Topological photoaffinity labeling of the rabbit ileal Na+/bile-salt-cotransport system.
For the investigation of the topology of the rabbit ileal Na+/bile-salt-cotransport system, composed of a 93-kDa integral membrane protein and a peripheral 14-kDa bile-acid-binding protein (ILBP), we have synthesized photolabile dimeric bile-salt-transport inhibitors (photoblockers), G1-X-G2, where two bile acid moieties (G1 and G2) are tethered together via a spacer, X, and where one of the two bile acid moieties carries a photoactivatable group. These photoblockers specifically interact with the ileal Na+/bile-salt-cotransport system as demonstrated by a concentration-dependent inhibition of [3H]cholyltaurine uptake by rabbit ileal brush-border membrane vesicles and by inhibition of photolabeling of the 93-kDa and 14-kDa bile-salt-binding proteins by 7,7-azo and 3,3-azo derivatives of cholyltaurine. Ileal bile-salt uptake was specifically inhibited by the photoblockers, which were not taken up themselves by the small intestine as demonstrated by in vivo ileal perfusion. Dependent on the photoblocker used several polypeptides in the molecular-mass range of 14-130 kDa were labeled. The cytoplasmically attached 14-kDa ILBP was significantly labeled only by inhibitors that are photoactivatable in bile acid moiety G1, suggesting that during binding and translocation of a bile-salt molecule by the ileal bile-salt-transport system the steroid nucleus gets access to the cytoplasmic site of the ileal brush-border membrane first. Photoaffinity labeling in the frozen state with the transportable 3,3-azo and 7,7-azo derivatives of cholyltaurine revealed a time-dependent increase in the extent of labeling of the 14-kDa and 93-kDa proteins, suggesting a labeling of these proteins from the cytoplasmic site of the ileal brush-border membrane. By photoaffinity labeling in the frozen state with the various photoblockers time-dependent changes in the extent of photoaffinity labeling of bile-salt-binding proteins were observed, demonstrating the possibility of topological analysis of the rabbit ileal Na+/bile-salt-cotransport system.
Kramer
W
W
Hoechst Marion Roussel, DG Metabolic Diseases, Frankfurt am Main, Germany.
Wess
G
G
Bewersdorf
U
U
Corsiero
D
D
Girbig
F
F
Weyland
C
C
Stengelin
S
S
Enhsen
A
A
Bock
K
K
Kleine
H
H
Le Dreau
M A
MA
Schäfer
H L
HL
D000345:Affinity Labels; D000818:Animals; D001647:Bile Acids and Salts / Q000737:chemistry / Q000378:metabolism; D001692:Biological Transport; D002352:Carrier Proteins / Q000302:isolation & purification / Q000378:metabolism*; D007082:Ileum; D007413:Intestinal Mucosa / Q000378:metabolism*; D007700:Kinetics; D008297:Male; D008871:Microvilli / Q000378:metabolism*; D015394:Molecular Structure; D008970:Molecular Weight; D029363:Organic Anion Transporters, Sodium-Dependent; D011817:Rabbits; D027981:Symporters
D000345:Affinity Labels; D001647:Bile Acids and Salts; D002352:Carrier Proteins; D029363:Organic Anion Transporters, Sodium-Dependent; D027981:Symporters; C071908:sodium-bile acid cotransporter