Purification and Properties of Three Separate Menadione Reductases from Hog Liver

Three separate menadione reductases—one specific for DPNH, another specific for TPNH, and a third equally reactive with either DPNH or TPNH—have been purified from the soluble fraction of hog liver homogenates. The DPNH-menadione reductase displayed two pH optima, one at pH 5.5 and another at pH 7.5. The TPNH enzyme had a pH optimum between 6.5 and 7.5, while the DPNH-TPNH enzyme showed a pH optimum at pH 5.5. The apparent Km for the DPNH enzyme was 1.2 x 10-5 m for DPNH and 1.0 x 10-4 m for menadione. Similar values for the TPNH- and the DPNH-TPNH enzymes are 4.6 x 10-5 m for TPNH, 1.1 x 10-5 m for menadione, 4.2 x 10-4 m for DPNH, and 1.0 x 10-5 m for menadione, respectively. DPNH-menadione reductase is not stimulated by added flavin adenine dinucleotide, but the TPNH and the DPNH-TPNH enzymes are almost completely dependent on added FAD for activity. Concentrations of dicumarol which inhibited the TPNH enzyme 100% were without effect on the DPNH enzyme. Similarly, concentrations of 2,4-dinitrophenol which inhibited the DPNH enzyme 100% were without effect on the TPNH enzyme. The DPNH-TPNH enzyme is highly sensitive to dicumarol and other uncoupling agents. The DPNH enzyme is inhibited by AMP, but not by any of the other nucleotides. The DPNH-TPNH enzyme is inhibited by high concentrations of nucleoside di- and triphosphates, but the inhibition is observed at acidic but not at basic pH values. The TPNH and the DPNH-TPNH enzymes are inhibited by folic acid and some of its derivatives. The DPNH enzyme is not inhibited by these pteridine derivatives.