The purification of dihydrofolate reductase (5, 6, 7, 8 tetrahydrofolate: NADP+ oxidoreductase, E.C.: 1.5.1.3) from Daucus carota to apparent homogeneity, is described. The enzyme is a soluble protein with a molecular weight of 183 000k +-2500, composed of identical subunits of 58 400+-1000. The enzyme is only weakly recognized by antibodies against human DHFR. The carrot DHFR is characterized by a pH optimum of 5.9, Km values for dihydrofolate and NADPH of 3.7 µM and 2.2 µM, respectively and a turnover number of 4750 or 1500 when referring to the 183 K form or the 58 K monomer, respectively. Molecular and kinetic properties are remarkably different from those reported for the soybean enzyme. Sensitivity to methotrexate is similar to that of bacterial and mammalian enzymes while sensitivity to trimethoprim and dihydrotriazine is intermediate between the two groups of organisms.
Dihydrofolate reductase from Daucus carota cell suspension cultures: purification, molecular and kinetic characterization.
CELLA, RINO
1985-01-01
Abstract
The purification of dihydrofolate reductase (5, 6, 7, 8 tetrahydrofolate: NADP+ oxidoreductase, E.C.: 1.5.1.3) from Daucus carota to apparent homogeneity, is described. The enzyme is a soluble protein with a molecular weight of 183 000k +-2500, composed of identical subunits of 58 400+-1000. The enzyme is only weakly recognized by antibodies against human DHFR. The carrot DHFR is characterized by a pH optimum of 5.9, Km values for dihydrofolate and NADPH of 3.7 µM and 2.2 µM, respectively and a turnover number of 4750 or 1500 when referring to the 183 K form or the 58 K monomer, respectively. Molecular and kinetic properties are remarkably different from those reported for the soybean enzyme. Sensitivity to methotrexate is similar to that of bacterial and mammalian enzymes while sensitivity to trimethoprim and dihydrotriazine is intermediate between the two groups of organisms.I documenti in IRIS sono protetti da copyright e tutti i diritti sono riservati, salvo diversa indicazione.
