Purification, localisation and characterisation of glucose-6-phosphate dehydrogenase of Trypanosoma brucei.
Cell-fractionation and digitonin titration of procyclic trypomastigotes of Trypanosoma brucei, revealed that almost half of the total NADP+ -dependent glucose-6-phosphate dehydrogenase (G6PDH) activity, the first enzyme of the pentose phosphate pathway (PPP), is associated with glycosomes. The specific activity of G6PDH in the purified organelles was increased 4-fold relative to a total cell extract and showed latency. Moreover, in the absence of detergents this activity was totally resistant to the action of trypsin. The cytosolic counterpart was neither latent, nor was it resistant to trypsin. Both cytosolic and glycosomal G6PDH activities behaved identically on phenyl-, CM-, heparin-, and Affigel-blue-Sepharose columns. Both isoenzymes had a subunit Mr of 62 000 and an isoelectric point of 6.85, while kinetic studies carried out on the partially purified G6PDH from both cell compartments did not reveal any differences. The purified enzyme had an apparent Km of 138 and 5.3 microM for glucose 6-phosphate (G6P), and for NADP+, respectively, and had a specific activity of 14 micromol. (min mg of protein)(-1). We conclude that while in procyclic stages of T. brucei G6PDH activity is present in two different cell compartments, i.e. the cytosol and the glycosomes, these two activities most likely represent one and the same isoenzyme.
Heise
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Research Unit for Tropical Diseases, Christian de Duve Institute of Cellular Pathology (ICP) and Laboratory of Biochemistry, Catholic University of Louvain, Brussels, Belgium.
Opperdoes
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D000818:Animals; D002458:Cell Fractionation; D002847:Chromatography, Agarose; D003600:Cytosol / Q000201:enzymology; D004072:Digitonin / Q000378:metabolism; D005954:Glucosephosphate Dehydrogenase / Q000737:chemistry / Q000302:isolation & purification* / Q000378:metabolism*; D006593:Hexokinase / Q000378:metabolism; D015388:Organelles / Q000201:enzymology; D014346:Trypanosoma brucei brucei / Q000201:enzymology* / Q000254:growth & development; D014357:Trypsin / Q000378:metabolism
D005954:Glucosephosphate Dehydrogenase; D006593:Hexokinase; D014357:Trypsin; D004072:Digitonin