Phosphorylation of Na,K-ATPase by acetyl phosphate and inorganic phosphate. Sidedness of Na+, K+ and nucleotide interactions and related enzyme conformations.
The effects of K+, Na+ and nucleotides (ATP or ADP) on the steady-state phosphorylation from [32P]Pi (0.5 and 1 mM) and acetyl [32P]phosphate (AcP) (5 mM) were studied in membrane fragments and in proteoliposomes with partially purified pig kidney Na,K-ATPase incorporated. The experiments were carried out at 20 degrees C and pH 7.0. In broken membranes, the Pi-induced phosphoenzyme levels were reduced to 40% by 10 mM K+ and to 20% by 10 mM K+ plus 1 mM ADP (or ATP); in the presence of 50 mM Na+, no E-P formation was detected. On the other hand, with AcP, the E-P formation was reduced by 10 mM K+ but was 30% increased by 50 mM Na+. In proteoliposomes E-P formation from Pi was (i) not influenced by 5-10 mM K+cyt or 100 mM Na+ext, (ii) about 50% reduced by 5, 10 or 100 mM K+ext and (iii) completely prevented by 50 mM Na+cyt. Enzyme phosphorylation from AcP was 30% increased by 10 mM K+cyt or 50 mM Na+cyt; these E-P were 50% reduced by 10-100 mM K+ext. However, E-P formed from AcP without K+cyt or Na+cyt was not affected by extracellular K+. Fluorescence changes of fluorescein isothiocyanate labelled membrane fragments, indicated that E-P from AcP corresponded to an E2 state in the presence of 10 mM Na+ or 2 mM K+ but to an E1 state in the absence of both cations. With pNPP, the data indicated an E1 state in the absence of Na+ and K+ and also in the presence of 20 mM Na+, and an E2 form in the presence of 5 mM K+. These results suggest that, although with some similarities, the reversible Pi phosphorylation and the phosphatase activity of the Na,K-ATPase do not share the whole reaction pathway.
Berberián
G
G
División de Biofisica, Instituto de Investigación Médica Mercedes y Martín Ferreyra, Córdoba, Argentina.
Beaugé
L
L
D000818:Animals; D002462:Cell Membrane / Q000201:enzymology; D016650:Fluorescein-5-isothiocyanate; D005452:Fluoresceins; D005453:Fluorescence; D005456:Fluorescent Dyes; D007668:Kidney / Q000201:enzymology; D008081:Liposomes / Q000378:metabolism; D010755:Organophosphates / Q000378:metabolism*; D010710:Phosphates / Q000378:metabolism*; D010766:Phosphorylation; D011487:Protein Conformation; D000254:Sodium-Potassium-Exchanging ATPase / Q000378:metabolism*; D013552:Swine; D013861:Thiocyanates
D005452:Fluoresceins; D005456:Fluorescent Dyes; D008081:Liposomes; D010755:Organophosphates; D010710:Phosphates; D013861:Thiocyanates; C011632:acetyl phosphate; D000254:Sodium-Potassium-Exchanging ATPase; D016650:Fluorescein-5-isothiocyanate