Expression, Purification and Characterization of the Rat Luteal 20 -Hydroxysteroid Dehydrogenase

Abstract

The enzyme, rat ovarian 20α-hydroxysteroid dehydrogenase (20αHSD), plays a central role in luteolysis and parturition. It catalyzes the reduction of progesterone, leading to the formation of progestationally inactive steroid, 20α-hydroxypregn-4-ene-3-one (20α-hydroxyprogesterone). Recently, we reported the cloning, sequencing, and deduced amino acid sequence of the rat luteal 20αHSD. To further investigate whether phosphorylation and/or glycosylation affect the activity of 20αHSD and to study its kinetic and biochemical properties, we established both bacterial and insect expression systems for obtaining large quantities of enzyme. The recombinant (rec) 20αHSD expressed as glutathione-S-transferase-20αHSD fusion protein was purified from bacterial lysates by affinity binding to glutathione-Sepharose beads followed by thrombin digestion, whereas the rec enzyme expressed in baculovirus-insect cell system was purified to apparent homogeneity by ion exchange chromatography, followed by dye affinity chromatographies. Both rec preparations of 20αHSD demonstrated a single polypeptide chain of 37 kDa with similar Km values for 20α-hydroxyprogesterone and NADP, although the corresponding maximum velocity values were slightly lower for the rec 20αHSD expressed in the insect cells. The rec 20αHSD showed preference for progesterone/20α-hydroxyprogesterone. 17α-Hydroxyprogesterone was only 30% as effective. The enzyme also used various substrates specific for aldo-keto reductases, although with much less efficiency. The rec enzyme preparations showed an absolute requirement for NADP(H). In vitro phosphorylation of rec bacterial enzyme with either protein kinase A or protein kinase C had no demonstrable effect on its activity. Finally, no differences in enzyme activity were noted between glycosylated (expressed in insect cells) and nonglycosylated (expressed in bacteria) forms of the enzyme. In conclusion, these studies demonstrate that rat luteal 20αHSD can be prepared in large amounts from either bacterial or insect expression systems in a catalytically active form. Indirect evidence also suggests that the catalytic activity of 20αHSD may be independent of phosphorylation and glycosylation states of the enzyme protein, i.e. posttranslational modification of 20αHSD may not be required for the maximal expression of enzyme activity.