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          Institute: MPI für Biophysik     Collection: Abt. Biophysikalische Chemie     Display Documents



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ID: 12029.0, MPI für Biophysik / Abt. Biophysikalische Chemie
Mechanism of the rate-determining step of the Na+,K+-ATPase pump cycle
Authors:Humphrey, P. A.; Lüpfert, C.; Apell, H. J.; Cornelius, F.; Clarke, R. J.
Date of Publication (YYYY-MM-DD):2002
Title of Journal:Biochemistry
Journal Abbrev.:Biochem.
Volume:41
Issue / Number:30
Start Page:9496
End Page:9507
Review Status:not specified
Audience:Experts Only
Abstract / Description:The kinetics of the E-2 --> E-1 conforinational change of unphosphorylated Na+, K+-ATPase from rabbit kidney and shark rectal -land were investigated via the stopped-flow technique using the fluorescent label RH421 (pH 7.4, 24 degreesC). The enzyme was pre-equilibrated in a solution containing 25 mM histidine and 0.1 mM EDTA to stabilize initially the E-, conformation. When rabbit kidney enzyme was mixed 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) in the case of rabbit kidney enzyme. The experimental behavior could be described by a binding of Na+ to the enzyme in the E-1 state with a dissociation constant of 31 +/- 7 mM. which induces a subsequent rate-limiting conformational chanue 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. Analogous experiments performed with enzyme from shark rectal gland showed similar effects, but with a significantly 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 that of ATP, in enhancing the rate of the rate-limiting E-2 --> E-1 conforinational transition by interaction with the E-2 state.
Free Keywords:yeast glyceraldehyde-3-phosphate dehydrogenase; fluorescein 5'-
; isothiocyanate modification; nicotinamide-adenine dinucleotide;
; co-operative binding; conformational-changes; sodium-pump;
; na+/k+-atpase; adenosine-triphosphatase; kidney na+;k+-atpase;
; nucleotide-binding
External Publication Status:published
Document Type:Article
Communicated by:N. N.
Affiliations:MPI für Biophysik/Abteilung Biophysikalische Chemie
External Affiliations:Clarke RJ Univ Sydney, Sch Chem, Sydney, NSW 2006, Australia
Identifiers:ISI:000177111600026 [ID No:1]
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