Protein-protein interaction affinity chromatography of leukotriene C4 synthase.
A novel affinity chromatography purification for human leukotriene C4 synthase is described. It is based on a specific interaction between leukotriene C4 synthase and microsomal glutathione S-transferase which occurs in the presence of magnesium ion. Microsomal glutathione S-transferase was immobilized on NHS-activated Sepharose 4B and used as an affinity matrix. Microsomes from 12-O-tetradecanoyl phorbol 13-acetate-treated human erythroleukemia cells were solubilized with taurocholic acid and applied on the affinity matrix at 0.1 M Mg2+ concentration. After washing with a buffer containing Mg2+, the enzyme was eluted with a glutathione-containing buffer lacking Mg2+. This facile one-step procedure gave a 166-fold purification of leukotriene C4 synthase with a yield of 44%. Analyses of proteins specifically adsorbed to the affinity matrix revealed components with apparent molecular weights of 18, 37, 48, and 60 kDa.
Söderström
M
M
Department of Biochemistry, Wallenberg Laboratory, Stockholm University, Sweden.
Morgenstern
R
R
Hammarström
S
S
D000818:Animals; D002478:Cells, Cultured; D002846:Chromatography, Affinity / Q000379:methods*; D005982:Glutathione Transferase / Q000302:isolation & purification* / Q000378:metabolism; D006801:Humans; D008861:Microsomes / Q000201:enzymology; D011485:Protein Binding; D051381:Rats
D005982:Glutathione Transferase; C041116:leukotriene-C4 synthase