Functional expression of the intestinal peptide-proton co-transporter in Xenopus laevis oocytes.
The expression of the intestinal peptide-proton cotransporter was examined in Xenopus laevis oocytes by microinjection of poly(A)+ mRNA prepared from rabbit intestinal mucosal cells. The concomitant expression of the glucose-sodium co-transporter was used as the control for the effectiveness of the expression technique. There was significant endogenous activity of Gly-Sar uptake in water-injected oocytes, but the uptake activity increased nearly 3-fold in poly(A)+ mRNA-injected oocytes. The expression of the peptide transporter was time-dependent. There was no detectable expression on day 1 after injection. The expression became noticeable on day 2 and increased with time, reaching a maximum on day 4. There was no further change on days 5 and 6. The endogenous uptake rate measured in water-injected oocytes, on the contrary, showed a slight decrease during this time. The expressed peptide transporter retained its substrate specificity, having affinity for the dipeptides, Gly-Sar and Gly-Pro, and no or little affinity for the free amino acids, Gly and Sar. The expressed peptide transporter also showed a dependence on a transmembrane H+ gradient for maximal activity. These data demonstrate that the mammalian intestinal peptide-proton co-transporter can be successfully expressed in Xenopus laevis oocytes. This expression system can provide an effective assay procedure to clone the gene encoding the transporter.
Miyamoto
Y
Y
Department of Cell and Molecular Biology, Medical College of Georgia, Augusta 30912-2100.
Thompson
Y G
YG
Howard
E F
EF
Ganapathy
V
V
Leibach
F H
FH
D000818:Animals; D001692:Biological Transport; D002352:Carrier Proteins / Q000235:genetics*; D002462:Cell Membrane / Q000378:metabolism; D005947:Glucose / Q000378:metabolism*; D007413:Intestinal Mucosa / Q000378:metabolism; D026901:Membrane Transport Proteins; D010063:Ovum; D011061:Poly A / Q000235:genetics / Q000302:isolation & purification; D011522:Protons; D012333:RNA, Messenger / Q000235:genetics / Q000302:isolation & purification; D011817:Rabbits; D013379:Substrate Specificity; D014982:Xenopus laevis
D002352:Carrier Proteins; D026901:Membrane Transport Proteins; D011522:Protons; D012333:RNA, Messenger; C067802:intestinal peptide-proton cotransporter; D011061:Poly A; D005947:Glucose