Molecular and Biochemical Parasitology, 40 (1990) 147-150 147 Elsevier

MOLBIO 01326

Shor t C o m m u n i c a t i o n

Occurrence of pyrophosphate:fructose 6-phosphate 1-phosphotransferase in Giardia lamblia trophozoites

E m m a n u e l M e r t e n s

The Rockefeller University, New York, NY, USA

(Received 10 November 1989; accepted 17 December 1989)

Key words: Pyrophosphate, inorganic; Phosphofructokinase; Giardia lamblia

The most important regulatory step of glycol- ysis in most eukaryotes is the formation of fruc- tose 1,6-bisphosphate (Fru-l,6-P2) from fructose 6-phosphate (Fru-6-P), catalyzed by ATP-de- pendent phosphofructokinase (PFK1) [1]. A ma- jor exception to this rule was found in the mid- seventies when Entamoeba histolytica was shown to contain a pyrophosphate-dependent phosphof- ructokinase (pyrophosphate:D-fructose 6-phos- phate 1-phosphotransferase; PPi-PFK) [2] but no detectable PFK1 [3]. This feature has also been detected recently in the ciliate Isotricha prostoma and in two flagellates, Tritrichomonas foetus and Trichomonas vaginalis [4]. Another notable property of these protists is the absence of fruc- tose-2,6-bisphosphate (Fru-2,6-P2) [4], a power- ful stimulator of glycolysis and inhibitor of glu- coneogenesis present in all other eukaryotes tested so far [5,6]. The above listed protists be- long to distant taxons, but have in common the absence of electron transport-linked phosphoryl- ation and of mitochondria [7]. It was of interest to extend this study to the intestinal parasite

Correspondence address: Emmanuel Mertens, The Rocke- feller University, 1230 York Avenue, New York, NY 10021, U.S.A.

Abbreviations: Fru-6-P, fructose 6-phosphate; Fru-l,6-P2, fructose 1,6-bisphosphate; Fru-2,6-P2, fructose 2,6-bisphos- phate; PPi, inorganic pyrophosphate; PPi-PFK, pyrophos- phate:fructose 6-phosphate 1-phosphotransferase; PFK1, ATP:fructose 6-phosphate 1-phosphotransferase; PEG, poly(ethylene glycol).

Giardia lamblia, another fermentative protist [8] that lacks mitochondria and hydrogenosomes [7], which is currently regarded as a representative of the earliest extant branch of the eukaryotic evo- lutionary tree [9]. This organism catabolizes car- bohydrates by an extended glycolytic pathway [8]. Of the glycolytic enzymes the presence of hexo- kinase, aldolase and pyruvate kinase has been re- ported [8]. This work shows that G. larnblia con- tains an active PPi-PFK, but no detectable PFK1 nor Fru-2,6-P2.

Frozen G. lamblia (strain Portland-l) tropho- zoites collected from axenic cultures were pro- vided by F. Opperdoes (Brussels) and by D.G. Lindmark (Cleveland). Homogenization proce- dures and enzyme assays were as described pre- viously [4]. PPi-PFK activity was enriched about three-fold by poly(ethylene glycol) (PEG) frac- tionation. An ice-cold solution containing 5 mM dithiothreitol, 5 p,g m1-1 leupeptin, 5 mM MgC12 and 50 mM Tris-HCl, pH 7.5 (buffer A) was added to the cell pellets which were then homog- enized in a Potter-Elvejhem device and centri- fuged for 10 min at 35 000 x g. Solid PEG 10 000 was added to the supernatant to a final concen- tration of 10%. After 5 min, this preparation was centrifuged again and PEG was added to the su- pernatant to 20% final concentration. After 5 min, the mixture was centrifuged, and the pellet was resuspended in 5 ml of buffer A. In this 10-20% PEG fraction, 79% of the initial activity of PPi- PFK was recovered with a specific activity of 2.2 p~mol min -1 (mg protein) -1. Molecular mass of

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the enzyme was determined by gel filtration on a Sephacryl S-300 column (1.4 x 30 cm), equili- brated with buffer A containing 100 mM NaCI. 500 ixl of the 10-20% PEG fraction was diluted 3-fold with buffer A with NaC1 100 raM, and ap- plied at a rate of 0.3 ml min -1.

Extracts of G. lamblia catalyzed the formation of Fru-l,6-P 2 PPi and from Fru-6-P, at a rate of about 0.67 ixmol rain -1 (mg protein) -1. They also catalyzed an ATP-dependent formation of Fru- 1,6-P2, but at a much lower rate, about 3% of that observed with PPi. This PFKl-like activity is in- sufficient to account for the glycolytic flux, esti- mated to be 100 nmol hexose equivalent min -1 (mg protein) -1 from data on glucose supported respiration [8]. The ATP-dependent activity was markedly enhanced by the addition of glucose 1- phosphate or of UTP and almost completely sup- pressed by the addition of i U of commercial yeast pyrophosphatase (200 U (mg protein)-1; Boeh- ringer). These results suggest that the ATP-de- pendent formation of Fru-l,6-P 2 was probably not due to PFK1 but to PPi-PFK which utilized PPi generated from ATP by UDPG pyrophosphor- ylase, an activity detectable in the extract (not shown). This explanation was already suggested for the low levels of PFKl-like activities detected in E. histolytica [10], trichomonads and L pros- toma [4]. Kinetic properties of PPi-PFK were studied with the use of the enriched preparation. Saturation curves for substrates were hyperbolic in either the forward (glycolytic) or reverse di- rection of the reaction. They were not affected by the presence of up to 2 IxM Fru-2,6-P2 which markedly stimulates PPi-PFK of plants [11] and of the photosynthetic protist Euglena gracilis [12]. The K m values determined from double-recipro- cal plots were: 80 IxM for Fru-6-P, 11 txM for PPi, 8 ixM for Fru-l,6-P2 and 510 ixM for Pi. Gel fil- tration of the enriched preparation gave an Mr value of 92 000 for the enzyme.

We were also interested in the possible pres- ence of Fru-2,6-P2 in G. lamblia. With the exper- imental procedures, used to study the above listed anaerobic protists, we were unable to detect the presence of this regulator (limit of detection, 2 pmol (rag protein)-1), or the activity of phos- phofructo-2-kinase, the enzyme that produces Fru-2,6-P 2 [5], or of its hypothetical PPi-linked

counterpart (limit of detection, 1 pmol Fru-2,6-P2 rain -1 (mg protein)-l). Furthermore, Fru-2,6-P2 had no effect on G. lamblia PPi-PFK or on its py- ruvate kinase activity (not shown). These data ar- gue against the presence of this regulatory com- pound in G. lamblia, too.

The results reported here give evidence for the presence of an active PPi-dependent PFK in place of PFK1 in G. lamblia. PPi-PFK of this organism had affinity constants and a molecular mass sim- ilar to other Fru-2,6-Pz-insensitive PPi-PFKs [4,13,14]. Detection of PPi-PFK confirms the presence of the Embden-Meyerhof pathway of glycolysis which has been inferred from the exist- ence of hexokinase, aldolase and pyruvate kinase [81.

In conclusion, in the characteristics of its phos- phofructokinase activity and lack of its regula- tion, G. lamblia is similar to the previously stud- ied fermentative protists, E. histolytica [2], I. prostoma, and the two trichomonads [4]. It is no- teworthy that recent data on small 16S-like rRNA sequences indicate that these organisms are phy- logenically very distant from each other and be- long to separate branches of the evolutionary tree [9]. To be mentioned at this point is the presence of PPi-PFK also in higher plants [15] and the pro- tist, E. gracilis [12], again representing independ- ent branches of the tree. Thus the distribution of PPi-PFK in the living world follows no discerni- ble evolutionary pattern. Data reported here give additional support to a correlation between gly- colysis as main source of ATP and a dependency of this pathway on PPi-PFK. As mentioned ear- lier [4], this relationship could reflect a potential advantage to the organism of the use of PPi-PFK instead of PFK1, since it can significantly im- prove the ATP yield of fermentative glycolysis [14], thank to the use of a by-product of biosyn- thetic reactions.

Acknowledgements

The author expresses his thanks to Dr. M. MOiler (The Rockefeller University) for his guid- ance, discussions and comments on the manu- script; Dr. E. Van Schaftingen (University of Louvain and International Institute of Cellular and Molecular Pathology, Brussels) for introduc-

ing him to the compara t ive explora t ion of glycol-

ysis; and to Dr . D . G . L i n d m a r k (Cleve land State Univers i ty , Cleve land) and Dr. F. Opperdoes ( In t e rna t iona l Ins t i tu te of Cel lu lar and Molecular

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