Bernikov L R, Dzhandzhugazyan K N, Lutsenko S V, Modyanov N N
Shemyakin Institute of Bioorganic Chemistry, USSR Academy of Sciences, Moscow.
Eur J Biochem. 1990 Dec 12;194(2):413-21. doi: 10.1111/j.1432-1033.1990.tb15634.x.
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.
对猪肾中处于各种构象状态的Na +,K(+)-ATP酶与ATP的二醛类似物α,α-(9-腺苷基)-α'-D-(羟甲基)二乙醇醛三磷酸酯(oATP)之间的相互作用进行了研究。这种相互作用导致酶的修饰，根据以下标准表明其为亲和型修饰。1. oATP可被Na +,K(+)-ATP酶水解，并能防止γ-[4-(N-2-氯乙基-N-甲氨基)]苄酰胺ATP对ATP酶活性的抑制，这表明它在酶的活性位点与Na +,K(+)-ATP酶相互作用。2. oATP不可逆地抑制Na +,K(+)-ATP酶的ATP水解活性；在20 mM ATP存在时，失活程度降低，且取决于修饰介质的离子组成。在含Na +,Mg2(+)的缓冲液中，抑制作用和ATP保护作用最大。3. 在低浓度(oATP浓度小于0.1 mM)时，[14C]oATP掺入α亚基的值与酶的失活程度成正比，外推至完全抑制时，相当于每摩尔α亚基掺入1.05摩尔试剂。4. 对分离得到的oATP修饰的α亚基进行胰蛋白酶水解，随后分离肽段，仅发现一个分子量约为10 kDa的标记片段。讨论了修饰片段在α亚基多肽链中的定位。结果表明修饰后形成了类似吗啉的结构。
