Glyceraldehyde‐phosphate dehydrogenase from Trypanosoma brucei

Abstract

Trypanosoma bruceicontains two glyceraldehyde-phosphate (GAPDH; EC 1.2.1.12) isoenzymes; one is located in glycosomes and represents 80% of the total activity, whereas the other is present in the cytosol. The purification of the cytosolic GAPDH, which is identical in both bloodstream-form and insect-stage trypanosomes, is described, and the enzyme compared with its glycosomal counterpart. Cytosolic GAPDH is specific for NAD. It is a tetrameric enzyme with subunits of 33.5 kDa, 5 kDa smaller than those of the glycosomal GAPDH. The native enzyme has a pl of 7.9, which is 1.5 pH units less basic than the glycosobal enzyme. Both enzymes display maximal activity at pH 8 but the cytosolic enzyme has a much broader activity profile especially towards lower pH values.Sequence comparison of the first 85 amino acids reveals that the N-terminal parts of both isoenzymes differ by 52%. The N terminus of the cytosolic isoenzyme resembles the corresponding N termini of ten other known GAPDH sequences in that they all lack three amino-acid insertions, which so far only have been found in the glycosomal isoenzyme of T. brucei. This observation explains in part the great difference in subunit size between the two T. brucei isoenzymes and suggests that at least one of these insertions is responsible for import of the glycosomal isoenzyme into the organelle.In Trypanosoma brucei, glyceraldehyde-phosphate dehydrogenase (GAPDH) has been found in two separate cellular compartments: the cytosol and the glycosomes [l]. However, thus far no direct proof has been brought forward for the existence of two separate isoenzymes in this organism.Recently two tandemly linked identical genes coding for GAPDH have been identified in the genome of T. brucei [2]. From sequence data it was inferred that both genes code for the glycosomal enzyme; subunit molecular mass, isoelectric point 131, amino-acid composition and the N-terminal sequence of the isolated glycosomal protein [2] were all in excellent agreement with the properties predicted from the amino acid sequence as deduced from the gene. A gene coding for a possible cytosolic isoenzyme has not yet been identified.In this paper we now present evidence for the existence of a separate cytosolic isoenzyme of GAPDH in cultured procyclic (insect-stage) as well as in bloodstream-form trypanosomes. We describe its purification, characterization and N-terminal amino acid sequence and compare the properties of both the glycosomal and the cytosolic counterparts.

The phosphoglycerate kinases from Trypanosoma brucei

“…Fig. 1 in the accompanying paper [14]) [8] and [9]. However, the amount of PGK which is soluble in insect trypanosomes varies from experiment to experiment: 91 % in Opperdoes et al [9] against 59% here (cf.…”

“…Since we had previously purified the glycosomal enzymes using phenylSepharose chromatography and therefore knew their exact elution positions, we decided to use this technique for the purification of cytosolic isoenzymes as well (see also the accompanying paper [14]). Fig.…”

“…Subsequent additions were as follows: 150 p1 1 M dithiothreitol, 150 p1 10 mM leupeptin, 1.5 mlO.1 M phenylmethylsulfonyl fluoride (a stock solution in N,N-dimethyl formamide) and 1.5 ml 0.1 M NAD. The addition of NAD was to stabilize the cytosolic glyceraldehyde-phosphate dehydrogenase enzyme, the purification of which is described in the accompanying paper [14]. After removal and washing free from the nuclear fraction by centrifugation (10 min at 1500 x g ) , the combined supernatants (200 ml) were centrifuged at 48 000 x g for 30 min.…”

“…Cytosolic phosphoglycerate kinase (cPGK) elutes as the slower component in the ethyleneglycol gradient, well separated from a minor, but faster component eluting in the ammonium sulphate gradient and representing the glycosomal isoenzyme (gPGK) [I 31. The purification of glyceraldehyde-phosphate dehydrogenase is described in the accompanying paper [14]. The cPGK was further purified on a 2',3'-ATP-Sepharose column, a procedure we also employed for the glycosomal isoenzyme and which is generally applied for the purification of PGKs from various sources [18].…”

Purification and Partial Characterization of Trypanosoma cruzi Triosephosphate Isomerase

“…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.…”