Abstract
Antisecretory factor (AF) is a protein known to inhibit intestinal fluid secretion induced by cholera toxin. cDNA clones, expressing immunoreactivity to AF were isolated from a human pituitary gland library and sequenced. The sequence contained 1309 base pairs plus a poly(A) tail; Northern blot analysis of pituitary RNA confirmed this size. One large open reading frame was found to code for 382 amino acids. The protein was expressed in pGEX-lambda 1T/Escherichia coli and purified. The recombinant AF was extremely potent, 9 ng (2.10(-13) mol), giving a significant antisecretory activity against cholera toxin-induced fluid secretion in rat. Antiserum against recombinant AF was used in immunohistochemical and Western blot analysis. Sections from human pituitary glands manifested specific intracellular staining in cells exclusively located in the anterior part. Both recombinant AF and AF extracted from pituitary gland appeared in SDS-polyacrylamide to have a molecular mass of 60 kDa, although the renal value was 41 kDa. The protein sequence manifested homology (29% identity) with one protein, a putative Saccharomyces cerevisiae 30-kDa protein of unknown function.
Highlights
From the Departments of :r,Medical Microbiology and Immunology, 'fIClinical Bacteriology, IlAnatomy and Cell Biology, and **Surgery, Gothenburg University, Guldhedsgatan 10, S-41346 Goteborg, Sweden
Antisecretory factor (AF) is a protein known to inhibit intestinal fluid secretion induced by cholera toxin. eDNA clones, expressing immunoreactivity to AF were isolated from a human pituitary gland library and sequenced
We describe the cloning and sequencing of eDNA coding for a protein with the same immunoreactivity as AF
Summary
From the Departments of :r,Medical Microbiology and Immunology, 'fIClinical Bacteriology, IlAnatomy and Cell Biology, and **Surgery, Gothenburg University, Guldhedsgatan 10, S-41346 Goteborg, Sweden. Sections from human pituitary glands manifested specific intracellular staining in cells exclusively located in the anterior part. Both recombinant AF and AF extracted from pituitary gland appeared in SDS.polyacrylamide to have a molecular mass of 60 kDa, the real value was 41 kDa. The protein sequence manifested homology (29% identity) with one protein, a putative Saccharomyces cerevisiae 30-kDa protein of unknown function. A large number of regulatory peptides occur both in the gastrointestinal tract and in the central nervous system where they are predominantly confined to the nerves themselves or to endocrine cells [1, 2] Some of these peptides affect the transport of water and electrolytes across the intestinal mucosa. The nucleotide sequencers) reported in this paper has been submitted to the GenBankTM I EMBL Data Bank with accession numberis) U24704
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