title: Improved purification of ribulose 5-phosphate 3-epimerase from Saccharomyces cerevisiae and characterization of the enzyme.
abstract: D-Ribulose 5-phosphate 3-epimerase from Saccharomyces cerevisiae was purified to homogeneity by 1970-fold enrichment elaborating the following steps: disruption of fresh cells, polyethylene glycol precipitation, ion exchange chromatography, heat-treatment, size exclusion chromatography on Sephadex G-75 and Bio-Sil SEC 125 and hydrophobic interaction chromatography. A molecular mass of 50 +/- 4 kDa was determined for the native enzyme by sedimentation equilibrium experiments. Sodium dodecyl sulfate polyacrylamide gel electrophoresis revealed a single band with 26 kDa which has been characterized as an individual polypeptide chain. Thus, the enzyme is a dimer composed of two identical subunits. The specific activity of the purified enzyme with 7700 units/mg protein was found to be 30-fold higher than described in previous papers. The enzyme shows a hyperbolic dependence of the catalytic activity towards ribulose 5-phosphate with a KM-value of 1.5 mmol/l. The N-terminal amino acid sequence analysis of the native enzyme and of several peptides obtained by chemical and proteolytic fragmentation provided a part of the primary structure which fits to the primary structure deduced from the DNA sequence.
authors.affiliation: Institute of Biochemistry, Medical Faculty, University of Leipzig, Germany.
authors.forename: J
authors.initials: J
authors.lastname: Bär
authors.forename: M
authors.initials: M
authors.lastname: Naumann
authors.forename: R
authors.initials: R
authors.lastname: Reuter
authors.forename: G
authors.initials: G
authors.lastname: Kopperschläger
mesh_terms: D000595:Amino Acid Sequence; D002238:Carbohydrate Epimerases / Q000302:isolation & purification* / Q000378:metabolism; D002850:Chromatography, Gel; D002852:Chromatography, Ion Exchange; D004591:Electrophoresis, Polyacrylamide Gel; D008969:Molecular Sequence Data; D012441:Saccharomyces cerevisiae / Q000201:enzymology*; D017386:Sequence Homology, Amino Acid
chemical_list: D002238:Carbohydrate Epimerases; C022554:L-ribulosephosphate 4-epimerase