Purification and characterization of the antisecretory factor: a protein in the central nervous system and in the gut which inhibits intestinal hypersecretion induced by cholera toxin

Abstract

The antisecretory factors (ASF) are hormone-like proteins which inhibit cholera toxin-induced intestinal hypersecretion. Although ASF concentrations in young control rats were low, those in old control rats and toxin-treated rats were high. Toxin-treated rats had 200 ED50 units/g wet weight of ASF in the pituitary gland, while their intestinal mucosa, bile and milk contained 3, 0.5 and 0.5 units/g. In adult man and in 8-9-month-old pig the pituitary level was about 20 units/g. The isoelectric points of ASF from pig and rat were 4.8 and 5.0, respectively, while the molecular size as determined by gel filtration on Bio-Gel P-150 was the same in both cases (Kav 0.43). The molecular weight as determined by SDS-polyacrylamide gel electrophoresis was 60,000 for ASF from porcine pituitary gland. One ED50 unit of the purified porcine ASF corresponded to about 10(-13) mol (1-5 ng) of protein. There were two different ASF from human pituitary gland: pI 5.2, Kav 0.43; and pI 4.5, Kav 0.6. Since antibodies against porcine ASF failed to neutralize the latter protein, it may be unrelated to porcine ASF; the human pI 5.2-protein and rat ASF were both neutralized, but less effectively than was porcine ASF. All the ASF molecules attached to agarose gel, from which they dissociated again in methyl alpha-D-glucose: porcine and rat ASF were eluted at 0.3-0.9 M methyl alpha-D-glucose, human pI 5.2-ASF at 0.1-0.9 M, and human pI 4.5-ASF at 0.1-1.5 M methyl alpha-D-glucose.