NH₃ Passive Transport And Ca²⁺ Active Transport Across Artificial Cell Membrane

Passive transport and active transport are important ways for cell extracting nutrients.Some disease would be caused due to the abnormal transport and developing drugs to regulate are the treatment strategy.Due to the complexity of cell,it is hard to study the function of only one kind of mass transport.Artificial cell models provide good tools for this issue.Up to now,the most popular artificial cell models include cell-sized emulsion,and liposome.In this study,both of emulsion and giant liposome were used as artificial cell models.The passive transport of ammonia was studied in emulsion model,and this model was used to remove colonic ammonia to prevent hepatic encephalopathy（HE）.Ca²⁺ active transport was studied in giant liposome model.Electric attraction force was suggested to induce fusion of giant liposome with proteoliposome for the incorporation of Ca²⁺ pumping protein SERCA and this model was used to study the regulation of compound on SERCA.This method provides a new approach for membrane protein with low stability and high MW.And giant liposome was used to measure the Ca²⁺ pumping activity of SERCA,which provides a new evaluation method for new drugs.The abnormal NH₃ transport is the direct cause of Hepatic Encephalopathy（HE）.Aimed at the lack of effective and little side effects drug for HE,emulsion model was built to study NH₃ passive transport,and absorb NH₃ in colon to prevent HE.Citric acid loaded W1/O/W2 with encapsulation more than 95%was prepared and polymer surfactant Abil EM90 was suggested to limit the formation of reverse micelles,reduce the water transport and improve the stability of emulsion（17%citric acid was released within 8h）and encapsulation.In vitro,90%ammonia was removed within 1.5 h5 among which 70%was absorbed into W]and 20%was netrulized by citric released from W1.In vivo,high dose emulsion has similar performance on the decreasing of blood ammonia with lactulose,and has little effect on the water content in faeces.Giant liposome is the idea model to study Ca²⁺ active transport.Solution with a certain salinity is necessary to maintain the activity of membrane protein,while giant liposome could not be formed in such high salinity solution via electro format ion assay.To solve this problem,surfactant triton x-100 was added in giant lipsome formation to make lipid membrane ion permeable and absorbed by bio bead SM-2 after ion balance to recover its ion impermeability after ionic balance.Giant liposome with diameter 10-100 μm was electroformed.The effect of salt such as KC1 and MgCl2 was studied.Giant liposome could not form in the buffer containing over 10mM KCl or 5mM MgCl2.Triton x-100（<200 μM）has little or no effect on the formation of giant liposome,and it would insert in the lipid bilayer of giant liposome and make it permeable for ion instead of macromolecular Fluo-5f.Fluo-5f was encapsulated in giant liposome via absorption by Bio beads SM-2.Ca²⁺ pumping protein SERCA（110kD）is the main protein channel.SERCA was purified from rabbit muscle.Green fluorescent probe FITC was used to label SERCA to make it visualable.When the mole ratio FITC/SERCA=10:1,a SERCA could be labeled with 2.76 FITC moleculars after 1h incubation at room temperature.For the pure SERCA,membrane filtration and dialysis and desalt column were used to remove unbound FITC.About 94%free FITC could be removed by Desalt column and 75-80%SERCA was still left.For the SERCA sample in nano sized liposome,about 96%free FITC could be removed by ultrocentrifugation,and 50%SERCA was left,and more importantly,the ratio of lipid and SERCA in nano sized liposome is unchanged.Due to the high MW and complicate structure,SERCA is hard to be incorporated into giant liposome via the current methods.To solve this problem,electrostatic attraction was suggested to induce giant liposome fuse with protein intensituted nano sized liposome for the incorporation of protein in giant liposome.SERCA was incorporated in nano sized liposome by detergent mediated assay and nano sized liposome was nagetively charged.Giant liposome was positively charged by addition of cationic phospholipid EDOPC.When the electrostatic attraction force reaches 4.49×10-6N,fusion between positive charged giant liposome and negatively charged nano sized liposome happened and SERCA was incorporated in giant liposome,and ATPase assay and Ca²⁺ transporting assay demonstrated that incorporated SERCA remains functionally active.Finally,this model was used to study the regulation of drug thapsigargin on SERCA and similar result with the current most used ATPase assay was gotten,showing the potential application of giant liposome in drug evaluation.