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Size selected mRNA induces expression of P-aminohippurate transport in Xenopus oocytes
Abstract:
Xenopus oocytes were injected with size-fractionated mRNA isolated from the renal cortex of rabbit kidney and after 4 days incubation, PAH uptake in oocytes injected with mRNA (0.7-1.3 kb) was 8 to 45 fold that of the water injected controls. The oocyte to medium ratio of accumulated PAH was 1.95. The Km and Vmax for transport were 333 microM and 66.6 nmoles.oocyte-1.min-1, respectively. This Km is similar to that reported for PAH transport in intact kidneys and slices. The uptake of PAH was unaffected by the absence of Na+ or the presence of probenecid. Expression of the transport represents the first step in an effort to clone and identify the gene for PAH transport.
Insights
Researchers identified mRNA in rabbit kidney that enables Xenopus oocytes to transport para-aminohippuric acid (PAH). This finding is a key step toward cloning the gene responsible for PAH transport.
Area of Science:
- Molecular Biology
- Renal Physiology
- Xenopus Oocyte Expression Systems
Background:
- Para-aminohippuric acid (PAH) is a crucial substance handled by the kidneys.
- Understanding the molecular mechanisms of PAH transport is vital for renal physiology research.
- Identifying the specific gene responsible for PAH transport remains a challenge.
Purpose of the Study:
- To investigate the potential of rabbit kidney mRNA to direct the synthesis of functional PAH transporters in Xenopus oocytes.
- To characterize the kinetic properties of PAH transport mediated by expressed mRNA.
- To lay the groundwork for cloning the gene encoding the PAH transporter.
Main Methods:
- Isolation and size-fractionation of mRNA from rabbit renal cortex.
- Injection of fractionated mRNA into Xenopus oocytes.
- Incubation of injected oocytes and measurement of para-aminohippuric acid (PAH) uptake.
- Kinetic analysis of PAH transport (Km and Vmax determination).
- Assessment of transport dependency on sodium (Na+) and inhibition by probenecid.
Main Results:
- mRNA fractions (0.7-1.3 kb) significantly increased PAH uptake in oocytes (8-45 fold compared to controls).
- The oocyte to medium ratio for accumulated PAH reached 1.95.
- Determined kinetic parameters: Km of 333 microM and Vmax of 66.6 nmoles/oocyte/min.
- PAH uptake was independent of Na+ and unaffected by probenecid.
- These results indicate successful expression of a functional PAH transporter.
Conclusions:
- Rabbit kidney mRNA encodes factors capable of mediating PAH transport in Xenopus oocytes.
- The characterized transport kinetics are consistent with those observed in native kidney tissues.
- This study represents a significant advancement in the effort to clone and identify the gene responsible for renal PAH transport.