Simultaneous purification of hexokinase, class-I fructose-bisphosphate aldolase, triosephosphate isomerase and phosphoglycerate kinase from Trypanosoma brucei.
A method is presented for the simultaneous purification of hexokinase, fructose-bisphosphate aldolase, triosephosphate isomerase and phosphoglycerate kinase, and the partial purification of glycerol-3-phosphate dehydrogenase (NAD+), 6-phosphofructokinase, glucosephosphate isomerase, and glycerol kinase from Trypanosoma brucei. As a first step, the glycosomes, microbody-like organelles of Trypanosomatidae, containing almost exclusively enzymes involved in glucose and glycerol metabolism [Opperdoes, F. R. and Borst, P. (1977) FEBS Lett. 80, 360-364], were purified eightfold from homogenates with an average yield of 38%. Subsequently, the glycosomal content was subjected to hydrophobic interaction chromatography on phenyl-Sepharose. This step results in pure hexokinase (15% final yield) and almost pure triosephosphate isomerase, while the other glycosomal enzymes elute as mixtures of two or three enzymes. Triosephosphate isomerase was further purified to homogeneity on CM-cellulose (33% final yield), while phosphoglycerate kinase and fructose-bisphosphate aldolase were separated from each other and purified to homogeneity by affinity chromatography using ATP-Sepharose (25% and 30% final yields, respectively). Fructose-bisphosphate aldolase was further characterized as a typical class I enzyme.
D000818:Animals; D002238:Carbohydrate Epimerases / Q000302:isolation & purification*; D002850:Chromatography, Gel; D002852:Chromatography, Ion Exchange; D004591:Electrophoresis, Polyacrylamide Gel; D005634:Fructose-Bisphosphate Aldolase / Q000302:isolation & purification*; D005991:Glycerol Kinase / Q000302:isolation & purification; D005993:Glycerolphosphate Dehydrogenase / Q000302:isolation & purification; D006593:Hexokinase / Q000302:isolation & purification*; D008830:Microbodies / Q000201:enzymology; D008970:Molecular Weight; D010735:Phosphoglycerate Kinase / Q000302:isolation & purification*; D051381:Rats; D011919:Rats, Inbred Strains; D014305:Triose-Phosphate Isomerase / Q000302:isolation & purification*; D014346:Trypanosoma brucei brucei / Q000201:enzymology*
D005993:Glycerolphosphate Dehydrogenase; D006593:Hexokinase; D005991:Glycerol Kinase; D010735:Phosphoglycerate Kinase; D005634:Fructose-Bisphosphate Aldolase; D002238:Carbohydrate Epimerases; D014305:Triose-Phosphate Isomerase