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Occurrence of pyrophosphate:fructose 6-phosphate 1-phosphotransferase in Giardia lamblia trophozoites
Molecular and Biochemical Parasitology, 40 (1990) 147-150 Elsevier
147
MOLBIO01326 Short Communication
Occurrence of pyrophosphate:fructose 6-phosphate 1-phosphotransferase in Giardia lamblia trophozoites Emmanuel Mertens 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 glycolysis in most eukaryotes is the formation of fructose 1,6-bisphosphate (Fru-l,6-P2) from fructose 6-phosphate (Fru-6-P), catalyzed by ATP-dependent phosphofructokinase (PFK1) [1]. A major exception to this rule was found in the midseventies when Entamoeba histolytica was shown to contain a pyrophosphate-dependent phosphofructokinase (pyrophosphate:D-fructose 6-phosphate 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 fructose-2,6-bisphosphate (Fru-2,6-P2) [4], a powerful stimulator of glycolysis and inhibitor of gluconeogenesis present in all other eukaryotes tested so far [5,6]. The above listed protists belong to distant taxons, but have in common the absence of electron transport-linked phosphorylation and of mitochondria [7]. It was of interest to extend this study to the intestinal parasite Correspondence address: EmmanuelMertens, The Rockefeller University,1230York 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-bisphosphate; PPi, inorganic pyrophosphate; PPi-PFK, pyrophosphate:fructose 6-phosphate 1-phosphotransferase; PFK1, ATP:fructose 6-phosphate 1-phosphotransferase; PEG, poly(ethyleneglycol).
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 evolutionary tree [9]. This organism catabolizes carbohydrates by an extended glycolytic pathway [8]. Of the glycolytic enzymes the presence of hexokinase, aldolase and pyruvate kinase has been reported [8]. This work shows that G. larnblia contains an active PPi-PFK, but no detectable PFK1 nor Fru-2,6-P2. Frozen G. lamblia (strain Portland-l) trophozoites collected from axenic cultures were provided by F. Opperdoes (Brussels) and by D.G. Lindmark (Cleveland). Homogenization procedures and enzyme assays were as described previously [4]. PPi-PFK activity was enriched about three-fold by poly(ethylene glycol) (PEG) fractionation. 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 homogenized in a Potter-Elvejhem device and centrifuged for 10 min at 35 000 x g. Solid PEG 10 000 was added to the supernatant to a final concentration of 10%. After 5 min, this preparation was centrifuged again and PEG was added to the supernatant 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 PPiPFK 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), equilibrated 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 applied 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 Fru1,6-P2, but at a much lower rate, about 3% of that observed with PPi. This PFKl-like activity is insufficient to account for the glycolytic flux, estimated 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 1phosphate or of UTP and almost completely suppressed by the addition of i U of commercial yeast pyrophosphatase (200 U (mg protein)-1; Boehringer). These results suggest that the ATP-dependent 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 pyrophosphorylase, 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 prostoma [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 direction 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-reciprocal 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 filtration of the enriched preparation gave an Mr value of 92 000 for the enzyme. We were also interested in the possible presence of Fru-2,6-P2 in G. lamblia. With the experimental 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 phosphofructo-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 pyruvate kinase activity (not shown). These data argue against the presence of this regulatory compound 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 similar 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 existence of hexokinase, aldolase and pyruvate kinase [81. In conclusion, in the characteristics of its phosphofructokinase activity and lack of its regulation, G. lamblia is similar to the previously studied fermentative protists, E. histolytica [2], I. prostoma, and the two trichomonads [4]. It is noteworthy that recent data on small 16S-like rRNA sequences indicate that these organisms are phylogenically very distant from each other and belong 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 protist, E. gracilis [12], again representing independent branches of the tree. Thus the distribution of PPi-PFK in the living world follows no discernible evolutionary pattern. Data reported here give additional support to a correlation between glycolysis as main source of ATP and a dependency of this pathway on PPi-PFK. As mentioned earlier [4], this relationship could reflect a potential advantage to the organism of the use of PPi-PFK instead of PFK1, since it can significantly improve the ATP yield of fermentative glycolysis [14], thank to the use of a by-product of biosynthetic reactions.
Acknowledgements The author expresses his thanks to Dr. M. MOiler (The Rockefeller University) for his guidance, discussions and comments on the manuscript; Dr. E. Van Schaftingen (University of Louvain and International Institute of Cellular and Molecular Pathology, Brussels) for introduc-
149 ing h i m to the c o m p a r a t i v e e x p l o r a t i o n of glycolysis; a n d to D r . D . G . L i n d m a r k ( C l e v e l a n d State U n i v e r s i t y , C l e v e l a n d ) a n d Dr. F. O p p e r d o e s ( I n t e r n a t i o n a l I n s t i t u t e of C e l l u l a r a n d M o l e c u l a r
P a t h o l o g y , Brussels) for p r o v i d i n g G. lamblia cells. This w o r k was s u p p o r t e d by U S Public H e a l t h Service g r a n t A I 11942.
References 1 Bloxham, D.P. and Lardy, H.A. (1973) Phosphofructokinase. In: The Enzymes, Vol. 8, (Boyer, P.B., Ed.), pp. 239-278, Academic Press, New York. 2 Reeves, R.E., South, D.J., Blytt, H.J. and Warren, L.G. (1974) Pyrophosphate:D-fructose 6-phosphate 1-phosphotransferase. A new enzyme with the glycolytic function of 6-phosphofructokinase. J. Biol. Chem. 249, 7737-7741. 3 Reeves, R.E. (1984) Metabolism of Entamoeba histolytica Schaudinn, 1903. Adv. Parasitol. 23, 105-142. 4 Mertens, E., Van Schaftingen, E. and Miiller, M. (1989) Presence of a fructose-2,6-bisphosphate-insensitive pyrophosphate:fructose-6-phosphate phosphotransferase in the anaerobic protozoa Tritrichomonas foetus, Trichomonas vaginalis and lsotricha prostoma. Mol. Biochem. Parasitol. 37, 183-190. 5 Van Schaftingen, E. (1987) Fructose-2,6-bisphosphate. Adv. Enzymol. Relat. Areas Mol. Biol. 57, 315-395. 6 Van Schaftingen, E., Mertens, E. and Opperdoes, F.R. (In Press) Fructose 2,6-bisphosphate in primitive systems. In: Fructose 2,6-bisphosphate, an Unique Bisphosphate Ester (Pilkis, S.J., Ed.), CRC Press, Cleveland, OH. 7 Miiller, M. (1988) Energy metabolism of protozoa without mitochondria. Annu. Rev. Microbiol. 42, 465-488. 8 Lindmark, D.G. (1980) Energy metabolism of the anaerobic protozoon Giardia lamblia. Mol. Biochem. Parasitol. 1, 1-12. 9 Sogin, M.L. (1989) Evolution of eukaryotic microorganisms and their small subunit ribosomal RNAs. Am. Zool. 29,487-499.
10 Wood, H.G., O'Brien, W.E. and Michaels, G. (1977) Properties of carboxytransphosphorylase, pyruvate, phosphate dikinase; pyrophosphate-phosphofructokinase and pyrophosphate-acetate kinase and their roles on the metabolism of inorganic pyrophosphate. Adv. Enzymol. 45, 85-155. 11 Sabularse, D.C. and Anderson, R.L. (1981) o-Fructose 2,6bisphosphate: a naturally occurring activator for inorganic pyrophosphate:D-fructose 6-phosphate 1-phosphotransferase in plants. Biochem. Biophys. Res. Commun. 103, 845-855. 12 Miyatake, K., Enomoto, T. and Kitakoa, S. (1986) Fructose-2,6-bisphosphate activates pyrophosphate:D-fructose 6-phosphate 1-phosphotransferase from Euglena gracilis. Agric. Biol. Chem. 50, 2417-2418. 13 Reeves, R.E., Serrano, R. and South, D.J. (1976) 6-Phosphofructokinase (pyrophosphate). Properties of the enzyme from Entamoeba histolytica and its reaction mechanism. J. Biol. Chem. 251, 2958-2962. 14 O'Brien, W.E., Bowien, S. and Wood, H.G. (1975) Isolation and characterization of a pyrophosphate-dependent phosphofructokinase from Propionibacterium shermanii. J. Biol. Chem. 250, 8690-8695. 15 Carnal, N.W. and Black, C.C. (1979) Pyrophosphate-dependent 6-phosphofructokinase, a new glycolytic enzyme in pineapple leaves. Biochem. Biophys. Res. Commun. 86, 20-26.
147
MOLBIO01326 Short Communication
Occurrence of pyrophosphate:fructose 6-phosphate 1-phosphotransferase in Giardia lamblia trophozoites Emmanuel Mertens 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 glycolysis in most eukaryotes is the formation of fructose 1,6-bisphosphate (Fru-l,6-P2) from fructose 6-phosphate (Fru-6-P), catalyzed by ATP-dependent phosphofructokinase (PFK1) [1]. A major exception to this rule was found in the midseventies when Entamoeba histolytica was shown to contain a pyrophosphate-dependent phosphofructokinase (pyrophosphate:D-fructose 6-phosphate 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 fructose-2,6-bisphosphate (Fru-2,6-P2) [4], a powerful stimulator of glycolysis and inhibitor of gluconeogenesis present in all other eukaryotes tested so far [5,6]. The above listed protists belong to distant taxons, but have in common the absence of electron transport-linked phosphorylation and of mitochondria [7]. It was of interest to extend this study to the intestinal parasite Correspondence address: EmmanuelMertens, The Rockefeller University,1230York 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-bisphosphate; PPi, inorganic pyrophosphate; PPi-PFK, pyrophosphate:fructose 6-phosphate 1-phosphotransferase; PFK1, ATP:fructose 6-phosphate 1-phosphotransferase; PEG, poly(ethyleneglycol).
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 evolutionary tree [9]. This organism catabolizes carbohydrates by an extended glycolytic pathway [8]. Of the glycolytic enzymes the presence of hexokinase, aldolase and pyruvate kinase has been reported [8]. This work shows that G. larnblia contains an active PPi-PFK, but no detectable PFK1 nor Fru-2,6-P2. Frozen G. lamblia (strain Portland-l) trophozoites collected from axenic cultures were provided by F. Opperdoes (Brussels) and by D.G. Lindmark (Cleveland). Homogenization procedures and enzyme assays were as described previously [4]. PPi-PFK activity was enriched about three-fold by poly(ethylene glycol) (PEG) fractionation. 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 homogenized in a Potter-Elvejhem device and centrifuged for 10 min at 35 000 x g. Solid PEG 10 000 was added to the supernatant to a final concentration of 10%. After 5 min, this preparation was centrifuged again and PEG was added to the supernatant 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 PPiPFK 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), equilibrated 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 applied 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 Fru1,6-P2, but at a much lower rate, about 3% of that observed with PPi. This PFKl-like activity is insufficient to account for the glycolytic flux, estimated 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 1phosphate or of UTP and almost completely suppressed by the addition of i U of commercial yeast pyrophosphatase (200 U (mg protein)-1; Boehringer). These results suggest that the ATP-dependent 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 pyrophosphorylase, 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 prostoma [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 direction 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-reciprocal 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 filtration of the enriched preparation gave an Mr value of 92 000 for the enzyme. We were also interested in the possible presence of Fru-2,6-P2 in G. lamblia. With the experimental 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 phosphofructo-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 pyruvate kinase activity (not shown). These data argue against the presence of this regulatory compound 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 similar 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 existence of hexokinase, aldolase and pyruvate kinase [81. In conclusion, in the characteristics of its phosphofructokinase activity and lack of its regulation, G. lamblia is similar to the previously studied fermentative protists, E. histolytica [2], I. prostoma, and the two trichomonads [4]. It is noteworthy that recent data on small 16S-like rRNA sequences indicate that these organisms are phylogenically very distant from each other and belong 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 protist, E. gracilis [12], again representing independent branches of the tree. Thus the distribution of PPi-PFK in the living world follows no discernible evolutionary pattern. Data reported here give additional support to a correlation between glycolysis as main source of ATP and a dependency of this pathway on PPi-PFK. As mentioned earlier [4], this relationship could reflect a potential advantage to the organism of the use of PPi-PFK instead of PFK1, since it can significantly improve the ATP yield of fermentative glycolysis [14], thank to the use of a by-product of biosynthetic reactions.
Acknowledgements The author expresses his thanks to Dr. M. MOiler (The Rockefeller University) for his guidance, discussions and comments on the manuscript; Dr. E. Van Schaftingen (University of Louvain and International Institute of Cellular and Molecular Pathology, Brussels) for introduc-
149 ing h i m to the c o m p a r a t i v e e x p l o r a t i o n of glycolysis; a n d to D r . D . G . L i n d m a r k ( C l e v e l a n d State U n i v e r s i t y , C l e v e l a n d ) a n d Dr. F. O p p e r d o e s ( I n t e r n a t i o n a l I n s t i t u t e of C e l l u l a r a n d M o l e c u l a r
P a t h o l o g y , Brussels) for p r o v i d i n g G. lamblia cells. This w o r k was s u p p o r t e d by U S Public H e a l t h Service g r a n t A I 11942.
References 1 Bloxham, D.P. and Lardy, H.A. (1973) Phosphofructokinase. In: The Enzymes, Vol. 8, (Boyer, P.B., Ed.), pp. 239-278, Academic Press, New York. 2 Reeves, R.E., South, D.J., Blytt, H.J. and Warren, L.G. (1974) Pyrophosphate:D-fructose 6-phosphate 1-phosphotransferase. A new enzyme with the glycolytic function of 6-phosphofructokinase. J. Biol. Chem. 249, 7737-7741. 3 Reeves, R.E. (1984) Metabolism of Entamoeba histolytica Schaudinn, 1903. Adv. Parasitol. 23, 105-142. 4 Mertens, E., Van Schaftingen, E. and Miiller, M. (1989) Presence of a fructose-2,6-bisphosphate-insensitive pyrophosphate:fructose-6-phosphate phosphotransferase in the anaerobic protozoa Tritrichomonas foetus, Trichomonas vaginalis and lsotricha prostoma. Mol. Biochem. Parasitol. 37, 183-190. 5 Van Schaftingen, E. (1987) Fructose-2,6-bisphosphate. Adv. Enzymol. Relat. Areas Mol. Biol. 57, 315-395. 6 Van Schaftingen, E., Mertens, E. and Opperdoes, F.R. (In Press) Fructose 2,6-bisphosphate in primitive systems. In: Fructose 2,6-bisphosphate, an Unique Bisphosphate Ester (Pilkis, S.J., Ed.), CRC Press, Cleveland, OH. 7 Miiller, M. (1988) Energy metabolism of protozoa without mitochondria. Annu. Rev. Microbiol. 42, 465-488. 8 Lindmark, D.G. (1980) Energy metabolism of the anaerobic protozoon Giardia lamblia. Mol. Biochem. Parasitol. 1, 1-12. 9 Sogin, M.L. (1989) Evolution of eukaryotic microorganisms and their small subunit ribosomal RNAs. Am. Zool. 29,487-499.
10 Wood, H.G., O'Brien, W.E. and Michaels, G. (1977) Properties of carboxytransphosphorylase, pyruvate, phosphate dikinase; pyrophosphate-phosphofructokinase and pyrophosphate-acetate kinase and their roles on the metabolism of inorganic pyrophosphate. Adv. Enzymol. 45, 85-155. 11 Sabularse, D.C. and Anderson, R.L. (1981) o-Fructose 2,6bisphosphate: a naturally occurring activator for inorganic pyrophosphate:D-fructose 6-phosphate 1-phosphotransferase in plants. Biochem. Biophys. Res. Commun. 103, 845-855. 12 Miyatake, K., Enomoto, T. and Kitakoa, S. (1986) Fructose-2,6-bisphosphate activates pyrophosphate:D-fructose 6-phosphate 1-phosphotransferase from Euglena gracilis. Agric. Biol. Chem. 50, 2417-2418. 13 Reeves, R.E., Serrano, R. and South, D.J. (1976) 6-Phosphofructokinase (pyrophosphate). Properties of the enzyme from Entamoeba histolytica and its reaction mechanism. J. Biol. Chem. 251, 2958-2962. 14 O'Brien, W.E., Bowien, S. and Wood, H.G. (1975) Isolation and characterization of a pyrophosphate-dependent phosphofructokinase from Propionibacterium shermanii. J. Biol. Chem. 250, 8690-8695. 15 Carnal, N.W. and Black, C.C. (1979) Pyrophosphate-dependent 6-phosphofructokinase, a new glycolytic enzyme in pineapple leaves. Biochem. Biophys. Res. Commun. 86, 20-26.