Pyridoxal 5'-phosphate probes at Lys-480 can sense the binding of ATP and the formation of phosphoenzymes in Na+,K(+)-ATPase.
The Lys-480 in the alpha-subunits of Na+,K(+)-ATPase from pig kidneys was specifically modified with pyridoxal 5'-phosphate (PLP) or pyridoxal 5'-diphospho-5'-adenosine (AP2PL) probes in the presence of NaCl. The site was shown to be the same as the ATP-protectable binding of these probes (Hinz, H.R., and Kirley, T.L. (1990) J. Biol. Chem. 265, 10260-10265). Modifications strongly reduced both Na+,K(+)-ATPase activity and the amount of Na(+)-dependent phosphoenzyme from [32P]ATP but not from [32P]acetyl-phosphate (AcP). Addition of AcP to the enzyme induced a slight decrease in the fluorescence of the PLP probe in the presence of 2 M NaCl and 4 mM MgCl2 but a single exponential increase in the presence of 16 mM NaCl and 4 mM MgCl2. The addition of ATP induced single exponential fluorescence increases at both Na+ concentrations. The data show that these probes can sense molecular events related to the formation of phosphoenzymes induced by AcP and presumably to the formation of Mg-Na-ATP-enzyme complex. The data also suggest that PLP or AP2PL probes at Lys-480 in the presence of Na+ and Mg2+ do not affect the transphosphorylation from AcP to Asp-369 to form phosphoenzymes but that they inhibit the transphosphorylation from the gamma-phosphoryl group of ATP and also ATP binding in the absence of Mg2+.
Kaya
S
S
Department of Chemistry, Faculty of Science, Hokkaido University, Sapporo, Japan.
Tsuda
T
T
Hagiwara
K
K
Fukui
T
T
Taniguchi
K
K
D000255:Adenosine Triphosphate / Q000378:metabolism*; D000595:Amino Acid Sequence; D000818:Animals; D001665:Binding Sites; D007668:Kidney / Q000201:enzymology; D008239:Lysine / Q000737:chemistry / Q000378:metabolism*; D015335:Molecular Probes; D008969:Molecular Sequence Data; D010755:Organophosphates; D011487:Protein Conformation; D011732:Pyridoxal Phosphate / Q000737:chemistry*; D000254:Sodium-Potassium-Exchanging ATPase / Q000037:antagonists & inhibitors / Q000737:chemistry / Q000378:metabolism*; D013050:Spectrometry, Fluorescence; D013552:Swine
D015335:Molecular Probes; D010755:Organophosphates; C011632:acetyl phosphate; D011732:Pyridoxal Phosphate; D000255:Adenosine Triphosphate; D000254:Sodium-Potassium-Exchanging ATPase; D008239:Lysine