散人笔记

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猪脑！

又得重复以前的工作。 市场上买回猪脑52个，弃去小脑，细胞破碎；离心，取上层清液，加30%硫酸铵盐析；再离心，取上层清液，加至70%硫酸铵继续盐析；再次离心，取下层沉淀；buffer溶解之，透析（MWCO=12,000~14,0000），上柱...... 过程差不多，不过这次要纯化的目标protein是Gluthione Reductase。 多谢Kevin和Alan的鼎力相助。



EC 1.8.1.7
NC-IUBMB view  •  ENZYME view
 
Names 
Accepted name: glutathione-disulfide reductase
Other name(s): GSH reductase
 GSSG reductase
 NADPH-GSSG reductase
 NADPH-glutathione reductase
 NADPH:oxidized-glutathione oxidoreductase
 glutathione S-reductase
 glutathione reductase
 glutathione reductase (NADPH)
Systematic name: glutathione:NADP+ oxidoreductase
Reaction 
2 glutathione + NADP+ = glutathione disulfide + NADPH + H+
Cofactors 
FAD
Comments 
A dimeric flavoprotein (FAD); activity is dependent on a redox-active disulfide in each of the active centres.
Links to other databases 
BRENDA, CSA, ERGO, ENZYME@ExPASy, GO, KEGG, OMIM, NIST 74, PDB, PROSITE

CAS Registry Number: 9001-48-3


UniProt (30):
[show] 		 
P48641GSHRC_ARATH O04955GSHRC_BRARP P48642GSHRC_ORYSA
Q43621GSHRC_PEA P42770GSHRP_ARATH P27456GSHRP_PEA
P48640GSHRP_SOYBN Q43154GSHRP_SPIOL P80461GSHRP_TOBAC
P48638GSHR_ANASP Q74ZK4GSHR_ASHGO P48639GSHR_BURCE
P30635GSHR_CAEEL Q6FRV2GSHR_CANGA Q6BPI1GSHR_DEBHA
P06715GSHR_ECOLI P43783GSHR_HAEIN P00390GSHR_HUMAN
Q6HA23GSHR_KLULA P47791GSHR_MOUSE Q873E8GSHR_NEUCR
O15770GSHR_PLAF7 Q94655GSHR_PLAFK P23189GSHR_PSEAE
P70619GSHR_RAT P78965GSHR_SCHPO P11804GSHR_SPISP
Q60151GSHR_STRTR Q6C5H4GSHR_YARLI P41921GSHR_YEAST
References 
1.Pai, E.F., Schirmer, R.H. and Schulz, G.E. Structural studies on crystalline glutathione reductase from human erythrocytes. In: Singer, T.P. and Ondarza, R.N. (Eds.), Mechanisms of Oxidizing Enzymes, Elsevier North Holland, New York, 1978, pp. 17-22.
2.Pigiet, V.P. and Conley, R.R. Purification of thioredoxin, thioredoxin reductase, and glutathione reductase by affinity chromatography. J. Biol. Chem. 252 (1977) 6367-6372. [PMID: 330529]
3.Racker, E. Glutathione reductase from bakers' yeast and beef liver. J. Biol. Chem. 217 (1955) 855-865.
4.van Heyningen, R. and Pirie, A. Reduction of glutathione coupled with oxidative decarboxylation of malate in cattle lens. Biochem. J. 53 (1953) 436-444.
5.Worthington, D.J. and Rosemeyer, M.A. Glutathione reductase from human erythrocytes. Catalytic properties and aggregation. Eur. J. Biochem. 67 (1976) 231-238. [PMID: 9277]
6.Böhmé, C.C., Arscott, L.D., Becker, K., Schirmer, R.H. and Williams, C.H., Jr. Kinetic characterization of glutathione reductase from the malarial parasite Plasmodium falciparum. Comparison with the human enzyme. J. Biol. Chem. 275 (2000) 37317-37323. [PMID: 10969088]
7.Libreros-Minotta, C.A., Pardo, J.P., Mendoza-Hernandez, G. and Rendon, J.L. Purification and characterization of glutathione reductase from Rhodospirillum rubrum. Arch Biochem Biophys 298 (1992) 247-253. [PMID: 1524433]
作者:散人 | 日期: 2006年7月14日 | 分类:散人札记
  1. 散人   2006-7-15 17:57:00  回复该留言

    其实，只用了一半猪脑，能拿到的protein太少了。所以我不太想继续做下去——因为那样做到最后能拿到的pure的protein肯定不够用——那就等于白做了。

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