Type Ⅰ,Ⅲ and Ⅴ collagens were extracted from bovine dermis and cornea by using pepsin treatment in acetic acid solution,followed by salt precipitation and dialysis,to purify and isolate each type of collagens.The p...Type Ⅰ,Ⅲ and Ⅴ collagens were extracted from bovine dermis and cornea by using pepsin treatment in acetic acid solution,followed by salt precipitation and dialysis,to purify and isolate each type of collagens.The preparation process was analyzed by using sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE).A reducing agent,2-mercaptoethanol,was used to remove disulfide bonds and analyze the structure of the bonds involved between α chains in some types of collagens.The use of delayed reducing methods resulted in the difference between α1(Ⅲ) and α1(Ⅰ) chains in a mixture containing type Ⅰ and Ⅲ collagens.The structure of disulfide bonds among α chains exists potentially in type Ⅴ collagen prepared from the pepsin-treatment extraction at 4℃,which differs from type Ⅲ collagen in relation to the locations of disulfide bonds.Compared with pepsin-treated collagen at 4℃,the relative molecular weights of α1(Ⅴ) and α2(Ⅴ) chains treated at room temperature decrease by 4.6% and 6.0%,respectively.It is concluded that type Ⅰ,Ⅲ and Ⅴ collagens can be prepared from bovine dermis and cornea by the use of pepsin treatment,salt precipitation and dialysis.The interchain disulfide bonds lie potentially near the edges of termini of type Ⅴ collagen molecules in extracellular matrix,and a small number of interchain crosslinks exist in type Ⅴ collagen.展开更多
Membrane proteins were extracted from eggs, schistosomulum, adult male and female worms of Schistosoma japonicum in order to analyze the differently expressed profile by two dimensional electrophoresis. Schistosomulum...Membrane proteins were extracted from eggs, schistosomulum, adult male and female worms of Schistosoma japonicum in order to analyze the differently expressed profile by two dimensional electrophoresis. Schistosomulum and adult worms were obtained from rabbits infected with 1 500 cercariaes on 14 and 42 days after challenge, respectively. Adult male and female worms on 42 days were manually detached and stored into liquid nitrogen until use. Eggs were collected by Percoll TM from the liver of rabbits. ProteoPrep Membrane Extraction Kit TM was employed to extracted membrane proteins by reducing and alkylating with TBP and iodoacetamide from 200 mg of eggs, schistosomulums, adult male worm and female worms, respectively. Immobilized pH gradient strips with a linear pH range of 3-10 (130 mm) were rehydrated together with membrane proteins (30 μg) in 250 μl solution containing 7 mol urea, 2 mol thiourea, 2% SB3-10, 4% CHAPS, 40 mmol Tris, 30 mmol DTT, then separated on 12.5% SDS polyacrylamide gel for the second dimensional electrophoresis. Gels were stained with silver, scanned by Labscan, and analyzed using ImageMaster TM Analysis software. The 2D maps of egg, schistosomulum, adult female worm and male worm were showed 78±3、67±3、108±4 and 122±4 spots respectively. There were 35±1 spots which showed specific expression in female worm as compared with male worm, but 45±2 spots were in male worms. Most differently expressed spots between male and female worms were located in the area of 40-70 kD and pI 4-7. The large number of unique spots from schistosomulum was located in the area of alkalescence. The 2D map of for adult male worms uniquely showed 5 spots as compared with that of schistosomulum and female worm. The female worm showed 4 unique spots as compared with that of schistosomulum, egg and male worm. The unique spots between male and female worms were identified by the database of SWISS 2D-PAGE according to the molecular weight and isoelectronic point. Calreticulin 1, methyltransferase, outer membrane protein tolc and oxygen-evolving enhancer protein 1-1 were uniquely showed in adult male worm after pairing. On the other hand, enolase, outer membrane protein X, ferrienterobactin receptor and heat shock cognate 70 kD protein 3 were uniquely showed in adult female worm. In conclusion, there are different expressions of membrane proteins from egg, schistosomulum, adult male worm and female worms of Schistosoma japonicum. These proteins, which were uniquely expressed between adult male and female worms after pairing, were involved in signal transduction and metabolism展开更多
基金Supported by National Natural Science Foundation of China (No.30970724)Natural Science Foundation of Tianjin (No.08JCYBJC03400)
文摘Type Ⅰ,Ⅲ and Ⅴ collagens were extracted from bovine dermis and cornea by using pepsin treatment in acetic acid solution,followed by salt precipitation and dialysis,to purify and isolate each type of collagens.The preparation process was analyzed by using sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE).A reducing agent,2-mercaptoethanol,was used to remove disulfide bonds and analyze the structure of the bonds involved between α chains in some types of collagens.The use of delayed reducing methods resulted in the difference between α1(Ⅲ) and α1(Ⅰ) chains in a mixture containing type Ⅰ and Ⅲ collagens.The structure of disulfide bonds among α chains exists potentially in type Ⅴ collagen prepared from the pepsin-treatment extraction at 4℃,which differs from type Ⅲ collagen in relation to the locations of disulfide bonds.Compared with pepsin-treated collagen at 4℃,the relative molecular weights of α1(Ⅴ) and α2(Ⅴ) chains treated at room temperature decrease by 4.6% and 6.0%,respectively.It is concluded that type Ⅰ,Ⅲ and Ⅴ collagens can be prepared from bovine dermis and cornea by the use of pepsin treatment,salt precipitation and dialysis.The interchain disulfide bonds lie potentially near the edges of termini of type Ⅴ collagen molecules in extracellular matrix,and a small number of interchain crosslinks exist in type Ⅴ collagen.
基金国家高技术研发 863项目 (No 2 0 0 1AA2 15 15 1)上海市重大科技攻关项目 (No 0 3DZ192 3 1)资助~~
文摘Membrane proteins were extracted from eggs, schistosomulum, adult male and female worms of Schistosoma japonicum in order to analyze the differently expressed profile by two dimensional electrophoresis. Schistosomulum and adult worms were obtained from rabbits infected with 1 500 cercariaes on 14 and 42 days after challenge, respectively. Adult male and female worms on 42 days were manually detached and stored into liquid nitrogen until use. Eggs were collected by Percoll TM from the liver of rabbits. ProteoPrep Membrane Extraction Kit TM was employed to extracted membrane proteins by reducing and alkylating with TBP and iodoacetamide from 200 mg of eggs, schistosomulums, adult male worm and female worms, respectively. Immobilized pH gradient strips with a linear pH range of 3-10 (130 mm) were rehydrated together with membrane proteins (30 μg) in 250 μl solution containing 7 mol urea, 2 mol thiourea, 2% SB3-10, 4% CHAPS, 40 mmol Tris, 30 mmol DTT, then separated on 12.5% SDS polyacrylamide gel for the second dimensional electrophoresis. Gels were stained with silver, scanned by Labscan, and analyzed using ImageMaster TM Analysis software. The 2D maps of egg, schistosomulum, adult female worm and male worm were showed 78±3、67±3、108±4 and 122±4 spots respectively. There were 35±1 spots which showed specific expression in female worm as compared with male worm, but 45±2 spots were in male worms. Most differently expressed spots between male and female worms were located in the area of 40-70 kD and pI 4-7. The large number of unique spots from schistosomulum was located in the area of alkalescence. The 2D map of for adult male worms uniquely showed 5 spots as compared with that of schistosomulum and female worm. The female worm showed 4 unique spots as compared with that of schistosomulum, egg and male worm. The unique spots between male and female worms were identified by the database of SWISS 2D-PAGE according to the molecular weight and isoelectronic point. Calreticulin 1, methyltransferase, outer membrane protein tolc and oxygen-evolving enhancer protein 1-1 were uniquely showed in adult male worm after pairing. On the other hand, enolase, outer membrane protein X, ferrienterobactin receptor and heat shock cognate 70 kD protein 3 were uniquely showed in adult female worm. In conclusion, there are different expressions of membrane proteins from egg, schistosomulum, adult male worm and female worms of Schistosoma japonicum. These proteins, which were uniquely expressed between adult male and female worms after pairing, were involved in signal transduction and metabolism
