title: Purification and crystallization of yeast hexokinase isoenzymes. Characterization of different forms by chromatofocusing.
abstract: The yeast hexokinase isoenzymes PI and PII have been purified in large amounts (20 mg) from overproducing yeast strains. The purification procedures of hexokinase PI and PII include anion-exchange chromatography on DEAE-Sephacel and chromatofocusing on PBE 94, hydrophobic interaction chromatography on phenyl-Sepharose (necessary for the isolation of the isoenzyme PI); in the final step either a Mono Q HR 5/5 or a Fractogel EMD TMAE 650(S) column was used. Hexokinase preparations were characterized before crystallization by chromatofocusing on a Mono P HR 5/20 FPLC column, where different forms of hexokinase can be rapidly distinguished by their elution behaviour. From both purified hexokinase PI and PII, large crystals were grown that diffract X-rays to high resolution.
authors.affiliation: Max-Planck-Society, Research Unit for Structural Molecular Biology, Hamburg, Germany.
authors.forename: L
authors.initials: L
authors.lastname: Jacob
authors.forename: V
authors.initials: V
authors.lastname: Beecken
authors.forename: L J
authors.initials: LJ
authors.lastname: Bartunik
authors.forename: M
authors.initials: M
authors.lastname: Rose
authors.forename: H D
authors.initials: HD
authors.lastname: Bartunik
mesh_terms: D002852:Chromatography, Ion Exchange; D003460:Crystallization; D004271:DNA, Fungal / Q000235:genetics; D004591:Electrophoresis, Polyacrylamide Gel; D006593:Hexokinase / Q000302:isolation & purification* / Q000378:metabolism; D006863:Hydrogen-Ion Concentration; D007527:Isoenzymes / Q000302:isolation & purification* / Q000378:metabolism; D007700:Kinetics; D010957:Plasmids; D012441:Saccharomyces cerevisiae / Q000201:enzymology*; D014961:X-Ray Diffraction
chemical_list: D004271:DNA, Fungal; D007527:Isoenzymes; D006593:Hexokinase