Kinetic investigations of the mechanism of the rate-determining step of the Na+,K+-ATPase pump cycle.
The kinetics of the E(2) --> E(1) conformational change of unphosphorylated Na(+),K(+)-ATPase from rabbit kidney were investigated via the stopped-flow technique using the fluorescent label RH421 (pH 7.4, 24 degrees C). The enzyme was preequilibrated in a solution containing 25 mM histidine and 0.1 mM EDTA to initially stabilize the E(2) conformation. On mixing enzyme with NaCl alone, tris-ATP alone, or NaCl and tris-ATP simultaneously, a fluorescence decrease was observed. The reciprocal relaxation time, 1/tau, of the fluorescent transient was found to increase with increasing NaCl concentration and reached a saturating value in the presence of 1 mM tris-ATP of 54 (+/-3) s(-1). The experimental behavior could be described by a binding of Na(+) to the enzyme in the E(2) state with a dissociation constant of 31 (+/-7) mM, which induces a subsequent rate-limiting conformational change to the E(1) state. Similar behavior, but with a decreased saturating value of 1/tau, was found when NaCl was replaced by choline chloride. Experiments performed with enzyme from shark rectal gland showed similar effects, but with a lower amplitude of the fluorescence change and a higher saturating value of 1/tau for both the NaCl and choline chloride titrations. The results suggest that Na(+) ions or salt in general play a regulatory role, similar to ATP, in enhancing the rate of the rate-limiting E(2) --> E(1) conformational transition by interaction with the E(2) state.
Clarke
Ronald J
RJ
School of Chemistry, University of Sydney, NSW 2006, Australia. r.clarke@chem.usyd.edu.au
Humphrey
Paul A
PA
Lüpfert
Christian
C
Apell
Hans-Jürgen
HJ
Cornelius
Flemming
F
D000818:Animals; D005456:Fluorescent Dyes; D015640:Ion Channel Gating / Q000502:physiology; D007668:Kidney / Q000201:enzymology; D007700:Kinetics; D008954:Models, Biological; D011485:Protein Binding; D011487:Protein Conformation; D011726:Pyridinium Compounds; D011817:Rabbits; D012964:Sodium / Q000378:metabolism; D000254:Sodium-Potassium-Exchanging ATPase / Q000737:chemistry / Q000378:metabolism*; D013343:Styrenes
D005456:Fluorescent Dyes; D011726:Pyridinium Compounds; D013343:Styrenes; C070534:RH 421; D012964:Sodium; D000254:Sodium-Potassium-Exchanging ATPase