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 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 A2 inhibitors. 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 appearance 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.
Paz
C
C
Department of Biochemistry, School of Medicine, University of Buenos Aires, Argentina.
Dada
L A
LA
Cornejo Maciel
M F
MF
Mele
P G
PG
Cymeryng
C B
CB
Neuman
I
I
Mendez
C F
CF
Finkielstein
C V
CV
Solano
A R
AR
Park
M
M
D000302:Adrenal Cortex / Q000378:metabolism; D000595:Amino Acid Sequence; D000818:Animals; D015153:Blotting, Western; D002848:Chromatography, DEAE-Cellulose; D002850:Chromatography, Gel; D002851:Chromatography, High Pressure Liquid; D002852:Chromatography, Ion Exchange; D004591:Electrophoresis, Polyacrylamide Gel; D008928:Mitochondria / Q000378:metabolism; D024101:Mitochondrial Proteins; D008969:Molecular Sequence Data; D008970:Molecular Weight; D010170:Palmitoyl-CoA Hydrolase; D010446:Peptide Fragments / Q000737:chemistry / Q000302:isolation & purification; D011374:Progesterone / Q000096:biosynthesis; D011506:Proteins / Q000737:chemistry / Q000302:isolation & purification* / Q000378:metabolism*; D051381:Rats; D017208:Rats, Wistar; D013256:Steroids / Q000096:biosynthesis*; D013869:Thiolester Hydrolases; D015383:Zona Fasciculata / Q000378:metabolism*
D024101:Mitochondrial Proteins; D010446:Peptide Fragments; D011506:Proteins; D013256:Steroids; D011374:Progesterone; D013869:Thiolester Hydrolases; C546434:Acot2 protein, rat; D010170:Palmitoyl-CoA Hydrolase