Characterization of cloned rat liver Na(+)-bile acid cotransporter using peptide and fusion protein antibodies.
A cDNA encoding a rat liver Na(+)-bile acid cotransporter (Ntcp) has recently been cloned (Hagenbuch, B., B. Steiger, M. Fouget, H. Lubbert, and P. J. Meier. Proc. Natl. Acad. Sci. USA 88: 10629, 1991) using expression cloning in Xenopus laevis oocytes. Although the open reading frame coded for a protein of 39 kDa, in vitro translation experiments produced a 35-kDa protein which increased to a product of 41 kDa after glycosylation by pancreatic microsomes. To more clearly characterize the native protein in rat liver, we have raised antipeptide and anti-fusion protein antibodies to the COOH-terminal part of the cloned transporter. On Western blot analysis both antisera but not preimmune serum specifically detected a protein of approximately 50 kDa in isolated rat liver basolateral plasma membranes (BLPM). The reactivity was abolished when the antiserum was preincubated with the synthetic alpha-337 peptide. Deglycosylation of BLPM with N-glycanase followed by antibody probing led to decrease of the molecular mass to 34.5 kDa, suggesting that the protein is N-glycosylated in vivo. Two-dimensional immunoblotting indicated that the Ntcp protein had an isoelectric point of approximately 6.0. The antibody did not react with any proteins in rat ileal and kidney cortex brush-border membranes, human liver basolateral plasma membranes, or rat hepatoma tissue culture cell homogenates. Immunofluorescence localization studies with both antibodies revealed specific staining of the sinusoidal membrane domain but not of intracellular or bile canalicular membranes. Moreover, there was no acinar gradient in the pattern of staining.(ABSTRACT TRUNCATED AT 250 WORDS)
Ananthanarayanan
M
M
Department of Pediatrics, Yale University School of Medicine, New Haven, Connecticut 06510.
Ng
O C
OC
Boyer
J L
JL
Suchy
F J
FJ
D000818:Animals; D000906:Antibodies / Q000276:immunology*; D001483:Base Sequence; D015153:Blotting, Western; D002352:Carrier Proteins / Q000737:chemistry* / Q000235:genetics* / Q000378:metabolism; D003001:Cloning, Molecular; D005455:Fluorescent Antibody Technique; D006801:Humans; D007106:Immune Sera; D015151:Immunoblotting; D008099:Liver / Q000378:metabolism; D008297:Male; D015335:Molecular Probes / Q000235:genetics; D008969:Molecular Sequence Data; D029363:Organic Anion Transporters, Sodium-Dependent; D010446:Peptide Fragments / Q000276:immunology*; D051381:Rats; D017207:Rats, Sprague-Dawley; D011993:Recombinant Fusion Proteins / Q000276:immunology*; D027981:Symporters
D000906:Antibodies; D002352:Carrier Proteins; D007106:Immune Sera; D015335:Molecular Probes; D029363:Organic Anion Transporters, Sodium-Dependent; D010446:Peptide Fragments; D011993:Recombinant Fusion Proteins; D027981:Symporters; C071908:sodium-bile acid cotransporter