Molecular cloning and expression of a pituitary gland protein modulating intestinal fluid secretion.
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.
Johansson
E
E
Department of Medical Microbiology, Gothenburg University, Göteborg, Sweden.
Lönnroth
I
I
Lange
S
S
Jonson
I
I
Jennische
E
E
Lönnroth
C
C
D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D015152:Blotting, Northern; D002772:Cholera Toxin / Q000494:pharmacology; D003001:Cloning, Molecular; D017931:DNA Primers; D018076:DNA, Complementary; D004926:Escherichia coli; D005656:Fungal Proteins / Q000737:chemistry; D015870:Gene Expression; D015723:Gene Library; D006801:Humans; D066298:In Vitro Techniques; D007413:Intestinal Mucosa / Q000187:drug effects / Q000378:metabolism*; D008969:Molecular Sequence Data; D009479:Neuropeptides / Q000096:biosynthesis* / Q000737:chemistry / Q000494:pharmacology; D010902:Pituitary Gland / Q000378:metabolism*; D010903:Pituitary Gland, Anterior / Q000166:cytology / Q000378:metabolism*; D051381:Rats; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000737:chemistry / Q000494:pharmacology; D012441:Saccharomyces cerevisiae / Q000378:metabolism; D017386:Sequence Homology, Amino Acid
D017931:DNA Primers; D018076:DNA, Complementary; D005656:Fungal Proteins; D009479:Neuropeptides; D011994:Recombinant Proteins; C049628:antisecretory factor; D002772:Cholera Toxin