Glycolytic enzymes of Trypanosoma brucei. Simultaneous purification, intraglycosomal concentrations and physical properties

Abstract

We have developed a method for the simultaneous purification of hexokinase, glucosephosphate isomerase, phosphofructokinase, fructose-l,6-bisphosphate aldolase, triosephosphate isomerase, D-glyceraldehyde-phosphate dehydrogenase, phosphoglycerate kinase, glycerol-3-phosphate dehydrogenase and glycerol kinase from Trypanosoma brucei in yields varying over 8 -55%.Crude glycosomes were prepared by differential centrifugation of cell homogenates. Subsequent hydrophobic interaction chromatography on phenyl-Sepharose resulted in six pools containing various mixtures of enzymes. These pools were processed via affinity chromatography (immobilized ATP), hydrophobic interaction chromatography (octyl-Sepharose) and ion-exchange chromatography (CM-and DEAE-cellulose) which resulted in the purification of all nine enzymes.The native enzyme and subunit molecular masses, as determined by gel filtration and gel electrophoresis under denaturing conditions, were compared with those of their homologous counterparts from other organisms. Trypanosomal hexokinase is a hexamer and differs in subunit composition from the mammalian enzymes (monomers) as well as in subunit size (51 kDa versus 96-100 kDa, respectively). Phosphofructokinase only differs in subunit size (51 kDa for T. brucei versus 80 -90 kDa for mammals) but had identical subunit composition (tetrameric). The others all have the same subunit composition as their mammalian counterparts. Except for triosephosphate isomerase, all Trypanosoma enzymes have subunits which are 1 -5 kDa larger in size.Together these nine enzymes contribute 3.3 f 1.6% to the total cellular protein of T. brucei and at least 90% to the total glycosomal protein. A comparison of calculated intraglycosomal concentrations of the enzymes with the glycosomal metabolite concentrations shows that in the case of aldolase, glyceraldehyde-phosphate dehydrogenase and phosphoglycerate kinase, the concentration of active sites is of the same order of magnitude as that of their reactants.A common feature of the glycosomal glycolytic enzymes (with the exception of glucosephosphate isomerase) is that they are highly basic proteins with PI values between 8.8 and 10.2, values which are 1-4 higher than in the case of their mammalian cytosolic counterparts and 3 -6 higher than in the case of the various unicellular organisms.It is suggested that both the larger subunit size and the basic character of the T. brucei glycolytic proteins are involved in the routing of the enzymes from their site of biogenesis (the cytosol) towards their site of action (the glycosome).

Purification and Partial Characterization of Trypanosoma cruzi Triosephosphate Isomerase

“…Highly active preparation of TPI has been obtained from T. brucei isolated glycosomes (Misset & Opperdoes 1984, Misset et al 1986, however the number of this organelle in this parasite is four times greater than in T. cruzi (Soares & De Souza 1988, Tetley & Vickerman 1990. These observations may indicate difficulty to obtain the TPI directly from T. cruzi purified organelle.…”

“…Most of these proteins are encoded by different genes (Sullivan et al 1985, Huang et al 1989) and the glycosomal enzymes are synthesised in the cytoplasm by free ribosomes and imported post-translationally without apparent cleavage or modification. Several of these glycosomal and cytosolic isoenzymes have been purified from T. brucei (Misset et al 1986(Misset et al , 1987 and T. cruzi (Bourguignon et al 1997) and were distinguished structurally from their mammalian counterparts (Michels & Opperdoes 1991). In view of this and based on the observation that the inhibition of some glycosomal enzymes from T. brucei (Durieux et al 1991) may result in the death of the parasite (Opperdoes et al 1990, Willson et al 1993) these enzymes have suggested as promising targets for drug design.…”

The phosphoglycerate kinases from Trypanosoma brucei

“…The elution profile of GAPDH is further discussed in the accompanying paper [ 141 total volume). Packing of the column, flow rates and fraction size were identical to what has been described previously [6].…”

“…The glycosomal isoenzyme was purified from bloodstream-form trypanosomes as described by Misset and Opperdoes [13] and Misset et al [6].…”

“…hexokinase, glucosephosphate isomerase, phosphofructokinase, aldolase, triosephosphate isomerase, glyceraldehyde-phosphate dehydrogenase, phosphoglycerate kinase, glycerol-3-phosphate dehydrogenase and glycerol kinase). The glycosomes of the vertebrate stage represent 3 -4% of the cellular volume [5] and an equal percentage of its total protein whereas more than 90% of all the protein contained within this organelle is involved in glycolysis [6].…”