Phorbol ester receptors in bovine luteal cells: relationship to protein kinase C.
We investigated the binding kinetics of the tumor-promoting phorbol ester, phorbol-12,13-dibutyrate (PBt2) to dispersed total bovine luteal cells, purified small luteal cells, and purified luteal protein kinase C (PKC). Saturation analysis and competitive displacement techniques were used. Binding of [3H]PBt2 to total luteal cell preparations resulted in two distinct affinities. The high affinity component was characterized by a Kd of 4.5 +/- 1.5 nM. Analysis of [3H]PBt2 binding to total cells using competitive displacement demonstrated that the low affinity binding was specific and displaceable but dependent on concentrations of [3H]PBt2 far above the Kd for the high affinity binding. In contrast to the total cell preparations, only high affinity binding was observed in intact purified small luteal cells (Kd = 0.96 +/- 0.04 nM). Partial purification of luteal cytosolic PKC by DEAE-Sephadex chromatography resulted in co-elution of PKC enzyme activity and the [3H]PBt2 binding activity. Under conditions of saturating calcium (0.1 mM) and phosphatidylserine (PS) (100 micrograms/tube) concentrations, binding to the partially purified PKC preparation was found to be of a single high affinity and exhibited a Kd (1.3 +/- 0.2 nM) similar to the high affinity binding observed in intact cells. These results suggest that the primary phorbol ester receptor in luteal cells is PKC. However, a low affinity, high capacity [3H]PBt2 binding site also exists within the corpus luteum, either in the large cells or in the accessory cell fraction which consists mainly of endothelial cells.
Dowd
J P
JP
Department of Physiology, NYS College of Veterinary Medicine, Cornell University, Ithaca 14853.
Alila
H W
HW
Hansel
W
W
D000818:Animals; D001667:Binding, Competitive; D029742:Caenorhabditis elegans Proteins; D002118:Calcium / Q000494:pharmacology; D002352:Carrier Proteins; D002417:Cattle; D003338:Corpus Luteum / Q000378:metabolism*; D003600:Cytosol / Q000378:metabolism; D005260:Female; D007700:Kinetics; D008184:Luteal Cells / Q000378:metabolism*; D015240:Phorbol 12,13-Dibutyrate / Q000378:metabolism; D010718:Phosphatidylserines / Q000494:pharmacology; D011493:Protein Kinase C / Q000302:isolation & purification / Q000378:metabolism*; D011955:Receptors, Drug / Q000378:metabolism*
D029742:Caenorhabditis elegans Proteins; D002352:Carrier Proteins; D010718:Phosphatidylserines; D011955:Receptors, Drug; C083141:phorbol ester binding protein; C027776:phorbol ester receptor; D015240:Phorbol 12,13-Dibutyrate; D011493:Protein Kinase C; D002118:Calcium