Isozymes of cAMP-dependent protein kinase present in the rat corpus luteum.
Regulatory (R) subunits and their association with catalytic subunits to form cAMP-dependent protein kinase holoenzymes were investigated in corpora lutea of pregnant rats. Following separation by DEAE-cellulose chromatography, R subunits were identified by labeling with 8-N3[32P]cAMP and autophosphorylation on one and two-dimensional gel electrophoresis and by reactivity with antisera. DEAE-cellulose elution of R subunits with catalytic subunits as holoenzymes or without catalytic subunits was determined by sedimentation characteristics on sucrose density gradient centrifugation and by cAMP-stimulated kinase activation characteristics on Eadie-Scatchard analysis. We identified the presence of a type I holoenzyme containing RI alpha (Mr 47,000) subunits, a prominent type II holoenzyme containing RII beta (Mr 52,000) subunits, and a second more acidic type II holoenzyme peak containing both RII beta and RII alpha (Mr 54,000) subunits. However, the majority of total R subunit activity was associated with a catalytic subunit-free peak of RI alpha protein which on elution from DEAE-cellulose was associated with cAMP. This report establishes the more basic elution position from DEAE-cellulose of the prominent rat luteal RII beta holoenzyme in very close proximity to free RI alpha and presents one of the few reports of a normal tissue containing a large percentage of catalytic subunit-free RI alpha.
Hunzicker-Dunn
M
M
Department of Cell, Molecular and Structural Biology, Northwestern University Medical School, Chicago, Illinois 60611.
Cutler
R E
RE
Maizels
E T
ET
DeManno
D A
DA
Lamm
M L
ML
Erlichman
J
J
Sanwal
B D
BD
LaBarbera
A R
AR
D000818:Animals; D002499:Centrifugation, Density Gradient; D002848:Chromatography, DEAE-Cellulose; D003338:Corpus Luteum / Q000201:enzymology*; D000242:Cyclic AMP / Q000378:metabolism; D004591:Electrophoresis, Polyacrylamide Gel; D005260:Female; D007527:Isoenzymes / Q000302:isolation & purification / Q000378:metabolism*; D007700:Kinetics; D046911:Macromolecular Substances; D008970:Molecular Weight; D011247:Pregnancy; D011494:Protein Kinases / Q000302:isolation & purification / Q000378:metabolism*; D051381:Rats; D011919:Rats, Inbred Strains
D007527:Isoenzymes; D046911:Macromolecular Substances; D000242:Cyclic AMP; D011494:Protein Kinases