Isolation and structural characterization of a cDNA clone encoding rat gastric intrinsic factor.
Rat intrinsic factor (IF) has been purified and proteolytic fragments were sequenced. A cDNA library was constructed from size-enriched gastric poly(A)+ RNA and screened for IF-positive clones by antibody and synthetic oligodeoxynucleotide probe hybridization. An IF clone was isolated and sequenced, revealing a predicted primary amino acid sequence in the coding region of 421 amino acids and a putative signal sequence of 22 amino acids. The primary translation product of IF produced in a cell-free translation system displayed cobalamin (Cbl)-binding activity without proteolytic processing or glycosylation. The amino-terminal region of IF showed significant secondary structural and hydropathic homologies with the nucleotide-binding domain in NAD-dependent oxidoreductases. Alignment of the first 80 residues of IF, following the signal peptide, demonstrated homology with the nucleotide-binding domain of cytoplasmic malate dehydrogenase. Based on these data, we propose a model of IF tertiary structure in which the Cbl-binding domain resides in the NH2-terminal half of the protein.
Dieckgraefe
B K
BK
Department of Medicine, Washington University School of Medicine, St. Louis, MO 63110.
Seetharam
B
B
Banaszak
L
L
Leykam
J F
JF
Alpers
D H
DH
D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D003001:Cloning, Molecular; D003062:Codon; D004247:DNA / Q000235:genetics* / Q000302:isolation & purification; D007437:Intrinsic Factor / Q000235:genetics*; D008969:Molecular Sequence Data; D010295:Parietal Cells, Gastric / Q000378:metabolism; D014176:Protein Biosynthesis; D011487:Protein Conformation; D012333:RNA, Messenger / Q000235:genetics / Q000302:isolation & purification; D051381:Rats
D003062:Codon; D012333:RNA, Messenger; D004247:DNA; D007437:Intrinsic Factor