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Biochemical characterization of a high affinity phosphate transporter (PiPT) from root endophyte fungus
Hemant Kumar1, Aayushi Bajaj2, Paras Kumar2
1School of Life Sciences, Jawaharlal Nehru University, New Delhi, 110067, India; Department of Biochemistry and Biophysics, University of California San Francisco, Mission bay, San Francisco, CA, 94158-2517, USA; Bapubhai Desaibhai Patel Institute of Paramedical Science, Charotar University of Science and Technology, Anand, Gujarat, 388421, India.
Abstract:
We have functionally characterized the high-affinity phosphate transporter (PiPT) from the root endophyte fungus Piriformospora indica. PiPT belongs to the major facilitator superfamily (MFS). PiPT protein was purified by affinity chromatography (Ni-NTA) and Size Exclusion Chromatography (SEC). The functionality of solubilized PiPT was determined in detergent-solubilized state by fluorescence quenching and in proteoliposomes. In the fluorescence quenching assay, PiPT exhibited a saturation concentration of approximately 2 μM, at a pH of 4.5. Proteoliposomes of size 121.6 nm radius, showed transportation of radioactive phosphate. Vmax was measured to be 232.2 ± 11 pmol/min/mg protein. We have found Km to be 45.8 ± 6.2 μM suggesting high affinity towards phosphate.
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