Purification by ceftibuten-affinity chromatography and the functional reconstitution of oligopeptide transporter(s) in rat intestinal brush-border membrane.
The transport activity of ceftibuten, a dianionic peptide-like compound, was extracted from rat intestinal brush-border membrane by n-octylglucoside and reconstituted into asolectin liposomes by dialysis. The proteoliposomes prepared from the membrane extract showed an inward H+-gradient-dependent uptake of ceftibuten and glycylsarcosine. Ceftibuten-immobilized affinity chromatography of the membrane extract permitted the isolation of two polypeptides (apparent molecular mass of 117 and 127 kDa) that can recognize the dianionic peptide structure of ceftibuten. Proteoliposomes prepared from reconstituting the isolated proteins into asolectin vesicles showed an overshooting uptake of ceftibuten in the presence of an inwardly directed H+ gradient, and this uptake could be inhibited by L-valyl-L-proline. N-glycanase digestion of the isolated proteins, 117 and 127 kDa, trimmed them into 78 and 120 kDa products, respectively. The protein core size of the smaller protein was in agreement with the calculated molecular mass of approximately 79 kDa for the rat PepT1 transporter obtained by other investigators.
Iseki
K
K
Department of Pharmacy, Hokkaido University Hospital, School of Medicine, Hokkaido University, Kita-14-jo, Nishi-5-chome, Sapporo 060, Japan.
Yonemura
K
K
Kikuchi
T
T
Naasani
I
I
Sugawara
M
M
Kobayashi
M
M
Kohri
N
N
Miyazaki
K
K
D000818:Animals; D001692:Biological Transport; D002352:Carrier Proteins / Q000302:isolation & purification* / Q000378:metabolism; D000077722:Ceftibuten; D002511:Cephalosporins / Q000378:metabolism*; D002846:Chromatography, Affinity; D004591:Electrophoresis, Polyacrylamide Gel; D007413:Intestinal Mucosa / Q000737:chemistry* / Q000378:metabolism / Q000648:ultrastructure*; D007583:Jejunum / Q000737:chemistry; D008081:Liposomes / Q000737:chemistry / Q000378:metabolism; D008297:Male; D008565:Membrane Proteins / Q000302:isolation & purification / Q000378:metabolism; D008871:Microvilli / Q000378:metabolism; D051381:Rats; D017208:Rats, Wistar; D012995:Solubility
D002352:Carrier Proteins; D002511:Cephalosporins; D008081:Liposomes; D008565:Membrane Proteins; D000077722:Ceftibuten