Cation transport by the neuronal K+-Cl− cotransporter KCC2: thermodynamics and kinetics of alternate transport modes

Both Cs+ and NH4+ alter neuronal Cl− homeostasis, yet the mechanisms have not been clearly elucidated. We hypothesized that these two cations altered the operation of the neuronal K+-Cl− cotransporter (KCC2). Using exogenously expressed KCC2 protein, we first examined the interaction of cations at the transport site of KCC2 by monitoring furosemide-sensitive 86Rb+ influx as a function of external Rb+ concentration at different fixed external cation concentrations (Na+, Li+, K+, Cs+, and NH4+). Neither Na+ nor Li+ affected furosemide-sensitive 86Rb+ influx, indicating their inability to interact at the cation translocation site of KCC2. As expected for an enzyme that accepts Rb+ and K+ as alternate substrates, K+ was a competitive inhibitor of Rb+ transport by KCC2. Like K+, both Cs+ and NH4+ behaved as competitive inhibitors of Rb+ transport by KCC2, indicating their potential as transport substrates. Using ion chromatography to measure unidirectional Rb+ and Cs+ influxes, we determined that although KCC2 was capable of transporting Cs+, it did so with a lower apparent affinity and maximal velocity compared with Rb+. To assess NH4+ transport by KCC2, we monitored intracellular pH (pHi) with a pH-sensitive fluorescent dye after an NH4+-induced alkaline load. Cells expressing KCC2 protein recovered pHi much more rapidly than untransfected cells, indicating that KCC2 can mediate net NH4+ uptake. Consistent with KCC2-mediated NH4+ transport, pHi recovery in KCC2-expressing cells could be inhibited by furosemide (200 μM) or removal of external [Cl−]. Thermodynamic and kinetic considerations of KCC2 operating in alternate transport modes can explain altered neuronal Cl− homeostasis in the presence of Cs+ and NH4+.

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Kinetics of bumetanide-sensitive Na+-K+ co-transport in erythrocytes of essential hypertensive patients

Clinical Science ◽

10.1042/cs0690607 ◽

1985 ◽

Vol 69 (5) ◽

pp. 607-611 ◽

Cited By ~ 6

Author(s):

Pietro Delva ◽

Mario De Gasperi ◽

Maurizio Degan ◽

Grazia Covi ◽

Alessandro Lechi

Keyword(s):

Kinetic Parameters ◽

Transport System ◽

Clinical Data ◽

Laboratory Data ◽

Cation Transport ◽

Hypertensive Patients ◽

Apparent Affinity ◽

Normotensive Subjects ◽

Kinetics Of

1. Outward bumetanide-sensitive Na+-K+ co-transport was studied in the erythrocytes of 51 subjects, 24 normotensive subjects and 27 hypertensive patients, matched for sex and age. 2. Three kinetic parameters of this cation transport system were considered: velocity of efflux at saturating internal sodium (Nai) concentrations (Vmax.), apparent affinity for sodium (K50%) and index of co-operativity among Nai sites (Hill's n). 3. We correlated these values with clinical and laboratory data determined routinely in hypertension. 4. There were no significant differences between normotensive and hypertensive subjects for the values considered and we did not find any significant correlations between co-transport and clinical data.

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Kinetics of the Inhibition of Plasmin in Acidified Human Plasma

Thrombosis and Haemostasis ◽

10.1055/s-0038-1657274 ◽

1982 ◽

Vol 48 (03) ◽

pp. 257-259 ◽

Cited By ~ 3

Author(s):

H R Lijnen ◽

M Maes ◽

M Castel ◽

M Samama ◽

D Collen

Keyword(s):

Human Plasma ◽

Plasma Proteins ◽

Competitive Inhibitor ◽

Normal Plasma ◽

Competitive Inhibitors ◽

Rate Of Hydrolysis ◽

Kinetics Of ◽

Hydrolysis Of

SummaryAcid-treated human plasma is a competitive inhibitor of the hydrolysis of D-Val-Leu-Lys-Nan (S-2251) by plasmin. The rate of hydrolysis is decreased to 50% by 750 fold diluted acidified normal plasma and by 60 fold diluted acidified α2-antiplasmin depleted plasma (α2-antiplasmin concentration less than 2%). These findings suggest that α2-antiplasmin is a contributary but not the main competitive inhibitor of acidified plasma. This interpretation is supported by the finding that α2-antiplasmin depleted plasma reconstituted with purified α2-antiplasmin inhibits the hydrolysis of S-2251 by plasmin at a 125 fold dilution following acidification and by the finding that in a purified system acid inactivated α2-antiplasmin inhibits the hydrolysis of S-2251 by plasmin with a Ki of 25 nM. Thus, besides α2-antiplasmin, other plasma proteins which are at least in part eliminated by the removal of α2-antiplasmin from plasma by immunoadsorption appear to be competitive inhibitors for plasmin in acidified plasma. It is suggested that several competitive inhibitors for plasmin are present and/or generated in acidified plasma and that these inhibitors may at least in part be responsible for the variability in the results of measurements of plasminogen and/or plasmin in plasma following acidification.

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Sarco/endoplasmic reticulum Ca2+-ATPase isoforms: diverse responses to acidosis

Biochemical Journal ◽

10.1042/bj3210545 ◽

1997 ◽

Vol 321 (2) ◽

pp. 545-550 ◽

Cited By ~ 39

Author(s):

Herman WOLOSKER ◽

Joao B. T. ROCHA ◽

Simone ENGELENDER ◽

Rogerio PANIZZUTTI ◽

Joari De MIRANDA ◽

...

Keyword(s):

Skeletal Muscle ◽

Cardiac Muscle ◽

Atp Hydrolysis ◽

Blood Platelets ◽

The Other ◽

Maximal Velocity ◽

Acidic Ph ◽

Apparent Affinity ◽

Maximal Activation ◽

Kinetics Of

The effects of acidic pH on the kinetics of Ca2+-ATPase isoforms from intracellular membranes of skeletal muscle, cardiac muscle, cerebellum and blood platelets were studied. At neutral pH, all four Ca2+-ATPase isoforms exhibited similar Ca2+-concentration requirements for half-maximal rates of Ca2+ uptake and ATP hydrolysis. A decrease in the pH from 7.0 to 6.0 promoted a decrease in both the apparent affinity for Ca2+ [increasing half-maximal activation (K0.5)] and the maximal velocity (Vmax) of Ca2+ uptake. With skeletal muscle vesicles these effects were 5 to 10 times smaller than those observed with all the other isoforms. Acidification of the medium from pH 7.0 to 6.5 caused the release of Ca2+ from loaded vesicles and a decrease in the amount of Ca2+ retained by the vesicles at the steady state. With the vesicles derived from skeletal muscle these effects were smaller than for vesicles derived from other tissues. The rate of passive Ca2+ efflux from skeletal and cardiac muscle vesicles, loaded with Ca2+ and diluted in a medium containing none of the ligands of Ca2+-ATPase, was the same at pH 7.0 and 6.0. In contrast, the rate of Ca2+ efflux from cerebellar and platelet vesicles increased 2-fold after acidification of the medium. The effects of DMSO, Mg2+ with Pi and arsenate on the rate of Ca2+ efflux varied among the different preparations tested. The differences became more pronounced when the pH of the medium was decreased from 7.0 to 6.0. It is proposed that the kinetic differences among the Ca2+-ATPase isoforms may reflect different adaptations to cellular acidosis, such as that which occurs during ischaemia.

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KINETICS OF NADH OXIDATION BY PIGEON-HEART MITOCHONDRIA

Canadian Journal of Biochemistry ◽

10.1139/o67-098 ◽

1967 ◽

Vol 45 (6) ◽

pp. 881-888 ◽

Cited By ~ 3

Author(s):

Thomas J. Griffith ◽

M. C. Blanchaer

Keyword(s):

Adenine Nucleotides ◽

Nadh Oxidation ◽

Heart Mitochondria ◽

Maximal Velocity ◽

Competitive Inhibitors ◽

Kinetics Of

The oxidation of 1-10 μM NADH by pigeon-heart mitochondria prepared by the method of Chance and Hagihara was studied. In a medium consisting of 0.07 M sucrose, 0.23 M mannitol, 0.02 M Tris–HCl, 0.02 mM EDTA, all at pH 7.2, the concentration of NADH required for half maximal velocity was found to be approximately 5 μM. ATP and ADP appear to be competitive inhibitors of NADH oxidation. Application of Michaelis–Menten kinetics to the data yielded a Ki of 0.7 mM for both ATP and ADP. Some possible sites of inhibition by adenine nucleotides are discussed.

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Irradiation behavior of pyrolytic silicon carbide

Proceedings, annual meeting, Electron Microscopy Society of America ◽

10.1017/s0424820100074288 ◽

1983 ◽

Vol 41 ◽

pp. 72-73

Author(s):

R. J. Lauf

Keyword(s):

Silicon Carbide ◽

Fission Products ◽

Thermodynamics And Kinetics ◽

Neutron Fluences ◽

Fuel Particles ◽

Sic Coatings ◽

Irradiation Behavior ◽

Kinetics Of ◽

Htgr Fuel

Fuel particles for the High-Temperature Gas-Cooled Reactor (HTGR) contain a layer of pyrolytic silicon carbide to act as a miniature pressure vessel and primary fission product barrier. Optimization of the SiC with respect to fuel performance involves four areas of study: (a) characterization of as-deposited SiC coatings; (b) thermodynamics and kinetics of chemical reactions between SiC and fission products; (c) irradiation behavior of SiC in the absence of fission products; and (d) combined effects of irradiation and fission products. This paper reports the behavior of SiC deposited on inert microspheres and irradiated to fast neutron fluences typical of HTGR fuel at end-of-life.

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Kinetic Aspects of the Interaction of Blood Clotting Enzymes

Thrombosis and Haemostasis ◽

10.1055/s-0038-1651214 ◽

1968 ◽

Vol 19 (03/04) ◽

pp. 364-367 ◽

Cited By ~ 4

Author(s):

H. C Hemker ◽

P. W Hemker

Keyword(s):

Enzyme Kinetics ◽

Blood Clotting ◽

Competitive Inhibition ◽

Competitive Inhibitor ◽

Kinetics Of

SummaryThe enzyme kinetics of competitive inhibition under conditions prevailing in clotting tests are developed and a method is given to measure relative amounts of a competitive inhibitor by means of the t — D plot.

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