Open AccessPurification of a Novel 43‐kDa Protein (p43) Intermediary in the Activation of Steroidogenesis from Rat Adrenal Gland
Author(s) -
Paz Cristina,
Dada Laura A.,
Maciel M. Fabiana Cornejo,
Mele Pablo G.,
Cymeryng Cora B.,
Neuman Isabel,
Mendez Carlos F.,
Finkielstein Carla V.,
Solano Angela R.,
Park Minkyu,
Fischer Wolfgang H.,
Towbin Harry,
Scartazzini Ricardo,
Podestá Ernesto J.
Publication year - 1994
Publication title -
european journal of biochemistry
Language(s) - English
Resource type - Journals
eISSN - 1432-1033
pISSN - 0014-2956
DOI - 10.1111/j.1432-1033.1994.00709.x
Subject(s) - steroid , arachidonic acid , stimulation , biology , in vitro , gel electrophoresis , biochemistry , microbiology and biotechnology , size exclusion chromatography , chemistry , endocrinology , enzyme , hormone
In previous reports we have demonstrated the presence of a soluble factor that responds to cAMP signals to induce steroid synthesis in adrenocortical tissue. Here, we describe the purification of this factor from 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 the capacity to stimulate steroid synthesis in an in vitro recombination assay. This activity is inhibited by the use of phospholipase A 2 , inhibitors. Antipeptide antibodies against the N‐terminal region recognize p43 as a double band on SDSPAGE that resolves in different spots on two‐dimensional gel electrophoresis. Adrenocorticotropin treatment of adrenal glands results in the appearence of multiple spots that migrated 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 similarities with other proteins, suggesting that p43 is a novel protein. We conclude from our data that the isolated protein (p43) is a novel, soluble protein that acts as intermediary in adrenocorticotropin‐induced stimulation of arachidonic acid release and steroid synthesis.