title: Purification and characterization of the cytosolic NADP(+)-dependent malic enzyme from human breast cancer cell line.
abstract: Cytosolic NADP(+)-dependent malic enzyme from a cultured human breast cancer cell line was purified to near homogeneity by two highly efficient chromatography systems: Pharmacia-LKB Q-Sepharose anion-exchange chromatography and adenosine-2',5'-bisphosphate-agarose affinity chromatography. The overall yield was 27%. The enzyme is presumably a tetramer composed of four probably identical subunits of Mr 65,000, which is similar to the enzyme from other sources. The pI and optimum reaction pH values for the tumor malic enzyme are 5.5 and 7.2, respectively. At pH 6.9, most of the enzyme exists as monomers. Activation energy for the enzyme-catalyzed oxidative-decarboxylation reaction is 57.4 kJ/mol. The enzyme is strictly NADP+ dependent, as NAD+ cannot support the oxidative-decarboxylation reaction. ATP at low concentration inhibits the enzyme activity. Fumarate at concentrations up to 5 mM does not affect the enzymatic reaction rate. Therefore the tumor cytosolic malic enzyme, unlike the mitochondrial malic enzyme, is not an allosteric regulatory enzyme.
authors.affiliation: Department of Biochemistry, National Defense Medical Center, Taipei, Taiwan, Republic of China.
authors.forename: G G
authors.initials: GG
authors.lastname: Chang
authors.forename: J K
authors.initials: JK
authors.lastname: Wang
authors.forename: T M
authors.initials: TM
authors.lastname: Huang
authors.forename: H J
authors.initials: HJ
authors.lastname: Lee
authors.forename: W Y
authors.initials: WY
authors.lastname: Chou
authors.forename: C L
authors.initials: CL
authors.lastname: Meng
mesh_terms: D001943:Breast Neoplasms / Q000201:enzymology*; D002846:Chromatography, Affinity; D002852:Chromatography, Ion Exchange; D003600:Cytosol / Q000201:enzymology*; D004591:Electrophoresis, Polyacrylamide Gel; D006801:Humans; D007525:Isoelectric Focusing; D007700:Kinetics; D008291:Malate Dehydrogenase / Q000302:isolation & purification* / Q000378:metabolism; D009249:NADP / Q000378:metabolism*; D010084:Oxidation-Reduction; D014407:Tumor Cells, Cultured
chemical_list: D009249:NADP; D008291:Malate Dehydrogenase