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(+)-dependen t glucose-6-phosphate dehydrogenase (G6PDH) activity, the first enzyme of t he pentose phosphate pathway (PPP), is associated with glycosomes. The spec ific activity of G6PDH in the purified organelles was increased 4-fold rela tive to a total cell extract and showed latency. Moreover, in the absence o f detergents this activity was totally resistant to the action of trypsin. The cytosolic counterpart was neither latent, nor was it resistant to tryps in. Both cytosolic and glycosomal G6PDH activities behaved identically on p henyl-, 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 G6PUH from both cell compartm ents did not reveal any differences. The purified enzyme had an apparent K- m of 138 and 5.3 mu M for glucose 6-phosphate (G6P), and for NADP(+), respe ctively, and had a specific activity of 14 mu mol. (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 glycos omes, these two activities most likely represent one and the same isoenzyme . (C) 1999 Elsevier Science B.V. All rights reserved.