Abstract
Quinonoid dihydropterin reductase (DHPR, EC 1.6.99.7) from bovine brain was purified 340-fold over the initial extract to an activity of 76 units/mg of protein with an overall recovery of 10 per cent. Analytical polyacrylamide gel electrophoresis, combined with protein staining, indicated that the single band of purified enzyme could have contained no more than 7% contaminants. Furthermore, using sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis, only one protein band was observed for the SDS-treated enzyme. Using a sucrose density gradient, the sedimentation coefficient and the molecular weight of the native enzyme were determined to be 3.70 and 47,000± 1,400 respectively. Molecular weight using 10% SDS-gel electrophoresis was found to be 22,400± 230, indicating that the brain reductase is composed of two equivalent subunits. Other characteristics of DHPR which were determined were the isoelectric point (pH 5.7), the K m, for quinonoid 6,7-dimethyl-dihydropterin (18 μM) and the K m for NADH (26 μM). Purified brain DHPR was used as an antigen to produce specific antibodies to the enzyme. The specificity of the antibody was established according to the following criteria: (1) the antibody was found to be a potent inhibitor of partially purified and homogeneous DHPR; (2) a single precipitin arc formed upon running either partially purified or homogeneous DHPR against the antibody on a double immunodiffusion plate, or by immunoelectrophoresis; (3) the antibody inhibited DHPR activity from rat brain, rat kidney and rat liver ; and (4) neither inhibition of activity, nor formation of precipitin arcs was exhibited by the antibody when reacted with other catecholamine-synthesizing enzymes partially purified from different rat tissues.
Published Version
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