Dye-ligand and immobilized metal ion interaction chromatography for the purification of enzymes of prenyl pyrophosphate metabolism.
Dye-ligand and immobilized metal ion interaction chromatography were shown to be efficient techniques for the rapid batchwise fractionation, from crude plant extracts, of a series of enzymes of prenyl pyrophosphate metabolism. Isopentenyl pyrophosphate isomerase, two prenyltransferases, and a number of terpene cyclases (synthases) were readily adsorbed to Matrex Gel Red A (a dimeric triazine dye coupled to cross-linked agarose beads), and desorbed in good yield with relatively high concentrations of KCl and increasing pH. Although all of these enzymes exhibit the common feature of employing a pyrophosphorylated substrate, selective elution could not be achieved with substrate or substrate analogues bearing a pyrophosphate function. Nor could the strong binding of these enzymes to triazine dyes be attributed solely to metal ion interactions or to hydrophobic effects. In a similar way, the isomerase, the prenyltransferases, and all of the terpene cyclases bound to a column of iminodiacetate-immobilized Ni(II) and were desorbed in relatively high fold purity with 15 mM imidazole. Although all of these enzymes bear accessible histidine residues, the interactions with the chelated metal ion were not sufficiently different to permit selective enzyme desorbtion by imidazole gradient elution. However, the use of columns charged with Zn(II) or Co(II) did allow some separation of the different cyclase and transferase types. While empirical in nature, these techniques offer simple, effective, and high-capacity methods for the preliminary concentration and purification of a group of enzymes that utilize prenyl pyrophosphate intermediates of isoprenoid biosynthesis.
Lanznaster
N
N
Institute of Biological Chemistry, Washington State University, Pullman 99164-6340.
Croteau
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R
D002846:Chromatography, Affinity / Q000379:methods*; D002852:Chromatography, Ion Exchange / Q000379:methods*; D004396:Coloring Agents; D004798:Enzymes / Q000302:isolation & purification*; D008024:Ligands; D008670:Metals; D010944:Plants / Q000378:metabolism; D011106:Polyisoprenyl Phosphates / Q000378:metabolism*
D004396:Coloring Agents; D004798:Enzymes; D008024:Ligands; D008670:Metals; D011106:Polyisoprenyl Phosphates