In Vitro SSAT Enzyme Activity Study Using Rat and Mouse Liver Tissue Preparations
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Polyamines such as spermidine (Spd) and spermine (Spm) are the organic compounds which have two or more amino groups. It serves multiple physiological functions including cell proliferation, cell adhesion, specific signalling and the repairing of extracellular matrix. Spermidine/spermine N1-acetyltransferase (SSAT) is the enzyme which acetylates Spd to acetylspermidine (AcSpd). SSAT is also capable of acetylating polyamine analogues such as triethylenetetramine (TETA) to acetyltriethylenetetramine (MAT). However, this process is debatable because while the overexpression of Spermidine/spermine N1-acetyltransferase (SSAT1) increases the MAT level, the knockout of SSAT1 remains the same MAT level as the non treatment groups. One of the theories explaining this is that another enzyme, Spermidine/spermine N1-acetyltransferase 2 (SSAT2), is responsible for the acetylation of TETA because the knockout of SSAT2 significantly decreases the MAT level. Thus, this research was designed to establish a method to precisely measure the activity of SSAT via the quantification of the metabolites resulting from SSAT catalysis by using spermidine-D6 (Spd-D6), the stable isotope of Spd, to get rid of the disturbance from endogenous Spd. Through this method, it is possible to find out if SSAT2 is really active in acetylating TETA by comparing the data of Spd-D6 with TETA. It is required to be mentioned that although SSAT in vitro polyamine acetylation assay has been done by some studies already, the application of Spd-D6 is unprecedented. This research first of all used differential centrifugation method and Ca2+ sediment centrifugation method to prepare mouse and rat liver microsome and cytosol, which are also considered as the enzyme sources of SSAT enzyme. Then Bradford Protein Assay was used to determine the protein concentrations of these liver preparations. After that, in vitro polyamine acetylation assay was applied to measure the SSAT enzyme activity by using Spd-D6 as the substrate. Finally, this research developed a liquid chromatography-mass spectrometry (LC-MS) 10 method to detect the metabolites, acetylspermidine-D6 (AcSpd-D6). This research not only measured the acetylation activity of enzyme from different tissue origin with various polyamine substrates, but also compared them with the results of different non-enzymatic controls. This research also compared the standard curves of AcSpd-D6, AcSpd and MAT in different matrix including microsome, cytosol and water to find out their effects on LC-MS detection. The results presented that non-enzymatic controls were all showing polyamine acetylation activities, and such activities were similar to the enzymatic groups, which suggested the incapability of SSAT acetylating polyamines. Standard curves were not showing obvious differences, which means the enzyme sources were not influencing the LC-MS detection. In conclusion, spermidine acetylation happens spontaneously without needing SSAT catalysis. The role of SSAT may be: 1) SSAT inhibits AcCoA from reacting with other substances. 2) SSAT accelerates the transportation of AcCoA from mitochondria to the cytoplasmic matrix. 3) SSAT is a carrier protein which transports AcCoA to other cellular locations.