The non-gastric H,K-ATPase is oligomycin-sensitive and can function as an H+,NH4(+)-ATPase
| Autor: | Herman G.P. Swarts, Jan B. Koenderink, Jan Joep H.H.M. De Pont, Peter H.G.M. Willems |
|---|---|
| Rok vydání: | 2005 |
| Předmět: |
Oligomycin
Protein Conformation ATPase Membrane transport and intracellular motility [NCMLS 5] Biochemistry Ouabain H(+)-K(+)-Exchanging ATPase chemistry.chemical_compound Vanadate Enzyme Inhibitors Phosphorylation Cation Transport Proteins Sequence Deletion Adenosine Triphosphatases chemistry.chemical_classification biology Imidazoles Recombinant Proteins Turnover number Pathogenesis and modulation of inflammation [N4i 1] Mitochondrial medicine [IGMD 8] Electrophoresis Polyacrylamide Gel Baculoviridae Protein Binding medicine.drug Energy and redox metabolism [NCMLS 4] Blotting Western Spodoptera Dephosphorylation Ammonia medicine Animals Na+/K+-ATPase Molecular Biology Binding Sites Sodium Poverty-related infectious diseases [N4i 3] Cell Biology Molecular biology Rats Enzyme Activation Renal disorders [UMCN 5.4] Kinetics Protein Subunits Enzyme chemistry Mutagenesis Site-Directed Potassium biology.protein Oligomycins Vanadates Cellular energy metabolism [UMCN 5.3] |
| Zdroj: | Journal of Biological Chemistry, 280, 33115-22 Journal of Biological Chemistry, 280, 39, pp. 33115-22 |
| ISSN: | 1083-351X 0021-9258 |
| Popis: | Contains fulltext : 48560.pdf (Publisher’s version ) (Open Access) We used the baculovirus/Sf9 expression system to gain new information on the mechanistic properties of the rat non-gastric H,K-ATPase, an enzyme that is implicated in potassium homeostasis. The alpha2-subunit of this enzyme (HKalpha2) required a beta-subunit for ATPase activity thereby showing a clear preference for NaKbeta1 over NaKbeta3 and gastric HKbeta. NH4(+), K+, and Na+ maximally increased the activity of HKalpha2-NaKbeta1 to 24.0, 14.2, and 5.0 micromol P(i) x mg(-1) protein x h(-1), respectively. The enzyme was inhibited by relatively high concentrations of ouabain and SCH 28080, whereas it was potently inhibited by oligomycin. From the phosphorylation level in the presence of oligomycin and the maximal NH4(+)-stimulated ATPase activity, a turnover number of 20,000 min(-1) was determined. All three cations decreased the steady-state phosphorylation level and enhanced the dephosphorylation rate, disfavoring the hypothesis that Na+ can replace H+ as the activating cation. The potency with which vanadate inhibited the cation-activated enzyme decreased in the order K+ > NH4(+) > Na+, indicating that K+ is a stronger E2 promoter than NH4(+), whereas in the presence of Na+ the enzyme is in the E1 form. For K+ and NH4(+), the E2 to E1 conformational equilibrium correlated with their efficacy in the ATPase reaction, indicating that here the transition from E2 to E1 is rate-limiting. Conversely, the low maximal ATPase activity with Na+ is explained by a poor stimulatory effect on the dephosphorylation rate. These data show that NH4(+) can replace K+ with similar affinity but higher efficacy as an extracellular activating cation in rat nongastric H,K-ATPase. |
| Databáze: | OpenAIRE |
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