Purification and crystallization of yeast hexokinase isoenzymes. Characterization of different forms by chromatofocusing.
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.
Jacob
L
L
Max-Planck-Society, Research Unit for Structural Molecular Biology, Hamburg, Germany.
Beecken
V
V
Bartunik
L J
LJ
Rose
M
M
Bartunik
H D
HD
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
D004271:DNA, Fungal; D007527:Isoenzymes; D006593:Hexokinase