Large-scale purification of bovine brain lactate dehydrogenase by affinity chromatography on immobilized colchicine.
Lactate dehydrogenase (LDH) [EC 1.1.1.27] in a crude extract (40-80% ammonium sulfate fraction) of bovine brain was adsorbed on an immobilized colchicine column and specifically eluted by addition of 1 mM NADH. The purity and subunit composition of the pooled LDH were estimated by two-dimensional gel electrophoresis. With an increase of NaCl concentration from 0 to 2.0 M, ligand saturation of LDH on immobilized colchicine increased from 6.8 to 14%, whereas that on immobilized Cibacron blue F3GA decreased from 2.1 to 0%. In the presence of high NaCl concentration, immobilized colchicine enabled both large- and small-scale purification of LDH by affinity chromatography and resulted in a yield of 117 mg from 1 kg of bovine brain in the presence of 2.5 M NaCl or higher recoveries of 54-96% from various tissues of one rat in the presence of 1.0 M NaCl. These results indicate that immobilized colchicine is an excellent adsorbent for the isolation and purification of LDH by affinity chromatography and has a high LDH-adsorbing capacity dependent upon a high NaCl concentration. Kinetic studies revealed that colchicine apparently competed with cofactor NAD for the active site of LDH and the Ki values of colchicine decreased with an increase of NaCl concentration. The chemical specificity of the colchicine-binding site of LDH was studied by the use of colchicine analogues and it is concluded that both the tropolone moiety (C-ring) and the amido bond in a side chain of colchicine structure are essential to the colchicine-LDH interaction.
Kocha
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T
Department of Hygienic Chemistry, Showa College of Pharmaceutical Sciences, Tokyo.
Fukuda
T
T
Isobe
T
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Okuyama
T
T
D000818:Animals; D001667:Binding, Competitive; D001921:Brain / Q000201:enzymology*; D002417:Cattle; D055598:Chemical Phenomena; D002621:Chemistry; D002846:Chromatography, Affinity / Q000379:methods; D003078:Colchicine / Q000031:analogs & derivatives; D003600:Cytosol / Q000201:enzymology; D015180:Electrophoresis, Gel, Two-Dimensional; D004591:Electrophoresis, Polyacrylamide Gel; D007770:L-Lactate Dehydrogenase / Q000302:isolation & purification*; D009928:Organ Specificity; D051381:Rats; D012965:Sodium Chloride; D013329:Structure-Activity Relationship
D012965:Sodium Chloride; D007770:L-Lactate Dehydrogenase; D003078:Colchicine