Purification, identification, and characterization of peanut isocitrate lyase.
Isocitrate lyase (ICL, EC 4.1.3.1) is commonly present in oil-rich seeds in catalyzing the cleavage of isocitrate to glyoxylate and succinate and plays an essential role in lipid metabolism and gluconeogenesis. When peanut kernels (Tainan 14) were germinated at 30 degrees C, the cotyledon ICL activities increased substantially in the initial 4 days, and the 4-day-germinated cotyledons were subjected to ICL purification by Tris-HCl buffer extraction, heat treatment at 55 degrees C for 1 h, (NH4)2SO4 fractionation at 25-35% saturation, DEAE-cellulose chromatography, and Sephacryl S-300 gel filtration. A single 64 kDa SDS-PAGE protein band was obtained with 7.7% recovery and 37.5-fold purity. It was identified as ICL by LC-MS/MS analyses and Mascot Search with 494 as the highest Probability Based Mowse Score (PBMS). On the basis of the sequence of the homologous ICL of Glycine max, 26% of the peptide sequences of the peanut ICL were identified. During gel filtration, separation of peanut catalase (identified by LC-MS/MS and Mascot Search with 405 as the highest PBMS) from peanut ICL was achieved. The highest measured peanut ICL enzymatic activities were obtained at 45 degrees C and pH 7.0-7.8, respectively. The enzyme activities were stable (>80%) as stored for 8 h at 30 degrees C, 15 days at 4 degrees C, or 60 days at -25 degrees C. As affected by the supplements in the reactants for activity determinations, ICL activity was not affected by glucose up to 4%, sucrose up to 5%, or ethanol up to 8.33%.
Lin
Shing-Fei
SF
Department of Food Science, National Chiayi University, Chiayi, Taiwan.
Ong
Ping-Lin
PL
Jhou
Chun-Ru
CR
Chiou
Robin Y Y
RY
D010367:Arachis / Q000201:enzymology*; D002848:Chromatography, DEAE-Cellulose; D002850:Chromatography, Gel; D004795:Enzyme Stability; D006358:Hot Temperature; D006863:Hydrogen-Ion Concentration; D007522:Isocitrate Lyase / Q000302:isolation & purification* / Q000378:metabolism*; D012639:Seeds / Q000201:enzymology
D007522:Isocitrate Lyase