Biospecific adsorption of hepatic DT-diaphorase on immobilized dicoumarol: I. Purification of cytosolic DT-diaphorase from control and 3-methylcholanthrene-treated rats

Abstract

Liver cytosolic DT-diaphorase has been purified from both control and 3-methylcholanthrene (MC)-treated rats, employing a new efficient affinity chromatography gel azodicoumarol coupled to divinyl sulfone cross-linked Sepharose 6B. A subsequent gel filtration on Sephacryl S-200 results in a homogeneous enzyme preparation with a yield of 50–55%. The enhanced DT-diaphorase activity observed in MC-treated rats is most probably due to an increase in enzyme concentration. This conclusion is based on the following results: (a) cycloheximide, an inhibitor of protein synthesis, prevents the increase in DT-diaphorase activity normally caused by MC; (b) purifications of DT-diaphorase from control and MC-treated rats result in enzyme preparations exhibiting closely similar specific activities; 15 times higher amounts of enzyme are obtained from MC-treated rats as compared to controls; (c) immunological identity between DT-diaphorase isolated from control and MC-treated rats are found with the Ouchterlony immunodiffusion method employing antisera raised against either type of enzyme preparation; (d) rocket immunoelectrophoresis reveals a severalfold higher specific content of DT-diaphorase in cytosol from MC-induced rats as compared to controls. The investigated physicochemical properties of DT-diaphorase are not altered after MC treatment of rats. The minimum molecular weight based on the flavin content of the enzyme is close to that obtained with SDS-slab gel electrophoresis, i.e., approximately 28,000, indicating that the dimer of DT-diaphorase contains two molecules of FAD. The molecular activities of DT-diaphorase with various electron acceptors have been investigated; no significant differences between DT-diaphorase isolated from control and MC-treated rats are found. However, the molecular activity of the enzyme with 2,6-dichlorophenolindophenol and menadione varies considerably from one preparation to another, irrespective of source. Employing fused rocket immunoelectrophoresis, it has been possible to detect at least three antigenic forms of DT-diaphorase with different reactivities toward electron acceptors such as 2,6-dichlorophenolindophenol and menadione. The possible existence of several forms of DT-diaphorase is discussed.