Purpose: The first purpose of this project is to determine if Pem has DNA-binding properties. To determine Pem's function, the second purpose of this project to determine the expression pattern of Pem during development. To clarify how the mCAT proteins affect aa transport, the third purpose of this project is to characterize arginine transport in SL12.3 and SL12.4 cells.
Methods: Affinity chromatography and the electrophoretic mobility shift assay (EMSA) were used to detect Pem-DNA interactions. The presence of Pem during late embryonic and early postnatal development was detected by immunohistochemistry. To assay arginine uptake in SL12 cells, a suspension of cells was incubated with ³H-arginine, and the cell lysate was assayed for ³H.
Results: An EMSA did not reveal a GST-Pem-DNA complex. Also, the lack of enrichment of Pem-binding DNA during affinity chromatography indicated a lack of specific Pem-DNA interactions. Pem is detected in the nuclei of placental cells but not in other tissues of developing mice (17 dpc to 18 days old). Arginine transport in SL12.3 is a Na+ - independent, lysine inhibitable, and non-leucine inhibitable.
Conclusion: The hypothesis that Pem binds DNA was disproven. The data suggest that Pem may require cofactors to bind DNA. The hypothesis that pem expression is seen in invasive tissues was supported by the detection of Pem in the placenta. Pem is nuclear localized, indicating that it may be a transcriptional regulator of metastatic or angiogenic events. As hypothesized, arginine transport in SL12.3 cells is mediated primarily by mCAT-1. Future studies will address the issue of mCAT-2 expression in Sl12.4 cells.
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