Guinea Pig Liver 3-Hydroxyhexobarbital Dehydrogenase*

3-Hydroxyhexobarbital dehydrogenase, which catalyzes the reversible oxidation of 3-hydroxyhexobarbital to d-oxohexobarbital, has been purified 47%fold from the soluble fraction of guinea pig liver with a yield of 47%. The specific activity of the purified enzyme is 9.4 units/mg of protein. Results of polyacrylamide gel disc electrophoresis and isoelectric focusing indicated that the purified enzyme preparation is a single and homogeneous protein. NADP+ served as preferred cofactor, but NAD+ is also utilized in the presence of phosphate ion. The guinea pig liver enzyme possessed a relatively narrow substrate specificity in comparison with the rabbit liver enzyme. It is very distinctive that guinea pig liver d-hydroxyhexobarbital dehydrogenase catalyzes the dehydrogenation of 17&hydroxysteroids such as testosterone, C-androstene3p, 17/3-diol, 5a-androstane-3a, 17P-diol, 5cr-androstane-3p, 17/3-diol, Sa-androstan-17@-ol-3-one, and 5P-androstane3cu, 17p-diol.