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Abstract
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.
Highlights
The expression of the intestinal peptide-proton co- border membrane Na’-H+ exchanger, produces a transmemtransporter was examined in Xenopus laevis oocytes brane H+ gradient across the membrane which is necessary by microinjection of poly(A)+mRNA preparedfrom for the invivo energization of the transport system [10,11,12]
Absorption of peptides across theintestinalepithelium is mediated by a specific transport system localized inthebrush-bordermembrane, and this system is distinct from the transport systems described for free amino acids [3, 4].The characteristicsof the peptide transporter have been studied extensively using isotransporter [21],and the transport systemsfor bile acid [22] and inorganic phosphate and sulfate [23, 24]
We describe the functionalexpression of the intestinal peptide transporter in Xenopus oocytes following injection of exogenous poly(A)+ mRNA isolatefdrom rabbit intestinalmucosal cells
Summary
The expression of the intestinal peptide-proton co- border membrane Na’-H+ exchanger, produces a transmemtransporter was examined in Xenopus laevis oocytes brane H+ gradient across the membrane which is necessary by microinjection of poly(A)+mRNA preparedfrom for the invivo energization of the transport system [10,11,12]. 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-injectedoocytes. We describe the functionalexpression of the intestinal peptide transporter in Xenopus oocytes following injection of exogenous poly(A)+ mRNA isolatefdrom rabbit intestinalmucosal cells.
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