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. [...]
[...] The second phase of this experiment should be re-executed and coupled with the first, so that the isoenzymes of AAT could be further localized in specific chloroplast fractions. Methods Our experiment can effectively be divided into two phases: the initial isolation and fractionation of chloroplasts and subsequent analysis using SDS-PAGE; and the assay of AAT using these centrifugation and fractionation techniques coupled with Native-PAGE analysis. The first phase involved the analysis of proteins found in leaves of baby spinach and the second phase studied the differences in AAT between four species: Swiss chard, spinach, basil and romaine lettuce. [...]
[...] Swiss chard contained at least four isoenzymes of AAT, showing that even a single cell can require multiple variations on the same enzyme to complete cellular tasks in different compartments of the cell or in different conditions (such as temperature, pH, substrate concentration, etc.). Similarly, different species contain distinct isoenzymes of AAT while retaining the same function. This relates to the growth conditions of each plant species; a plant cannot change location when encountering changing climate or soil conditions. One strategy plants use to cope with possibly adverse conditions is by having multiple isoenzymes for the same process that activate at different temperatures (caused by climate) or pH (caused by changes in soil composition). [...]
[...] In this manner, we investigated the number of isoenzymes for AAT in Swiss chard and used this information to compare AAT isoenzymes of chard to other species' isoenzymes. The experiment showed that Swiss chard has at least four different isoenzymes of AAT in its whole cell. We say least” because there are four distinct bands in the gel lanes for Swiss chard, but other isoenzymes of AAT may have been in too low concentration to appear in our staining technique. [...]
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