Different susceptibility to phospholipase A2 treatment of the fluorescence intensity changes in the vicinity of Cys-964 and Lys-501 in the alpha-chain of probe-labeled Na+,K(+)-ATPase.
Phospholipase A2 [EC 3.1.1.4] treatment of pig kidney Na+,K(+)-ATPase [EC 3.6.1.3] labeled with fluorescence probes at the alpha-chain reduced the extent of the fluorescence intensity change of an N-[p-(2-benzimidazolyl)phenyl]maleimide (BIPM) probe at Cys-964 to below one-third of the control level accompanying the accumulation of phosphoenzymes. However, it only induced a slight decrease in that of a fluorescence isothiocyanate (FITC) probe at Lys-501 with a large decrease in the rate of change. The addition of phosphatidylserine (PS) or phosphatidylinositol (PI) to the phospholipase-treated BIPM-FITC-labeled enzyme increased the rate of the FITC fluorescence change. Phospholipase treatment of the BIPM-enzyme greatly reduced the Na+,K(+)-ATPase activity. The addition of PS or PI to the treated enzyme induced reactivation. These data and others suggest that Cys-964 and Glu-953 (Rb+ protectable dicyclohexyl carbodiimide binding site) are located in the vicinity of the surface area of the enzyme where hydrocarbon chains of phospholipids are present, and conserved H-bonding amino acids, Thr-955 and Ser-962, are located rather near the center of a domain forming a cation binding route or cage with other hydrophobic transmembrane segments. These data may indicate that the interaction between the BIPM probe and the hydrocarbon chains of phospholipids changes in such a way as to sense the change in the binding state of various ligands accompanying the sequential appearance of reaction intermediates of the enzyme.
Nakamura
Y
Y
Department of Chemistry, Faculty of Science, Hokkaido University, Sapporo.
Kai
D
D
Kaya
S
S
Adachi
Y
Y
Taniguchi
K
K
D000178:Acrylamides / Q000494:pharmacology; D000818:Animals; D001665:Binding Sites; D003545:Cysteine / Q000737:chemistry / Q000378:metabolism; D016650:Fluorescein-5-isothiocyanate; D005454:Fluorescence Polarization; D005456:Fluorescent Dyes; D006860:Hydrogen Bonding; D007668:Kidney / Q000187:drug effects*; D008239:Lysine / Q000737:chemistry / Q000378:metabolism; D008301:Maleimides; D008958:Models, Molecular; D010741:Phospholipases A / Q000494:pharmacology*; D054467:Phospholipases A2; D011092:Polyethylene Glycols / Q000494:pharmacology; D011487:Protein Conformation; D000254:Sodium-Potassium-Exchanging ATPase / Q000737:chemistry / Q000378:metabolism*; D013050:Spectrometry, Fluorescence; D013552:Swine
D000178:Acrylamides; D005456:Fluorescent Dyes; D008301:Maleimides; C017272:dodecyloctaethyleneglycol monoether; D011092:Polyethylene Glycols; C000245:N-(4-(2-benzimidazolyl)phenyl)maleimide; D010741:Phospholipases A; D054467:Phospholipases A2; D000254:Sodium-Potassium-Exchanging ATPase; D016650:Fluorescein-5-isothiocyanate; D008239:Lysine; D003545:Cysteine