Hofmeister effects of anions on the kinetics of partial reactions of the Na+,K+-ATPase.
The effects of lyotropic anions, particularly perchlorate, on the kinetics of partial reactions of the Na+,K+-ATPase from pig kidney were investigated by two different kinetic techniques: stopped flow in combination with the fluorescent label RH421 and a stationary electrical relaxation technique. It was found that 130 mM NaClO4 caused an increase in the Kd values of both the high- and low-affinity ATP-binding sites, from values of 7.0 (+/- 0.6) microM and 143 (+/- 17) microM in 130 mM NaCl solution to values of 42 (+/- 3) microM and 660 (+/- 100) microM in 130 mM NaClO4 (pH 7.4, 24 degrees C). The half-saturating concentration of the Na+-binding sites on the E1 conformation was found to decrease from 8-10 mM in NaCl to 2.5-3.5 mM in NaClO4 solution. The rate of equilibration of the reaction, E1P(Na+)3 left arrow over right arrow E2P + 3Na+, decreased from 393 (+/- 51) s-1 in NaCl solution to 114 (+/- 15) s-1 in NaClO4. This decrease is attributed predominantly to an inhibition of the E1P(Na+)3 --> E2P(Na+)3 transition. The effects can be explained in terms of electrostatic interactions due to perchlorate binding within the membrane and/or protein matrix of the Na+,K+-ATPase membrane fragments and alteration of the local electric field strength experienced by the protein. The kinetic results obtained support the conclusion that the conformational transition E1P(Na+)3 --> E2P(Na+)3 is a major charge translocating step of the pump cycle.
Ganea
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C
Department of Biophysics, C. Davila Medical University, 76241 Bucharest, Romania.
Babes
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A
Lüpfert
C
C
Grell
E
E
Fendler
K
K
Clarke
R J
RJ
D000255:Adenosine Triphosphate / Q000737:chemistry; D000818:Animals; D000838:Anions / Q000737:chemistry*; D001665:Binding Sites; D002384:Catalysis; D007668:Kidney / Q000201:enzymology; D007700:Kinetics; D010472:Perchlorates / Q000737:chemistry; D011188:Potassium / Q000737:chemistry; D011487:Protein Conformation; D011726:Pyridinium Compounds / Q000737:chemistry; D012964:Sodium / Q000737:chemistry; D017670:Sodium Compounds / Q000737:chemistry; D000254:Sodium-Potassium-Exchanging ATPase / Q000737:chemistry*; D055672:Static Electricity; D013343:Styrenes / Q000737:chemistry; D013552:Swine
D000838:Anions; D010472:Perchlorates; D011726:Pyridinium Compounds; D017670:Sodium Compounds; D013343:Styrenes; C070534:RH 421; D000255:Adenosine Triphosphate; C031068:sodium perchlorate; D012964:Sodium; D000254:Sodium-Potassium-Exchanging ATPase; D011188:Potassium