new linear tetrapyrroles, which we identified as isomers of sulfur-containing biliverdin (SBV), and that only H2S, but not GSH, cysteine, and polysulfides, induces SBV formation. As BV is converted to bilirubin (BR), SBV is enzymatically reduced to sulfur-containing bilirubin (SBR), which shares similar properties such as antioxidative effects with normal BR. SBR was detected in culture media of mouse macrophages, confirming the existence of this H2S-induced reaction in mammalian cells. H2S reacted specifically with a ferric verdoheme intermediate of HO, and verdoheme cleavage proceeded through an O2-independent hydrolysis-like mechanism. This change in activation mode diminished O2 dependence of the overall HO activity, circumventing the rate-limiting O2 activation of HO. We propose that H2S could largely affect O2 sensing by mammalian HO, which is supposed to relay hypoxic signals by decreasing CO output to regulate cellular functions. Moreover, the novel H2S-induced reaction identified here helps sustain HO's heme-degrading and antioxidant-generating capacity under highly hypoxic conditions.

Characterization of Holliday structures in FLP protein-promoted site-specific recombination

1990 ◽  
Vol 10 (1) ◽  
pp. 235-242
Author(s):  
L Meyer-Leon ◽  
R B Inman ◽  
M M Cox
Keyword(s):  
Reaction Rate ◽  
Reaction Pathway ◽  
Reaction Intermediates ◽  
Wild Type ◽  
Site Specific ◽  
Site Specific Recombination ◽  
Isomerization Process ◽  
Previous Demonstration ◽  
Rate Limiting

Holliday structures are formed in the course of FLP protein-promoted site-specific recombination. Here, we demonstrate that Holliday structures are formed in reactions involving wild-type substrates and that they are kinetically competent with respect to the overall reaction rate. Together with a previous demonstration of chemical competence (L. Meyer-Leon, L.-C. Huang, S. W. Umlauf, M. M. Cox, and R. B. Inman, Mol. Cell. Biol. 8:3784-3796, 1988), Holliday structures therefore meet all criteria necessary to establish that they are obligate reaction intermediates in FLP-mediated site-specific recombination. In addition, kinetic evidence suggests that two distinct forms of the Holliday intermediate are present in the reaction pathway, interconverted in an isomerization process that is rate limiting at 0 degree C.

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