Modulation of Soluble Ovarian Adenosine 3′,5′- Monophosphate-Dependent Protein Kinase Activity during Prepubertal Development of the Rat*

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Abstract
Studies were conducted to evaluate the ontogeny of cAMP-dependent protein kinases in the soluble fraction of rat ovaries obtained throughout prepubertal ovarian maturation (days 5-37). Protein kinase activity stimulated by cAMP and inhibited by the heat-stable protein kinase inhibitor, designated cAMP-dependent protein kinase activity, was already present in ovaries of 5 day old rats. This kinase activity subsequently increased to reach maximal values between days 16 and 18, then plunged 4-fold to a nadir between days 21 and 23. cAMP-dependent protein kinase activity began to rise again by day 24 increasing 5-fold to a maximum on day 27. Experiments were conducted to evaluate the basis for the 80% reduction of cAMP-dependent protein kinase activity in the soluble extract of ovaries of 21-23 day old rats. To determine whether the decrease in kinase activity was accompanied by a concomitant reduction of cAMP-binding activity, the ability of ovarian cytosol to bind [3H]cAMP was evaluated. At the time of decreased cAMP-dependent protein kinase activity, total soluble cAMP-binding activity was not significantly reduced. Additionally, the cAMP-binding activity of the regulatory subunits RI and RII(regulatory subunits of the type I and II isoenzyme forms of cAMP-dependent protein kinase), as detected by photoaffinity labeling with 8-N3-[32P]cAMP, was not changed. To determine whether the decline in kinase activity was due to an actual disappearance of the catalytic subunit from the soluble ovarian extracts of ovaries of 21-23 day old rats, the relative amounts of catalytic subunit were quantified by an enzyme-linked immunosorbent assay using an antiserum directed against bovine heart catalytic subunit. The decrease in protein kinase catalytic activity was not due to a reduction in the amount of catalytic subunits. Experiments were conducted to determine whether the reduction of protein kinase catalytic activity was not due to a reduction in the amount of catalytic subunits. Experiments were conducted to determine whether the reduction of protein kinase catalytic activity in unfractionated cytosol was expressed after anion exchange chromatography. The estimated total cAMP-stimulated protein kinase activity measured after DEAE-cellulose chromatography of the soluble ovarian extracts of 21-23 day old rats was no longer depressed. Since the 80% reduction of catalytic kinase activity in ovarian extracts of 21-23 day old rats was detectable in unfractionated cytosol before but not after DEAE-cellulose chromatography, tests were made for the presence of an endogenous inhibitor of catalytic kinase activity in the cytosol fraction. Determinations of levels of the heat-stable protein kinase inhibitor in 21-23 day old rats or at any other prepubertal age. However, when cytosol of 21-24 day old rats was tested for its ability to modulate the activity of exogenous catalytic activity, an apparent concentration-dependent inhibition of exogenous catalytic activity was observed. This inhibition was abolished on heat treatment of the cytosol and was absent in ovaries of older rats. The 80% reduction of cAMP-dependent protein kinase catalytic activity detected in soluble ovarian extracts of 21-23 day old rats before but not after DEAE-cellulose chromatography is the result of an as yet unidentified heat-labile inhibitor which is freed on anion exchange chromatography. Neither the identity nor the physiological significance of this inhibitor is currently known.

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