Modulation of Soluble Ovarian Adenosine 3′,5′-Monophosphate-Dependent Protein Kinase Activity during Prepubertal Development in the Rat. II. Evaluation of the Catalytic Subunit Inhibitor Activity1

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Abstract
In a previous report on the ontogeny of the ovarian adenosine 3'',5''-monophosphate (cAMP)-dependent protein kinase activity during prepubertal development of the rat, we concluded that the 4-fold decline in cAMP-dependent protein kinase activity observed in ovaries of 21- to 23-day-old rats was due to the presence of a heat-labile inhibitor in the ovarian extracts (Hunzicker-Dunn et al., 1984). We developed an assay for this ovarian kinase inhibitor activity that was based on the observation that ovarian cytosol added to an exogenous catalytic subunit of cAMP-dependent protein kinase caused a time-dependent and ovarian cytosol protein concentration-dependent inhibition of exogenous catalytic subunit phosphotransferase activity. The present studies were conducted to evaluate the basis for this catalytic subunit inhibitor present in soluble rat ovarian extracts of prepubertal-aged rats. This inhibitor activity was absent from cytosol extracts of rat corpora lutea, rat liver, rabbit follicles, and rabbit corpora lutea. Inhibitor activity present in rat ovarian cytosol was not attributable to insufficient levels of the phosphorylation substrate Kemptide. Inhibitor activity was also not related to the presence of the large amount of catalytic subunit-free regulatory subunit of the cAMP-dependent protein kinase present in ovarian extracts of late juvenile-aged rats. Inhibitor activity, however, did correlate with an endogenous adenosine triphosphatase (ATPase) activity that reduced assay ATP concentrations below levels needed to accurately measure phosphotransferase activity, despite the presence of sodium fluoride (an ATPase inhibitor) and ATP concentrations 5-to 15-fold greater than the Km of the kinase for ATP. Moreover, the 4-fold reduction is cAMP-dependent protein kinase activity in ovaries of 21- to 23-day-old rats was no longer detected when assay ATP levels were maintained. We conclude that "mature" levels of cAMP-dependent protein kinase activity are achieved in ovarian extracts by the beginning of the late juvenile phase of ovarian maturation.