Molecular cloning and expression of rat antisecretory factor and its intracellular localization

Antisecretory factor (AF) was identified as a pituitary protein that inhibi ts the intestinal fluid secretion induced by cholera toxin. One aim of this study waste elucidate whether AF is also synthesized in the intestine or i f AF produced in the pituitary is transported to the intestinal tract for i ts function there, cDNA clones encoding a protein proposed to be AF were is olated from rat pituitary gland and intestinal mucosa cDNA libraries. The n ucleotide sequences of clones isolated from the rat pituitary gland and int estinal mucosa were identical. The deduced amino acid sequence was highly h omologous to the sequence for Subunit 5a of the human 26S protease that exi sts abundantly in the cytosol and nucleus. The production of AF in the inte stine was confirmed by Northern blot and immunoblot analyses. Immunocytoche mical observations of cells transfected with the rat AF cDNA showed that th e AF protein was localized in the cytoplasm. These findings suggest that th e protein proposed to be AF may be a cytoplasmic protein, it exists in the intestine rather than being transported from the pituitary gland, and it ma y function in intestinal cells.