title: Aspartate aminotransferase is potently inhibited by copper complexes: Exploring copper complex-binding proteome.
abstract: Recent researches indicated that a copper complex-binding proteome that potently interacted with copper complexes and then influenced cellular metabolism might exist in organism. In order to explore the copper complex-binding proteome, a copper chelating ion-immobilized affinity chromatography (Cu-IMAC) column and mass spectrometry were used to separate and identify putative Cu-binding proteins in primary rat hepatocytes. A total of 97 putative Cu-binding proteins were isolated and identified. Five higher abundance proteins, aspartate aminotransferase (AST), malate dehydrogenase (MDH), catalase (CAT), calreticulin (CRT) and albumin (Alb) were further purified using a SP-, and (or) Q-Sepharose Fast Flow column. The interaction between the purified proteins and selected 11 copper complexes and CuCl2was investigated. The enzymes inhibition tests demonstrated that AST was potently inhibited by copper complexes while MDH and CAT were weakly inhibited. Schiff-based copper complexes 6 and 7 potently inhibited AST with the IC50value of 3.6 and 7.2μM, respectively and exhibited better selectivity over MDH and CAT. Fluorescence titration results showed the two complexes tightly bound to AST with binding constant of 3.89×106and 3.73×106M-1, respectively and a stoichiometry ratio of 1:1. Copper complex 6 was able to enter into HepG2 cells and further inhibit intracellular AST activity.
authors.affiliation: Institute of Molecular Science, Key Laboratory of Chemical Biology and Molecular Engineering of the Education Ministry, Shanxi University, Taiyuan, Shanxi 030006, People's Republic of China.
authors.forename: Yuqi
authors.initials: Y
authors.lastname: Jia
authors.affiliation: Institute of Molecular Science, Key Laboratory of Chemical Biology and Molecular Engineering of the Education Ministry, Shanxi University, Taiyuan, Shanxi 030006, People's Republic of China. Electronic address: luliping@sxu.edu.cn.
authors.forename: Liping
authors.initials: L
authors.lastname: Lu
authors.affiliation: Institute of Molecular Science, Key Laboratory of Chemical Biology and Molecular Engineering of the Education Ministry, Shanxi University, Taiyuan, Shanxi 030006, People's Republic of China.
authors.forename: Caixia
authors.initials: C
authors.lastname: Yuan
authors.affiliation: Institute of Molecular Science, Key Laboratory of Chemical Biology and Molecular Engineering of the Education Ministry, Shanxi University, Taiyuan, Shanxi 030006, People's Republic of China.
authors.forename: Sisi
authors.initials: S
authors.lastname: Feng
authors.affiliation: Institute of Molecular Science, Key Laboratory of Chemical Biology and Molecular Engineering of the Education Ministry, Shanxi University, Taiyuan, Shanxi 030006, People's Republic of China. Electronic address: miaoli@sxu.edu.cn.
authors.forename: Miaoli
authors.initials: M
authors.lastname: Zhu
mesh_terms: D000818:Animals; D001219:Aspartate Aminotransferases / Q000378:metabolism*; D002352:Carrier Proteins / Q000378:metabolism*; D003300:Copper / Q000378:metabolism*; D056945:Hep G2 Cells; D022781:Hepatocytes / Q000378:metabolism*; D006801:Humans; D020543:Proteome / Q000378:metabolism*; D051381:Rats
chemical_list: D002352:Carrier Proteins; D020543:Proteome; C023548:copper-binding protein; D003300:Copper; D001219:Aspartate Aminotransferases
keywords: Aspartate aminotransferase; Copper complex; Copper complex-binding proteins; Cu-binding proteins; Inhibition