[Purification and properties of NADP-specific malate dehydrogenase from bovine adrenal cortex mitochondria].
An electrophoretically homogeneous preparation of mitochondrial NADP-dependent malate dehydrogenase with a specific activity of 155 u./mg and a 67% yield has been obtained, using ammonium sulfate fractionation, gel filtration through Toyopearl HW-55 F, ion-exchange chromatography on DEAE-Toyopearl 650 M and affinity chromatography on 2',5'-ADP-Sepharose 4B. The molecular mass of native malate dehydrogenase is 260 kD; Mr of the SDS-treated enzyme is 61 kD, which is suggestive of a tetrameric structure of the protein. Malate dehydrogenase is active only in the presence of Mg2+ or Mn2+, but not Ca2+ or Ba2+. The Km' values for Mn2+ and Mg2+ are 50 and 66 microM, respectively. At low malate concentrations and NADP saturation, the enzyme is characterized by a sigmoidal kinetics which changes to hyperbolic at low concentrations of NADP. The Lineweaver--Burk plots for the dependence of the initial reaction rate on the concentration of one substrate at several fixed concentrations of the other substrate intersect to the left of the B-axis. NADPH competes with NADP:pyruvate inhibits malate dehydrogenase ++noncompetitively with respect to the coenzyme. NADPH and pyruvate inhibit the malate dehydrogenase-catalyzed reaction via a mixed type mechanism with respect to malate. The data obtained are consistent with a consecutive mechanism of reaction, whose first substrate is NADP and the last product is NADPH.
Senkevich
S B
SB
D000302:Adrenal Cortex / Q000201:enzymology*; D000818:Animals; D002417:Cattle; D007700:Kinetics; D008291:Malate Dehydrogenase / Q000302:isolation & purification* / Q000378:metabolism; D008928:Mitochondria / Q000201:enzymology*; D009249:NADP / Q000378:metabolism*
D009249:NADP; D008291:Malate Dehydrogenase
Oshistka i nekotorye svoĭstva NADP-spetsifichnoĭ malatdegidrogenazy iz mitokhondriĭ kory nadpochechnikov byka.