Glutamate Lab Report

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The aim of this experiment is to investigate the mechanism by which glutamate is transported, and to determine how much of this transport is dependent on sodium ions using a Na+, K-ATPase pump. As part of this practical, we will be looking at an example of secondary active transport (where a solute’s movement against a concentration gradient is tied to the movement of another molecule down its concentration gradient). [4] By investigating glutamate signaling, we will gain a greater understanding of its importance, and the problems that arise when this mechanism fails (excitotoxicity). By investigating the sodium dependent transport of glutamate, we will appreciate its role in the glutamate-glutamine cycle. Glutamate is a fast acting, excitatory neurotransmitter that activates ionotropic and metabotropic Glutamate receptors. It is the main excitatory neurotransmitter in the central nervous system. NMDA, AMPA and Kainate make up the ionotropic receptors activated by glutamate. In the brain, glutamate is the endogenous agonist of these receptors.[5] Glutamate can also be a potent neurotoxin, and…show more content…
Nine Eppendorf tubes were prepared, all of which contained 25μl of the radioactively labeled glutamate: [3H-glutamate]. I added 900μl of Kreb’s solution to the first three tubes, and made note of their incubation times: 0, 5 and 10 minutes respectively. Tube 4 instead contained 800μl of Kreb’s solution, while Tube 5 had 700μl. I then added 100μl and 200μl of D-aspartate to tubes 4 and 5 respectively. 800μl of Kreb’s solution, as well as 100μl of ouabain were added to tube 6. Tubes 4 to 6 had incubation times of 10 minutes. Instead of the Kreb’s HEPES buffer, 900μl of sodium-free Kreb’s solution was added to tubes 7, 8 and 9. Their incubation times were 0, 5 and 10 minutes respectively. I placed any of the tubes that did not require an incubatory period (0 minutes) into ice. The rest of the tubes were pre-incubated for 10 minutes at 25
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