Are pyridoxal and fluorescein probes in lysine residues of alpha-chain in Na+,K(+)-ATPase sensing ATP binding?
Na+,K(+)-ATPase preparations from pig kidneys were treated with 50 microM pyridoxal 5'-diphospho-5'-adenosine (AP2PL) in the presence of NaCl. The resulting preparations contained 0.5 mol of the AP2PL probe at the Lys-480/mol alpha-chain. This modification reduced both Na+,K(+)-ATPase activity and the amount of Na(+)-dependent phosphoenzyme from ATP to around 50% but not that from acetyl phosphate (AcP). The addition of 1 mM AcP to the modified enzyme in the presence of Mg2+ and Na+ induced phosphorylation (3.0/s) followed by an AP2PL fluorescence increase (1.2/s). The addition of 10 microM ATP instead of AcP induced rapid phosphorylation (28/s) followed by a slow increase in fluorescence (1.0/s). When modified enzyme preparations were treated with fluorescein 5'-isothiocyanate (FITC), the phosphorylation capacity from ATP was reduced to around 5% with little influence on either the AP2PL fluorescence change by ATP or phosphorylation from AcP. The addition of increasing concentrations of ATP with 160 mM NaCl to the K(+)-bound AP2PL-FITC-labeled enzyme showed different rates for each fluorescence change and different affinities for ATP of the changes. These data and others indicate that the AP2PL probe at Lys-480 can monitor ATP binding to high- and low-affinity sites and suggest the simultaneous presence of two different low-affinity sites for ATP detected by an AP2PL probe at Lys-480 and an FITC probe at Lys-501.
Tsuda
T
T
Graduate School of Science, Hokkaido University, Sapporo, Japan.
Kaya
S
S
Yokoyama
T
T
Taniguchi
K
K
D000244:Adenosine Diphosphate / Q000031:analogs & derivatives* / Q000378:metabolism; D000255:Adenosine Triphosphate / Q000378:metabolism*; D000345:Affinity Labels; D000818:Animals; D005456:Fluorescent Dyes; D007668:Kidney / Q000201:enzymology; D007700:Kinetics; D008239:Lysine; D046911:Macromolecular Substances; D010766:Phosphorylation; D011730:Pyridoxal / Q000378:metabolism*; D011732:Pyridoxal Phosphate / Q000031:analogs & derivatives* / Q000378:metabolism; D000254:Sodium-Potassium-Exchanging ATPase / Q000737:chemistry* / Q000378:metabolism*; D013050:Spectrometry, Fluorescence; D013552:Swine
D000345:Affinity Labels; D005456:Fluorescent Dyes; D046911:Macromolecular Substances; D011730:Pyridoxal; D011732:Pyridoxal Phosphate; D000244:Adenosine Diphosphate; D000255:Adenosine Triphosphate; D000254:Sodium-Potassium-Exchanging ATPase; C048236:pyridoxal 5'-diphospho-5'-adenosine; D008239:Lysine