PURIFICATION OF A NOVEL 43-KDA PROTEIN (P43) INTERMEDIARY IN THE ACTIVATION OF STEROIDOGENESIS FROM RAT ADRENAL-GLAND

In previous reports we have demonstrated the presence of a soluble fac tor that responds to cAMP signals to induce steroid synthesis in adren ocortical tissue. Here, we describe the purification of this factor fr om adrenal zona fasciculata cells by using a five-step procedure that includes DEAE-cellulose, gel filtration, Mono Q HPLC and Superose HPLC , and elution of the protein from SDS/PAGE. This procedure results in the purification to homogeneity of a protein of 43-kDa that retains th e capacity to stimulate steroid synthesis in an in vitro recombination assay. This activity is inhibited by the use of phospholipase A, inhi bitors. Antipeptide antibodies against the N-terminal region recognize p43 as a double band on SDS/PAGE that resolves in different spots on two-dimensional gel electrophoresis. Adrenocorticotropin treatment of adrenal glands results in the appearence of multiple spots that migrat ed towards a lower pH compared to controls, suggesting the presence of phosphorylated and dephosphorylated forms of p43. Sequencing of the N -terminal region and internal peptides reveals no significant similari ties with other proteins, suggesting that p43 is a novel protein. We c onclude from our data that the isolated protein (p43) is a novel, solu ble protein that acts as intermediary in adrenocorticotropin-induced s timulation of arachidonic acid release and steroid synthesis.