EXPRESSION, PURIFICATION AND CHARACTERIZATION OF THE RAT LUTEAL 20-ALPHA-HYDROXYSTEROID DEHYDROGENASE

The enzyme, rat ovarian 20 alpha-hydroxysteroid dehydrogenase (20 alph a HSD), plays a central role in luteolysis and parturition. Its cataly zes the reduction of progesterone, leading to the formation of progest ationally inactive steroid, 20 alpha-hydroxypregn-4-ene-3-one (20 alph a-hydroxyprogesterone). Recently, we reported the cloning, sequencing, and deduced amino acid sequence of the rat luteal 20 alpha HSD. To fu rther investigate whether phosphorylation and/or glycosylation affect the activity of 20 alpha HSD and to study its kinetic and biochemical properties, we established both bacterial and insect expression system s for obtaining large quantities of enzyme. The recombinant (rec) 20 a lpha HSD expressed as glutathione-S-transferse-20 alpha HSD fusion pro tein was purified from bacterial lysates by affinity binding to glutat hione-Sepharose beads followed by thrombin digestion, whereas the rec enzyme expressed in baculovirus-insect cell system was purified to app arent homogeneity by ion exchange chromatography, followed by dye affi nity chromatographies. Both rec preparations of 20 alpha HSD demonstra ted a single polypeptide chain of 37 kDa with similar K-m values for 2 0 alpha-hydroxyprogesterone and NADP, although the corresponding maxim um velocity values were slightly lower for the rec 20 alpha HSD expres sed in the insect cells. The rec 20 alpha HSD showed preference for pr ogesterone/20 alpha-hydroxyprogesterone. 17 alpha-Hydroxyprogesterone was only 20% as effective. The enzyme also used various substrates spe cific for aldo-keto reductases, although with much less efficiency. Th e rec enzyme preparations showed an absolute requirement for NADP(H). In vitro phosphorylation of rec bacterial enzyme with either protein k inase A or protein kinase C had no demonstrable effect on its activity . Finally, no differences in enzyme activity were noted between glycos ylated (expressed in insect cells) and nonglycosylated (expressed in b acteria) forms of the enzyme. In conclusion, these studies demonstrate that rat luteal 20 alpha HSD can be prepared in large amounts from ei ther bacterial or insect expression systems in a catalytically active form. Indirect evidence also suggests that the catalytic activity of 2 0 alpha HSD may be independent of phosphorylation and glycosylation st ates of the enzyme protein, i.e. posttranslational modification of 20 alpha HSD may not be required for the maximal expression of enzyme act ivity.