Dialdehyde ATP derivative as an affinity modifier of the Na+,K(+)-ATPase active site.
Interaction of Na+,K(+)-ATPase from pig kidney in various conformational states with the dialdehyde analogue of ATP, alpha,alpha-(9-adenyl)-alpha'-D-(hydroxymethyl)diglycolaldehyde triphosphate ester (oATP), has been studied. This interaction leads to an enzyme modification which was shown to be of the affinity type according to the following criteria. 1. oATP can be hydrolyzed by Na+,K(+)-ATPase and prevent inhibition of ATPase activity by gamma-[4-(N-2-chloroethyl-N-methylamino)]benzylamide ATP, indicating that it interacts with Na+,K(+)-ATPase in the enzyme active site. 2. oATP irreversibly inhibits ATP-hydrolyzing activity of Na+,K(+)-ATPase; the extent of inactivation is decreased in the presence of 20 mM ATP and depends on the ion composition of the modification medium. The inhibition and ATP protection are maximal in Na+,Mg2(+)-containing buffer. 3. The value of [14C]oATP incorporation into the alpha subunit is proportional to the degree of enzyme inactivation at low (less than 0.1 mM) concentration of oATP and, on extrapolation to complete inhibition, corresponds to incorporation of 1.05 mol reagent/mol alpha subunit. 4. Tryptic hydrolysis of the isolated oATP-modified alpha subunit and subsequent separation of the peptides revealed only one labelled fragment with a molecular mass of about 10 kDa. Localization of the modified fragment in the alpha-subunit polypeptide chain is discussed. A morpholine-like structure was shown to be formed as a result of the modification.
Bernikov
L R
LR
Shemyakin Institute of Bioorganic Chemistry, USSR Academy of Sciences, Moscow.
Dzhandzhugazyan
K N
KN
Lutsenko
S V
SV
Modyanov
N N
NN
D000255:Adenosine Triphosphate / Q000031:analogs & derivatives* / Q000494:pharmacology; D000345:Affinity Labels; D000818:Animals; D001665:Binding Sites; D002384:Catalysis; D002851:Chromatography, High Pressure Liquid; D007679:Kidney Medulla / Q000201:enzymology; D008274:Magnesium; D011188:Potassium; D012964:Sodium; D000254:Sodium-Potassium-Exchanging ATPase / Q000037:antagonists & inhibitors / Q000737:chemistry / Q000378:metabolism*; D013056:Spectrophotometry, Ultraviolet; D013552:Swine
D000345:Affinity Labels; C004282:periodate-oxidized adenosine 5'-triphosphate; D000255:Adenosine Triphosphate; D012964:Sodium; D000254:Sodium-Potassium-Exchanging ATPase; D008274:Magnesium; D011188:Potassium