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Isoenzymes of aspartate aminotransferase

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  1. Introduction.
  2. Results.
  3. Discussion.
  4. Methods.
  5. Conclusion.

The continued production of protein-based medicines and chemically-engineered enzymatic products necessitates further information about protein structure and function. An enzyme in a cell can have many functions or loci. To account for slight differences in reaction products or locations, an enzyme can exhibit different forms, or isoenzymes.1 One such enzyme, and an enzyme that is widely studied and documented, is aspartate aminotransferase (AAT). AAT has a wide variety of uses in a cell and is highly conserved across kingdoms. In fact, scientists have found thermophiles with AATs that can function normally up to 80 oC that contain the same active-site sequences as AATs that function normally at room temperature. AAT catalyses transamination in important Krebs' Cycle intermediates. This particular isoenzyme of AAT is therefore important in the proper function of metabolic respiration and in the energetic needs of organisms.

[...] Our research attempts to study differences in protein composition among cellular components, and to use this data as a building block to discovering the amount of AAT isoenzymes in a given plant species. We will use the techniques of differential and equilibrium density centrifugation, SDS-PAGE and Native-PAGE assay to explore this topic. Prominent research in our field has shown that these techniques are useful in isolating cellular organelles, such as chloroplasts, and of further isolating specific proteins.4 In research by Schultz et al., Native-PAGE assay has been used to separate isoenzymes in plant species such as Arabidopsis thaliana, consequently determining specific loci and functionality of said isoenzymes. [...]

[...] A small portion of the supernatant was saved and labeled containing all the proteins that exist in the chloroplast stroma, intermembrane space and thylakoid lumen. The pellet was resuspended in lysis buffer to wash off any remaining soluble protein. This resuspension was divided equally into three small centrifuge tubes. After a centrifugation at 2000rcf at 4 oC for 2 minutes, the supernatants for these three tubes were discarded. One tube was resuspended with lysis buffer and labeled ?Membrane? to be run later in SDS- PAGE. [...]

[...] We determined a loading strategy and incorporated the samples from other groups as well (spinach, basil, and romaine lettuce) into our gel. A consistent volume of sample was loaded into each well. The gel was stained in a Tupperware stain-bath overnight on a shaker, then photographed almost 24 hours later. Literature Cited 1. Kwok E. Lab instructions. 9/27/06, 9/20/06, 9/13/ Okamoto Kato Masui Yamagishi Oshima T & Kuramitsu A. An aspartate aminotransferase from an extremely thermophilic bacterium, Thermus thermophiles. J Biochem (Tokyo) 119: 135- Kirsch JF, Eichele Ford GC, Vincent MG, Jansonius JN, Gehring H & Christen P. (1984) [...]

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