We have previously reported the production of a monoclonal antibody (IZAb) which interacts with an antigen, found predominantly in rat adrenal inner zone tissue, which may have a role in steroidogenesis. Here we describe initial studies on its characterization.
Immunoblot analysis of rat adrenocortical proteins obtained from fresh tissue and separated by sodium dodecyl sulphate-polyacrylamide gel electrophoresis, showed that the IZAb interacted with a protein with a molecular mass of approximately 30 000 Da (IZAg1). This protein was found predominantly in rat adrenal inner zone tissue. Small amounts were seen in the zona glomerulosa, while no corresponding protein was seen in rat ovary, heart, liver, testis or kidney tissue. Subcellular fractionation of rat adrenocortical inner zone tissue and immunoblot analysis showed that the IZAg1 was present in the microsomal and mitochondrial fractions of the cell, but was absent from the cytosol. Invivo treatment with ACTH (100 μg/day) for more than 5 days also increased the expression of this protein by rat adrenal inner zone tissue, and this was coincident with increased corticosterone and 18-hydroxydeoxycorticosterone (18-OH-DOC) production in incubations of inner zone tissue in vitro.
In experiments involving the short-term culture of rat adrenal inner zone cells, IZAb interacted with two protein bands. IZAg1 was detected as a minor band in untreated control cells, while another protein with a molecular mass of approximately 60 000 Da, designated IZAg2, was present in greater amounts. Treatment of cells for 48 h with either ACTH (1 μmol/l) or dibutyryl-cAMP (100 μmol/l) resulted in apparent increased expression of IZAg1 and diminished levels of IZAg2. As in the in-vivo treatments, the increase in IZAg1 was associated with a corresponding increase in corticosterone and 18-OH-DOC production.
These findings suggest that the IZAb recognizes a protein (IZAg2) which occurs in unstimulated adrenal cells. On stimulation by steroidogenic agents, this protein becomes processed to yield a smaller protein (IZAg1) which is associated with enhanced adrenal steroidogenesis.