The Assay Of Pyruvate Kinase Activity In Exosomes

Background:In 2011,the medical community in the United States put forward the concept of "precision medicine" for the first time.In October 2015,Obama proposed the "Precise Medical Plan" again in the U.S.State of the Union Address,which indicates the advent of a new medical era.With the introduction of the concept of precision medicine,more and more scholars pay more attention to the accurate diagnosis and treatment of the disease.Because of its potential role in the diagnosis and treatment of diseases,exosomes have a bright future in the development of precision medicine.Exosomes are tiny vesicles with lipid bilayer membrane structure secreted by a variety of cells.They contain proteins,lipids,nucleic acids and metabolites that reflect the physiological and pathological conditions of the cells of origin.And they are widely distributed in the blood,pleural effusion,urine and other body fluids and have a longer half-life.Exosomes can be stored in body fluids for long periods of time and they can maintain their membrane integrity and osmotic pressure.Whether or not it has an energy harvesting system deserves further exploration.Sugars are the main energy-supplying substances in human body and can provide energy to cells by anaerobic glycolysis and aerobic oxidation.Pyruvate kinase,an important rate-limiting enzyme in glucose glycolysis,catalyzes the conversion of phosphoenolpyruvate and ADP to pyruvate and ATP.Therefore,the detection of pyruvate kinase activity in exosomes can further explore the ways in which exosomes can obtain energy and gain energy.Objective:Determining the activity of pyruvate kinase in exosomes by 2,4-dinitrophenylhydrazine colorimetric method and ICSH-recommended method respectively.Comparing the differences in pyruvate kinase activity between exosomes added with glucose group and PBS group and exploring whether the exosomes carry out glycolysis to provide energy for themselves.Methods:Extract the exosomes from DU145,HEPA1-6 and RGC-5 cells by ultracentrifugation and use transmission election microscopy to identify characteristics of exosomes.The activity of pyruvate kinase in exosomes was determined by 2,4-dinitrophenylhydrazine colorimetric method and ICSH method.The changes of absorbance were measured by enzyme standard instrument.The changes of pyruvate kinase activity in exosomes of glucose-added and PBS-added groups were compared over time,and the survival of exosomes was analyzed.Results:(1)The circular or oval double-layer lipid membrane vesicles with diameter of 30-120 nm were detected in the DU145,HEPA1-6 and RGC-5 cells,so we identified them as exosomes.(2)The pyruvate kinase activity of DU145,HEPA1-6,and RGC-5 exosomes was determined to be 27.570 IU/g,25.515 IU/g,and 23.752 IU/g,respectively,using the 2,4-dinitrophenylhydrazine colorimetric method.The pyruvate kinase activity of DU145,HEPA1-6,and RGC-5 exosomes was determined to be 26.426 IU/g,26.323 IU/g,and 22.257 IU/g,respectively,using the ICSH recommended method.The activity of pyruvate kinase in exosomes was detected in the 2,4-dinitrophenylhydrazine colorimetric method and the ICSH recommended method.The results obtained by the two methods were not significantly different(p>0.05).(3)When exosomes in glucose group and PBS control group were incubated at 4 °C for 4 days,the activity of pyruvate kinase in glucose group began to appear higher than that in PBS control group.After incubation for 6 days at 4°C,pyruvate kinase activity in exosomes was still higher in the glucose group than that in the PBS control group.Conclusion:(1)The study confirmed the presence of pyruvate kinase activity in DU 145,HEPA1-6,and RGC-5 exosomes,indicated the presence of pyruvate kinase in the exosomes,and suggested that exosomes can produce ATP to maintain membrane structure and osmotic pressure.(2)The study provides a basis for exploring whether exosomes can use glucose to provide energy for themselves.