[NADP-dependent malate dehydrogenase from bovine adrenal cortex cytoplasm. Isolation and properties].
The NADP-dependent decarboxylating malate dehydrogenase was isolated from the cytoplasmic fraction of bovine adrenal cortex and purified 3530-fold by 3-fold ammonium sulfate fractionation, ion-exchange chromatography on DEAE-Toyopearl 650 M and DEAE-Sephadex A-50 with subsequent two-fold gel filtration through Toyopearl HW-55. The specific activity of homogeneous enzyme preparations was equal to 60 U/mg protein with a 30% yield. The enzyme molecular weight as determined by gel filtration on Sephadex G-20 was 155000. Upon polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate malate dehydrogenase dissociated into two subunits with Mr 77000. The Arrhenius plot for the reaction rate showed a break at 30 degrees C. The values of activation energy and temperature coefficient above and below the breakpoint were equal to 45049 and 147188 J X mol-1; 1.68 and 2.63, respectively. Within the temperature range of 26-40 degrees C, malate dehydrogenase exhibited hyperbolic kinetics with respect to the substrate. At 30 degrees C, Km for malate was equal to 250 microM, whereas at 40 degrees C it was 130 microM. The curve for the dependence of the initial reaction velocity versus NADP concentration was S-shaped. The Hill coefficient was 1.4, which testifies to positive cooperativity of NADP interaction with malate dehydrogenase.
D000302:Adrenal Cortex / Q000201:enzymology*; D000818:Animals; D002417:Cattle; D002852:Chromatography, Ion Exchange; D003593:Cytoplasm / Q000201:enzymology; D004589:Electrophoresis, Disc; D066298:In Vitro Techniques; D007700:Kinetics; D008291:Malate Dehydrogenase / Q000302:isolation & purification / Q000378:metabolism*; D008928:Mitochondria / Q000201:enzymology; D008970:Molecular Weight
D008291:Malate Dehydrogenase; C031917:malate dehydrogenase (oxaloacetate-decarboxylating) (NADP+)
NADP-zavisimaia malatdegidrogenaza iz tsitoplazmy kory nadpochechnikov byka. Ochistka i nekotorye svoĭstva.