An investigation into the expression and physiological function of mitochondrial uncoupling proteins

THESIS 7419Our study provides evidence that rat thymus contains a functioning mitochondrial uncoupling protein (UCP 1). Using reverse transcriptase-polymerase chain reaction (RT-PCR), we showed that rat thymus contains UCP 1 mRNA. Antipeptide UCP 1 antibodies, that we show to be specific for UCP 1, detected UCP 1 protein in thymus and thymus cell (i.e. thymocyte) mitochondria. We also show that starvation (48-hour) induced no significant change in the abundance of UCP 1 in thymus and thymocyte mitochondria. Evidence for functional UCP 1 in rat thymus mitochondria was obtained using a radiolabeled binding assay in thymus and BAT mitochondria. UCP 1 in thymus mitochondria binds [3H] GDP with similar binding parameters, Bmax and KD values, as BAT mitochondria. In addition, a large component of the non-phosphorylating oxygen consumption by thymus mitochondria was inhibited by purine nucleotides and activated by nanomolar amounts of fatty-acids, akin to UCP 1 in BAT mitochondria. We also purified UCP 1 from thymus mitochondria by hydroxyapatite chromatography and identified the isolated protein by peptide mass mapping and tandem mass spectrometry using MALDI-TOF and LC-MS/MS, respectively. We conclude that rat thymus contains a functioning UCP 1 protein, which has the capacity to regulate metabolic flux and production and transport of reactive oxygen containing molecules in the thymus