The phosphoglycerate kinases from Trypanosoma brucei

Abstract

Trypanosoma brucei has two phosphoglycerate kinase (PGK) isoenzymes, one is particle-bound and localized in glycosomes while the other is present in the cytosol. The cytosolic isoenzyme (cPGK) was 900-fold purified from cultured procyclic trypanosomes by hydrophobic interaction chromatography on phenyl-Sepharose followed by affinity chromatography on 2,3'-ATP-Sepharose and had a specific activity of 275 units/mg protein. cPGK was compared with the purified glycosomal isoenzyme (gPGK) from bloodstream-form trypanosomes as well as with the commercially available PGKs from yeast, rabbit muscle and Spirulina platensis, a blue-green alga.Like all other PGKs, cPGK was a monomeric protein with a molecular mass of approximately 45 kDa similar to that of the PGKs from other organisms but 2 kDa smaller than that of gPGK. Despite thls difference in length and a great difference in isoelectric point, the two trypanosome isoenzymes strongly resembled each other in several respects. (a) The kinetic parameters did not differ significantly from each other or from the PGKs of other organisms. (b) Both trypanosome enzymes resembled the enzyme from S . platensis in that they had an almost absolute requirement for ATP, contrary to the enzymes from yeast and rabbit muscle, which were capable of utilizing GTP and ITP also. This difference in substrate specificity may be related to the amino acid substitutions, Trp 308-His and Ala 306-+Glu in the adenine-binding site, which are only found in the two Trypanosoma isoenzymes. Kinetic analysis showed that these substitutions do not prevent binding of the ATP analogoues, but probably prevent phosphoryl-group transfer. (c) Both isoenzymes displayed an activity optimum at pH 6.0 -9.0 similar to that for the enzyme of yeast.Both gPGK and cPGK were inhibited by the trypanocidal drug Suramin. This inhibition could be described as competitive both with ATP and 3-phosphoglycerate with two inhibitor molecules binding to one molecule of enzyme. The gPGK, however, was much more sensitive (KYPP, = 8.0 pM) to Suramin than either the cPGK (KrPP. = 20 pM) or the enzymes from rabbit muscle (KrPP. = 55 pM), yeast (KrPP. = 167 pM) or S . platensis (KrPP. = 250 pM). It is suggested that positive charges on the enzyme's surface may play an important role in the potentiation of the binding of the negatively charged Suramin molecule.Trypanosoma brucei, a protozoan haemoflagellate belonging to the order Kinetoplastida, undergoes a complex life cycle comprising vertebrate and insect stages. When the parasite grows in the midgut of the tsetse fly, mitochondrial metabolism is fully active, involving a functional Kreb's cycle and cytochromes [l]. These trypanosomes mainly utilize amino acids [2]. However, when introduced into the mammalian bloodstream upon the infective bite of a fly it is the long slender bloodstream form, completely lacking mitochondrial activity [l], that develops in the host. In the vertebrate stage glycolysis is the only ATP-generating pathway available, producing two molecules of ATP for each...

Control and Regulation of Gene Expression

“…PGKB and PGKC have distinct expression patterns as follows. The cytosolic PGKB protein and its mRNA are predominantly expressed in the procyclic form, and the glycosomally localized PGKC isoenzyme (and the PGKC mRNA) is the predominant isoenzyme in the bloodstream form (27,30). The total cellular PGK activity does not differ much between bloodstream-and insect form trypanosomes.…”

Contribution of Pyruvate Phosphate Dikinase in the Maintenance of the Glycosomal ATP/ADP Balance in the Trypanosoma brucei Procyclic Form

“…Immunodetection was performed as described (37,38) using as primary antibodies rabbit anti-PGK (diluted 1:500; gift from P. Michels, Edinburgh, UK, and M. Parsons, Seattle, WA) (39,40), rat anti-PEPCK (diluted 1:1000; gift from T. Seebeck, Bern, Switzerland) (22), rabbit anti-PPDK (diluted 1:500) (5), rabbit anti-TDH (diluted 1:500) (11), rabbit anti-GPDH (glycerol-3-phosphate dehydrogenase, EC 1.1.1.8; diluted 1:100) (41), mouse anti-hsp60 (diluted 1:10,000) (42), and mouse anti-AMP-dependent acetyl-CoA synthetase (EC 6.2.1.1; diluted 1:100) (43) and as secondary antibodies, anti-mouse, anti-rat, or anti-rabbit IgG conjugated to horseradish peroxidase (Bio-Rad, 1:5,000 dilution). Revelation was performed using the SuperSignal West Pico Chemiluminescent Substrate as described by the manufacturer (Thermo Scientific).…”

Suramin, an anti‐cancer drug, inhibits protein kinase C and induces differentiation in neuroblastoma cell clone NB2A

“…The inhibition of protein kinases in Trypanosomatida by suramin has been demonstrated [ 1,2]. In recent studies suramin was used in the chemotherapy of acquired immune deficiency syndrome (AIDS) [3] but host toxicity precluded its usefulness.…”

“…Suramin, a polysulphonated naphthylurea, was the first widely accepted antiparasitic drug to be developed and is still one of the most commonly prescribed antitrypanosomal drugs. The inhibition of protein kinases in Trypanosomatida by suramin has been demonstrated [ 1,2]. In recent studies suramin was used in the chemotherapy of acquired immune deficiency syndrome (AIDS) [3] but host toxicity precluded its usefulness.…”