Jf. Mao et al., EXPRESSION, PURIFICATION AND CHARACTERIZATION OF THE RAT LUTEAL 20-ALPHA-HYDROXYSTEROID DEHYDROGENASE, Endocrinology, 138(1), 1997, pp. 182-190
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.