Purification and crystallization of NADP+-specific isocitrate dehydrogenase from Escherichia coli using polyethylene glycol.
A simple and rapid method is presented for purifying the NADP+-dependent isocitrate dehydrogenase (threo-DS-isocitrate:NADP+ oxidoreductase (decarboxylating), from Escherichia coli, which relies on fractionation of the enzyme with polyethylene glycol. The shortened preparation results in a 32% relative recovery of purified enzyme at a specific activity of 127 micronmol/min per mg of protein. The Km values for threo-DS-isocitrate, NADP+, NAD+, Mg2+ and Mn2+ are 6.4, 36, 3000, 19.7 and 2.0 micronM, respectively. The stability of the enzyme as a function of dilution and temperature are also reported. Recrystallization of the purified enzyme under different conditions readily produces a variety of single crystals. Crystals grown from ammonium sulfate solutions belong to monoclinic space group C2 with a = 125 A, b = 111 A, c = 83.5 A and beta = 108degrees 45'. Density measurements of these crystals indicate there are two 80 000-dalton dimers per asymmetric unit.
D003460:Crystallization; D004926:Escherichia coli / Q000201:enzymology*; D007521:Isocitrate Dehydrogenase / Q000302:isolation & purification* / Q000378:metabolism; D008274:Magnesium / Q000494:pharmacology; D008345:Manganese / Q000494:pharmacology; D009249:NADP; D011092:Polyethylene Glycols
D011092:Polyethylene Glycols; D008345:Manganese; D009249:NADP; D007521:Isocitrate Dehydrogenase; D008274:Magnesium