Separation of the complexes formed between the regulatory and catalytic subunits of cyclic adenosine monophosphate-dependent protein kinase and topoisomerase I activity in preovulatory follicle-enriched immature rat ovaries.
Our previous studies have shown that the regulatory subunits of the type II form of cAMP-dependent protein kinase (RII) present in soluble extract of immature rat ovaries elute from diethylaminoethyl-cellulose as three separate peaks of activity, based on their association with the catalytic subunit (C) of this enzyme, as R2IIC2, an apparent R2IIC, and R2II. Based upon the existence of ovarian RII in three different subunit arrangements, the large amount of C subunit-free R2II in this tissue, and a previous report which indicated that RII exhibited intrinsic topoisomerase I activity, we determined whether DNA topoisomerase I activity was associated with any of these molecular complexes of the ovarian RII subunits. Cyclic AMP-binding activities in soluble extracts of preovulatory follicle-enriched immature rat ovaries were separated by diethylaminoethyl-cellulose chromatography and sucrose density gradient centrifugation. Topoisomerase I activity cosedimented with the apparent R2IIC and R2II obtained from sucrose gradients but was not detected in fractions containing R2IIC2. Upon cAMP affinity purification of the RII derived from fractions containing R2IIC2, R2IIC, and R2II, respectively, no topoisomerase I activity could be detected in any fraction. Phosphorylation of the affinity purified RIIs by the C subunit of beef heart cAMP-dependent protein kinase did not alter this result. These data indicate that none of the RII subunits in soluble extracts of preovulatory follicle-enriched ovaries exhibit intrinsic topoisomerase I activity.
Hunzicker-Dunn
M
M
Department of Molecular Biology, Northwestern University Medical School, Chicago, Illinois 60611.
Maizels
E T
ET
Kern
L C
LC
Ekstrom
R C
RC
Constantinou
A I
AI
D000818:Animals; D002499:Centrifugation, Density Gradient; D002846:Chromatography, Affinity; D002848:Chromatography, DEAE-Cellulose; D003600:Cytosol / Q000201:enzymology; D004264:DNA Topoisomerases, Type I / Q000302:isolation & purification* / Q000378:metabolism; D005260:Female; D046911:Macromolecular Substances; D008970:Molecular Weight; D006080:Ovarian Follicle / Q000201:enzymology*; D010053:Ovary / Q000201:enzymology*; D011494:Protein Kinases / Q000302:isolation & purification* / Q000378:metabolism; D051381:Rats; D011919:Rats, Inbred Strains; D012741:Sexual Maturation
D046911:Macromolecular Substances; D011494:Protein Kinases; D004264:DNA Topoisomerases, Type I