Encyclopedia of Experiments: Biology
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Transcript
Amino acid transporters are membrane-bound proteins that mediate amino acid transfer across cell membranes, regulating vital cellular processes.
To study amino acid uptake in vitro, take a culture of mouse bone marrow-derived stromal cells. Aspirate the growth medium. Add a pre-warmed buffer supplemented with glutamine — an amino acid — labeled with radioactive hydrogen. Incubate for the required duration.
The pre-warmed buffer provides physiological temperature and an appropriate pH required for the cellular uptake of glutamine. Glutamine transporters consist of transport domains — responsible for substrate translocation via sodium ion-coupled co-transport — and membrane-anchored scaffold domains.
The transport domain binds to radiolabeled glutamine along with sodium ions from the buffer. Upon binding, it releases the molecules into the cytoplasm. Simultaneously, the transport domain translocates intracellular sodium ions and neutral amino acids outside the cell, resulting in cellular homeostasis.
Wash the cells with ice-cold buffer, terminating the reaction and removing any extracellular radiolabeled glutamine. Add a detergent solution. The detergent lyses the cells, causing the release of intracellular radiolabeled glutamine.
Centrifuge the lysate. Add the glutamine-containing supernatant into a vial with a scintillation cocktail, and place the vial inside a scintillation counter.
The radiolabeled glutamine molecules emit high-energy beta particles. The scintillators in the cocktail absorb the particles and emit light pulses that are detected by the scintillation counter.
Measure the radioactivity to estimate the cellular glutamine uptake.
