Study Of Established SPT Inhibitor Screening Model By HPLC

Purpose:Serine palmitoyl transferase (SPT) [EC2.3.1.50] is the key enzyme in sphingolipid synthesis. plays a pivotal role in the regulation of sphingolipid metabolism, impact Various components of sphingolipids in vivo synthesis, distribution and function. Cordyceps cicadae is a Chinese traditional medicinal mushroom, which belongs to the class Ascomycetes and DongChongXiaCao group in Chinese herbs, the main active ingredient myriocin is a natural inhibitor of SPT, has strong immunosuppressive activity, but also has anti-atherosclerotic, anti-fungal and other pharmacological effects, such activity may regulate the same target-SPT, SPT find better inhibitors have broad clinical value. this paper has developed a kind of SPT inhibitor screening platform with simple, high precision and accuracy, lay the foundation and technical means for further study.Methods:Rat lung activity of SPT was higher in body, so enzyme source from the rat lungs. to prepare lysates containing enzyme by sonication and centrifuged, substrate reaction, adding the internal standard, liquid-liquid extraction of enzyme reaction products, HPLC determination of content of enzyme reaction in order to establish inhibitor screening platform, Through the inhibition rate of the internal standard method to evaluate inhibitory effect. experimental optimization of the experimental conditions of cracking, extraction, enzymatic reaction steps. we developed a method of determination for Sa (product), Phy (internal standard) with HPLC,based on the pre-column derivatization with OPA.Several factors influencing the buffer salt concentration and pH, OPA volume, ME volume, derivative reaction time, reaction temperature were investigated and optimized. enzyme reaction system of Adding myriocin standard to test the feasibility of the platform, and then by adding several different herbal extracts to a preliminary screening test.Results:The formed derivative was stable for more than 7h at 4℃in refrigerator, The detection wavelength was 230 nm, The der ivatizat ion react ion was performed at 25℃with 2h, The system offered the following analytical parameters: detection limi ts of 0.5μg/ml for Sa (signal-to-noise ratio S/N=3:1), the linear range response was from 3.125—100.0μg ml-1,0.9μg/mL for Phy (S/N=3:1), the linear range response was from 5.0—500. 0μg ml-1. rat lung lysates with Cracking uniform, protein stability, by optimizing the extraction process, different conditions of extraction rate remained at 70.0% to 85.0%, and the supernatant volume of 90μL, The HPLC injection volume 40μL (concentration of substrate: L-serine (200 mM), palmitoyl coenzyme A (5 mM)) for the optimized reaction conditions, in the inhibitor screening test, in addition to Cordyceps cicadae mycelium extracts inhibition rate of 69.20%, other herbal extracts no significant inhibition.conclusion: Experiment established the SPT activity detected by HPLC, On this basis, establishment of the SPT inhibitors screening system in vitro. application of the method were tested 4 of herbal extracts and found that all extracts no significant inhibition. in the future can increase screening efforts in order to find better inhibitors. In this study, based on the reference to foreign literature, for the first time to establish a SPT inhibitor screening system in China for general laboratory, features with high sensitivity, low cost, easy operation, and comparison with isotope labeling method, has advantages of no radiation risk and simple equipment. Carrying out Vigorously screening of new SPT inhibitor has broad prospects, especially, inhibitor from traditional Chinese medicine of obvious effect has a good of the theoretical value and economic value.