Isolation of a cDNA encoding a taurine transporter in the human retinal pigment epithelium
作者:
MiyamotoYusei,
LiouGregory I.,
SprinkleTerry Joe,
期刊:
Current Eye Research
(Taylor Available online 1996)
卷期:
Volume 15,
issue 3
页码: 345-349
ISSN:0271-3683
年代: 1996
DOI:10.3109/02713689609007631
出版商: Taylor&Francis
关键词: cloning;human retinal pigment epithelium;oocyte expression;reverse transcription-polymerase chain reaction (RT-PCR);taurine transporter
数据来源: Taylor
摘要:
Reverse transcription-polymerase chain reaction (RT-PCR) was performed to amplify a cDNA encoding a taurine transporter in the human retinal pigment epithelium (HRPE). The coding region of a PCR product was found to be 1863 bp long, predicting a 620-amino acid protein (69,826 Da). This cDNA sequence is almost identical to those taurine transporters recently determined in the human thyroid and placenta: 12 and 1 base pair(s) different from the reported thyroid and placenta transporter clones, respectively. The injection of mRNAin vitrotranscribed from the PCR product markedly increased taurine uptake inXenopus laevisoocytes. Taurine uptake is Na+and Cl-dependent. Unlabeled taurine,β-alanine andγ-amino-n-butyric acid at 100μM inhibited the uptake of radiolabeled taurine whereas 100μMα-alanine andα-aminoisobutyric acid did not. A kinetic study showed that taurine uptake is mediated by a single carrier system with the apparent Michaelis-Menten constant of approximately 2μM. These results suggest that the PCR product encodes a functional taurine transporter whose characteristics are similar to those of taurine uptake observed in the original HRPE cells. A DNA encoding the reported placental transporter was made from the PCR product by site-directed mutagenesis but it was not functional in the oocyte expression. A similar RT-PCR was performed with poly (A)+mRNA isolated from JAR human placenta choriocarcinoma cells. This PCR product was identical to that from the HRPE. In addition, the clone of the human thyroid transporter was obtained and re-sequenced. Its translation coding region was also identical to that of the PCR product from the HRPE, showing that taurine transporters are identical in the human RPE, thyroid and placenta.
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