Isolation And Purification Of Spirulina Kinase, Mass Spectrometry And The Effect Of Crude Extract On MEF Cell Senescence

Abstract Objective:To purify Spirulinakinase from crude fermentative Spirulina and analyze the information on protein of Spirulinakinase using mass spectrometry analysis, providing experimental basis for producing genetic engineering spirulinakinase and studying its structure and function further.METHODS:(1) Spirulinakinase crude extract was prepared by water extraction and was ultrafiltrated by the tangential ultrafiltration system, and its protein content was determined by CBB G-250(Coomassie brilliant blue G-250) staining.(2) Spirulinakinase crude extract were got by Sephadex G-75gel filtration chromatography futher purification. The filbrinolytic activity was screened by fibrin plate experiment.(3) The strip of spirulinakinase was got by SDS-PAGE electrophoresis after the concentration of the elution peak which had the fibrinolytic activity.(4) The information on protein of spirulinakinase was analyzed by MADLI-TOF-TOF-MS spectrometry.RESULT:The separation and purification of Spirulinakinase:(1) Spirulinakinase crude extract was prepared by water extraction and ultrafiltrated by the tangential ultrafiltration system, and its protein content was determine by CBB G-250(Coomassie brilliant blue G-250) staining, and the concent of spirulinakinase protein was1.735ug/ml.(2) Sephadex G-75gel filtration chromatography:Five elution peaks were got by separating spirulinakinase crude fermentation ultrafiltrate through Sephadex G-75gel filtration chromatography. The fibrinolytic activity of the fifth elution peak was determined by fibrin plate method with urokinase as positive group, and the obvious cracking ring was shown to improve thrombilysis activity, and the fifth elution peak was collected.(3) SDS-PAGE electrophoresis result:Five elution peaks were got by Sephadex G-75gel filtration separation,and the fifth peak which had thrombolysis activity was determined by SDS-PAGE electrophoresis and the single protein strip was obtained to show the molecular weight as45KD consulting standard protein content.2.The analysis on the information of spirulinakinase protein:(1) The result that molecular weight43705Dalton of urine glycine formamide and isoelectric point4.91were shown on the Mass report No.gi493675806. And the molecular weight of spirulinakinase was prompted to be close to43705Dalton. And the result of Mass spectrum identification that the fixed modification amino acids of spirulinakinase C-end acetamide, variable amino acid modified protein N acetyl,amide and dioxide oxidation were shown on the Mass report.(2) The result that the neutral peptides single isotope quality theoretical value1284.6754of spirulinakinase protein purification IYFPVYVEGAK peptides was shown on Mass report No.gi1493675806through protein which would had been purified and idetificated by trypsin banding gel with enzyme digestion and peptides mixture was determined by MALDI-TOF-TOF-MS. The observation value with the theoretical value of deviation was on hundred and twenty-one over one million,the peptide score was76relative to the entire protein amino acid and the modified site was urea methylation.(3) The result of bioinformatics analysis that the high similarity of protein coverage rate of35%between the amino acid sequence of Arthrospira maxima and urine glycine fomamide enzymes. And that was suggested they were homologous. Conclusion:The strip of spirulinakinase was purified from crude fermentative spirulina successfully, and its information that the homology between spirulinakinase and urine glycine formamide enzymes was confirmed by MADLI-TOF-TOF-MS which was used to get information about Spirulinakinase protein. And the difference between molecular weight43705Dalton of spirulinakinase and that of natto kinase or douche fibrinolytic enzyme was suggested that spirulinakinase was a new type fibrinolytic enzyme which was different from natto kinase and douche fibrinolytic enzyme. OBJECTIVE:To study on the effect of spirulinakinase crude extracting solution about cell morphology, cell activity and senescent cells positive influence by culturing wild type MEF cells in vitro, in order to discusse the effect of spirulinakinase on cells senescence about two types that wild type MEF cells and mutant Zmpste24-/-MEF cells from the aspects of anti-aging efficacy of spirulinakinase extracting solution,providing theoretical basis for developing new anti-aging drugs. METHODS:1、 Cell model building:Wild type MEF cells model was subcultured and set up from the normal pregnant rats after13.5days pregnancy. And mutant Zmpets24-/-MEF cells model was subcultured and set up from the Zmpets24+/-knockout heterozygous pregnant rats after13.5days pregnancy.2、Groups of experiment:Groups of experiment were divided into two parts,one was the blank control group with the solution of10%PBS and cells growth liquid, the other group was drugs control group withthe solution of10%PBS,cells growth liquid and10%Spirulinakinase crude extracting solution (0.036mg/ml), in order to research anti-aging effect of Spirulinakinase crude etracting solution on two cells types that wild type MEF cells and mutant Zmpets24-/-MEF cells.3、Testing items:(1) The change of the mutant24-/-MEF cells morphology was observed under a microscope.(2) The proliferation of wild type MEF cells and mutant24-/-MEF cells were tested by CCK-8method.(3) The cell positive rate of replication senescence of wild type MEF cells was detected by β-galactose glucoside enzyme staining method. RESULTS:1、Cells morphological changes of mutant Zmpets24-/-MEF cells:Cells blank control was observed with situation that cells arrangement was irregular,cells form was large and flat, cells endocrine particles increased obviously, cytoplasmic transparency was low,part of the nucleolus swell was broken and particles in nuclear released.On the contrary, drugs control was observed with situation that cells grew well,configuration was normal,cells form was a long fusiform or irregular shape,endocrine cells had few particles,cytoplasmic had a great transparency,nucleous was full, outline was clear, cells grew as a riverlike shape or radial.2、The proliferation of wild type MEF cells and mutant Zmpets24-/-MEF cells:From the cells vitality curve, wild type MEF cells from the mouse of the normal pregnant rats after13.5days pregnancy were treated by spirulinakinase crude extracting solution, and then were measured by CCK-8staining method,and the cells vitality106.46%(P(?)0.05) at96h processing time point and129.03%(P(?)0.05) at120h processing time point were determined during0-144h. And the maximum was the value of120h processing time point. The result was shown that the cells anti-aging effect of spirulinakinase crude extracting solution was obvious on wild type MEF cells. With comparation, the effect of anti-aging on mutant Zmpets24-/-MEF cells was negative because of the P value higher than0.05.(3) Cells senescence was detected by β-gal staning method:The positive rate of blank control was20.85%, and positive rate of drug group was8.74%. CONCLUSION:1、The growth state of wild type was better due to the reason that the young state of the mutant Zmpets24-/-MEF cells was maintained with spirulinakinase effect.2、The result of experiment CCK-8stainning that the biggest number and the best statue of cells growth of wild type MEF cells which was treated by drug spirulinakinase was at the processing time120h compared to that of the processing time0h because of anti-aging effect of spirulinakinase.3、The result of experiment β-gal stainning that the positive rate of drug control was lower than that of blank control was due to the reason that the activity of β-gal enzyme was downgraded, the cells aging was inhibited, the life of cells was prolonged, and the renewal of cells and appreciation were promoted.