Thyroid hormone regulation of perch ovarian 3beta-hydroxysteroid dehydrogenase/delta5-delta4-isomerase activity: involvement of a 52-kDa protein.

Ovarian follicles were collected from perch belonging to the prespawning (vitellogenic) stage and incubated in vitro for 5 h in the absence (control) and presence of 3, 5, 3'-triiodothyronine (T3). Addition of increasing concentrations of T3 from 12.5 to 100 ng/ml caused a linear increase of 3beta-hydroxysteroid dehydrogenase/delta5-delta4-isomerase (3 beta-HSD) activity to 50 ng and then it leveled off indicating a saturation of enzyme activity with 50 ng T3. T3 stimulation of 3beta-HSD activity could be blocked by cycloheximide indicating the involvement of T3-induced protein (TIP) isolated and purified earlier from this laboratory. Addition of fish TIP purified from perch ovarian follicle (fTIP) or rat granulosa cell TIP to ovarian follicular incubation at a dose of 5 microg/ml significantly increased (P < 0.01) 3beta-HSD activity. To observe whether TIP acts directly on the enzyme or not, 3beta-HSD from perch ovarian follicle was purified to homogeneity by the following steps: (i) Sephadex G 75 gel filtration, (ii) DEAE-Sephacel chromatography, and (iii) NAD-affinity column chromatography. Purified 3beta-HSD gave a clear single band on an SDS gel and its molecular weight is 45 kDa. Addition of fTIP to an assay mixture containing purified 3beta-HSD resulted in a fourfold increase of the enzyme activity. fTIP alone did not show enzyme activity when incubated with the radiolabeled substrate. Addition of T3 (50 ng) to the 3beta-HSD assay mixture had no effect on the enzyme activity. Determination of Vmax and Km of the purified enzyme in the absence (control) and presence of fTIP demonstrated a considerable increase of 3beta-HSD affinity and rate of enzyme reaction.