Thermotoga maritima phosphofructokinases: expression and characterization of two unique enzymes

A pyrophosphate-dependent phosphofructokinase (PP_i-PFK) and an ATP-dependent phosphofructokinase (ATP-PFK) from Thermotoga maritima have been cloned and characterized. The PP_i-PFK is unique in that the K_m and V_max values indicate that polyphosphate is the preferred substrate over pyrophosphate; the enzyme in reality is a polyphosphate-dependent PFK. The ATP-PFK was not significantly affected by common allosteric effectors (e.g., phosphoenolpyruvate) but was strongly inhibited by PP_i and polyphosphate. The results suggest that the control of the Embden-Meyerhof pathway in this organism is likely to be modulated by pyrophosphate and/or polyphosphate.     

PPi-Dependent Phosphofructokinase from Thermoproteus tenax,an Archaeal Descendant of an Ancient Line in Phosphofructokinase Evolution

Flux into the glycolytic pathway of most cells is controlled via allosteric regulation of the irreversible, committing step catalyzed by ATP-dependent phosphofructokinase (PFK) (ATP-PFK; EC 2.7.1.11), the key enzyme of glycolysis. In some organisms, the step is catalyzed by PP_i-dependent PFK (PP_i-PFK; EC 2.7.1.90), which uses PP_i instead of ATP as the phosphoryl donor, conserving ATP and rendering the reaction reversible under physiological conditions. We have determined the enzymic properties of PP_i-PFK from the anaerobic, hyperthermophilic archaeon Thermoproteus tenax, purified the enzyme to homogeneity, and sequenced the gene. The ∼100-kDa PP_i-PFK from T. tenax consists of 37-kDa subunits; is not regulated by classical effectors of ATP-PFKs such as ATP, ADP, fructose 2,6-bisphosphate, or metabolic intermediates; and shares 20 to 50% sequence identity with known PFK enzymes. Phylogenetic analyses of biochemically characterized PFKs grouped the enzymes into three monophyletic clusters: PFK group I represents only classical ATP-PFKs from Bacteria and Eucarya; PFK group II contains only PP_i-PFKs from the genus Propionibacterium, plants, and amitochondriate protists; whereas group III consists of PFKs with either cosubstrate specificity, i.e., the PP_i-dependent enzymes from T. tenax and Amycolatopsis methanolica and the ATP-PFK from Streptomyces coelicolor. Comparative analyses of the pattern of conserved active-site residues strongly suggest that the group III PFKs originally bound PP_i as a cosubstrate.As first discovered in Entamoeba histolytica (27), in some members of all three domains of life (Bacteria, Eucarya, and Archaea), the first committing step of glycolysis, the phosphorylation of fructose 6-phosphate (Fru 6-P), is catalyzed not by common ATP-dependent phosphofructokinase (PFK) (ATP-PFK; EC 2.7.1.11) but by an enzyme which uses PP_i as a phosphoryl donor (PP_i-PFK; EC 2.7.1.90) (22–34). The only archaeal PP_i-PFK described so far is the enzyme of Thermoproteus tenax, a hyperthermophilic, anaerobic archaeon which is able to grow chemolithotrophically with CO₂, H₂, and S⁰, as well as chemo-organothrophically in the presence of S⁰ and carbohydrates (11, 41). As shown by enzymatic and in vivo studies (pulse-labeling experiments), T. tenax metabolizes glucose mainly via a variation of the Embden-Meyerhof-Parnas pathway distinguished by the reversible PP_i-PFK reaction (34, 35).In contrast to the virtually irreversible reaction catalyzed by the ATP-PFK, the phosphorylation by PP_i is reversible. Thus, for thermodynamic reasons, the PP_i-PFK should be able to replace the enzymes of both the forward (ATP-PFK) and reverse (fructose-bisphosphatase [FBPase]) reactions. Consistent with the presumed bivalent function of the PP_i-dependent enzyme, in prokaryotes and parasitic protists which possess PP_i-PFK, little, if any, ATP-PFK or FBPase activity is present. Strikingly, the PP_i-PFKs of these organisms proved to be nonallosteric, suggesting that the control of the carbon flux through the pathway is no longer exerted by the PFK in these organisms. A considerably different situation has been described for higher plants and the green alga Euglena gracilis, showing comparable ATP-PFK, FBPase, and PP_i-PFK activities and allosteric regulation of their PP_i-dependent enzyme by fructose 2,6-bisphosphate (12, 22). However, in most cases it is not obvious which physiological role PP_i-PFK performs: reversible catalysis of glycolysis/gluconeogenesis, increase of the energy yield of glycolysis under certain conditions in which the energy charge is low, or ATP-conservation in obligately fermentative organisms (22).Closely related to questions concerning the biological function of PP_i-PFKs is the matter of their evolutionary origin: are these enzymes the result of a secondary adaptation from ATP-PFKs, or do they represent an original phenotype, as suggested by their specificity for PP_i, which is thought to be an ancient source of metabolic energy (9, 18, 19, 26). Indicated by sequence similarity (3, 4), all known PP_i- and ATP-PFKs are homologous and therefore originated from a common ancestral root. From more recent studies of Streptomyces coelicolor PFK (4), the previous assumption of a single event which separated PP_i- and ATP-PFKs had to be revised in favor of a multiple differentiation, leaving open, however, the question of the primary or secondary origin of PP_i-PFK.Understanding of PFK evolution has been impaired by a lack of knowledge concerning archaeal PFK, although the existence of ATP-PFK (31), PP_i-PFK (34), and also ADP-dependent PFK (16, 31) in Archaea has been described. To address the evolution of PFK, we describe the PP_i-PFK from T. tenax and compare its sequence and structure to those of known bacterial and eucaryal PFK enzymes.     

Glycolytic activity in embryos of Pisum sativum and of non-dormant or dormant seeds of Avena sativa L. expressed through activities of PFK and PPi-PFK

Summary A quantative cytochemical assay for PP_i-PFK activity in the presence of Fru-2,6-P₂ is described along with its application to determine levels of activity in embryos of Pisum sativum and Avena sativa. The activity of ATP-PFK has also been studied in parallel as have PFK activities during the switch from dormant to non-dormant embryos in Avena sativa. PP_i-PFK activity, has been demonstrated in all tissues of Pisum sativum embryos and of Avena sativa embryos including the scutellum and the aleurone layers. The PP_i-PFK activity was greater than that of ATP-PFK in both dormant and non-dormant seeds though with only marginally more activity in the dormant as opposed to the non-dormant state.Abbreviations AMP adenosine monophosphate - ATP adenosine triphosphate - Fru-1,6-P₂ fructose 1,6-bisphosphate - Fru-2,6-P₂ fructose 2,6-bisphosphate - Fru-6-P fructose 6-phosphate - FB Pase 2 fructose 2,6-bisphosphatase (EC 3.1.3.46) - Gl-3-PD glyceraldehyde-3-phosphate dehydrogenase - NAD nicotinamide adenine dinucleotide - NBT nitroblue tetrazolium - PEP phosphoenolpyruvate - PFK 6-phosphofructokinase (EC 2.7.1.11) - PFK₂ 6-phosphofructo-2-kinase (EC 2.7.1.105) - PP_i pyrophosphate - PP_i-PFK pyrophosphate: fructose 6-phosphate 1-phosphotransferase (EC 2.7.1.90) - PVA polyvinyl alcohol (G04/140 Wacke Chemical Company)     

The Pyrophosphate-Dependent Phosphofructokinase of the Protist, Trichomonas vaginalis, and the Evolutionary Relationships of Protist Phosphofructokinases

The pyrophosphate-dependent phosphofructokinase (PP_i-PFK) of the amitochondriate protist Trichomonas vaginalis has been purified. The enzyme is a homotetramer of about 50 kDa subunits and is not subject to allosteric regulation. The protein was fragmented and a number of peptides were sequenced. Based on this information a PCR product was obtained from T. vaginalis gDNA and used to isolate corresponding cDNA and gDNA clones. Southern analysis indicated the presence of five genes. One open reading frame (ORF) was completely sequenced and for two others the 5′ half of the gene was determined. The sequences were highly similar. The complete ORF corresponded to a polypeptide of about 46 kDa. All the peptide sequences obtained were present in the derived sequences. The complete ORF was highly similar to that of other PFKs, primarily in its amino-terminal half. The T. vaginalis enzyme was most similar to PP_i-PFK of the mitochondriate heterolobosean, Naegleria fowleri. Most of the residues shown or assumed to be involved in substrate binding in other PP_i-PFKs were conserved in the T. vaginalis enzyme. Direct comparison and phylogenetic reconstruction revealed a significant divergence among PP_i-PFKs and related enzymes, which can be assigned to at least four distantly related groups, three of which contain enzymes of protists. The separation of these groups is supported with a high percentage of bootstrap proportions. The short T. vaginalis PFK shares a most recent common ancestor with the enzyme from N. fowleri. This pair is clearly separated from a group comprising the long (>60-kDa) enzymes from Giardia lamblia, Entamoeba histolytica pfk2, the spirochaetes Borrelia burgdorferi and Trepomena pallidum, as well as the α- and β-subunits of plant PP_i-PFKs. The third group (``X') containing protist sequences includes the glycosomal ATP-PFK of Trypanosoma brucei, E. histolytica pfk1, and a second sequence from B. burgdorferi. The fourth group (``Y') comprises cyanobacterial and high-G + C, Gram-positive eubacterial sequences. The well-studied PP_i-PFK of Propionibacterium freudenreichii is highly divergent and cannot be assigned to any of these groups. These four groups are well separated from typical ATP-PFKs, the phylogenetic analysis of which confirmed relationships established earlier. These findings indicate a complex history of a key step of glycolysis in protists with several early gene duplications and possible horizontal gene transfers. Received: 5 December 1997 / Accepted: 18 March 1998     

Protein import into hydrogenosomes of Trichomonas vaginalis involves both N-terminal and internal targeting signals: a case study of thioredoxin reductases

The parabasalian flagellate Trichomonas vaginalis harbors mitochondrion-related and H₂-producing organelles of anaerobic ATP synthesis, called hydrogenosomes, which harbor oxygen-sensitive enzymes essential to its pyruvate metabolism. In the human urogenital tract, however, T. vaginalis is regularly exposed to low oxygen concentrations and therefore must possess antioxidant systems protecting the organellar environment against the detrimental effects of molecular oxygen and reactive oxygen species. We have identified two closely related hydrogenosomal thioredoxin reductases (TrxRs), the hitherto-missing component of a thioredoxin-linked hydrogenosomal antioxidant system. One of the two hydrogenosomal TrxR isoforms, TrxRh1, carried an N-terminal extension resembling known hydrogenosomal targeting signals. Expression of hemagglutinin-tagged TrxRh1 in transfected T. vaginalis cells revealed that its N-terminal extension was necessary to import the protein into the organelles. The second hydrogenosomal TrxR isoform, TrxRh2, had no N-terminal targeting signal but was nonetheless efficiently targeted to hydrogenosomes. N-terminal presequences from hydrogenosomal proteins with known processing sites, i.e., the alpha subunit of succinyl coenzyme A synthetase (SCSα) and pyruvate:ferredoxin oxidoreductase A, were investigated for their ability to direct mature TrxRh1 to hydrogenosomes. Neither presequence directed TrxRh1 to hydrogenosomes, indicating that neither extension is, by itself, sufficient for hydrogenosomal targeting. Moreover, SCSα lacking its N-terminal extension was efficiently imported into hydrogenosomes, indicating that this extension is not required for import of this major hydrogenosomal protein. The finding that some hydrogenosomal enzymes require N-terminal signals for import but that in others the N-terminal extension is not necessary for targeting indicates the presence of additional targeting signals within the mature subunits of several hydrogenosome-localized proteins.     

Regulation and roles for alternative pathways of hexose metabolism in plants

Plant cells have two cytoplasmic pathways of glycolysis and gluconeogenesis for the reversible interconversion of fructose 6-phosphate (F-6-P) and fructose 1,6-bisphosphate (F-1,6-P₂). One pathway is described as a maintenance pathway that is catalyzed by a nucleotide triphosphate-dependent phosphofructokinase (EC 2.7.1.11; ATP-PFK) glycolytically and a F-1,6 bisphosphatase (EC 3.1.3.11) gluconeogenically. These are non-equilibrium reactions that are energy consuming. The second pathway, described as an adaptive pathway, is catalyzed by a readily reversible pyrophosphate-dependent phosphofructokinase (EC 2.7.1.90; PP-PFK) in an equilibrium reaction that conserves energy through the utilization and the synthesis of pyrophosphate. A constitutive regulator cycle is also present for the synthesis and hydrolysis of fructose 2,6-bisphosphate (F-2,6-P₂) via a 2-kinase and a 2-phosphatase, respectively. The pathway catalyzed by the ATP-PFK and F-1,6-bisphosphatase, the maintenance pathway, is fairly constant in maximum activity in various plant tissues and shows less regulation by F-2,6-P₂. Plants use F-2,6-P₂ initially to regulate the adaptive pathway at the reversible PP_i-PFK step. The adaptive pathway, catalyzed by PP_i-PFK, varies in maximum activity with a variety of phenomena such as plant development or changing biological and physical environments. Plants can change F-2,6-P₂ levels rapidly, in less than 1 min when subjected to rapid environmental change, or change levels slowly over periods of hours and days as tissues develop. Both types of change enable plants to cope with the environmental and developmental changes that occur during their lifetimes. The two pathways of sugar metabolism can be efficiently linked by the cycling of uridylates and pyrophosphate required for sucrose breakdown via a proposed sucrose synthase pathway. The breakdown of sucrose via the sucrose synthase pathway requires half the net energy of breakdown via the invertase pathway. Pyrophosphate occurs in plant tissues as a substrate pool for biosynthetic reactions such as the PP_i-PFK or uridine diphosphate glucose pyrophosphorylase (EC 2.7.7.9; UDPG pyrophosphorylase) that function in the breakdown of imported sucrose. Also, pyrophosphate links the two glycolytic/gluco-neogenic pathways; and in a reciprocal manner pyrophosphate is produced as an energy source during gluconeogenic carbon flow from F-1,6-P₂ toward sucrose synthesis.     

The N‐Terminal Sequences of Four Major Hydrogenosomal Proteins Are Not Essential for Import into Hydrogenosomes of Trichomonas vaginalis

The human pathogen Trichomonas vaginalis harbors hydrogenosomes, organelles of mitochondrial origin that generate ATP through hydrogen‐producing fermentations. They contain neither genome nor translation machinery, but approximately 500 proteins that are imported from the cytosol. In contrast to well‐studied organelles like Saccharomyces mitochondria, very little is known about how proteins are transported across the two membranes enclosing the hydrogenosomal matrix. Recent studies indicate that—in addition to N‐terminal transit peptides—internal targeting signals might be more common in hydrogenosomes than in mitochondria. To further characterize the extent to which N‐terminal and internal motifs mediate hydrogenosomal protein targeting, we transfected Trichomonas with 24 hemagglutinin (HA) tag fusion constructs, encompassing 13 different hydrogenosomal and cytosolic proteins of the parasite. Hydrogenosomal targeting of these proteins was analyzed by subcellular fractionation and independently by immunofluorescent localization. The investigated proteins include some of the most abundant hydrogenosomal proteins, such as pyruvate ferredoxin oxidoreductase (PFO), which possesses an amino‐terminal targeting signal that is processed on import into hydrogenosomes, but is shown here not to be required for import into hydrogenosomes. Our results demonstrate that the deletion of N‐terminal signals of hydrogenosomal precursors generally has little, if any, influence upon import into hydrogenosomes. Although the necessary and sufficient signals for hydrogenosomal import recognition appear complex, targeting to the organelle is still highly specific, as demonstrated by the finding that six HA‐tagged glycolytic enzymes, highly expressed under the same promoter as other constructs studied here, localized exclusively to the cytosol and did not associate with hydrogenosomes.     

The expression of monocyte chemotactic protein (MCP-1) in human vascular endotheliumin vitro andin vivo

Total 6-phosphofructo-1-kinase (PFK) activity, amounts of each type of PFK subunit, and levels of fructose-2,6-P₂ in the cerebral cortex, midbrain, pons-medulla, and cerebellum of 3, 12, and 25 month rats were measured. Further, the role of fructose-2,6-P₂ in the regulation of brain PFK activity was examined. A positive correlation was found to exist between the reported losses of glucose utilization as measured by 2-deoxy-D-glucose uptake and PFK activity in each region. That is, both parameters decreased to their lowest level by 12 months of age and remained decreased and fairly constant thereafter. Fructose-2,6-P₂ levels did not appear to directly correlate with regional changes in glucose utilization. Also, region-specific and age-related alterations of the PFK subunits were found although these changes apparently did not correlate with decreased glucose utilization. Brain PFK is apparently saturated with fructose-2,6-P₂ due to the high endogenous levels, and it contains a large proportion of the C-type subunit which dampens catalytic efficiency. Consequently, brain PFK could exist in a conformational state such that it can readily consume fructose-6-P rather than in an inhibited state requiring activation. This may explain, in part, the ability of brain to efficiently but conservatively utilize available glucose in energy production.Abbreviations fructose-2,6-P₂ D-fructose 2,6-bisphosphate - fructose-6-P D-fructose 6-phosphate - PAGE Polyacrylamide Gel Electrophoresis - PFK 6-phosphofructo-1-kinase - PP_i-PFK Pyrophosphate-dependent Phosphofructokinase, ribose-1,5-P₂, ribose-1,5-bisphosphate - SDS Sodium Dodecyl Sulfate     

Different Physiological Roles of ATP- and PPi-Dependent Phosphofructokinase Isoenzymes in the Methylotrophic Actinomycete Amycolatopsis methanolica

Cells of the actinomycete Amycolatopsis methanolica grown on glucose possess only a single, exclusively PP(i)-dependent phosphofructokinase (PP(i)-PFK) (A. M. C. R. Alves, G. J. W. Euverink, H. J. Hektor, J. van der Vlag, W. Vrijbloed, D.H.A. Hondmann, J. Visser, and L. Dijkhuizen, J. Bacteriol. 176:6827-6835, 1994). When this methylotrophic bacterium is grown on one-carbon (C(1)) compounds (e.g., methanol), an ATP-dependent phosphofructokinase (ATP-PFK) activity is specifically induced, completely replacing the PP(i)-PFK. The two A. methanolica PFK isoenzymes have very distinct functions, namely, in the metabolism of C(6) and C(1) carbon substrates. This is the first report providing biochemical evidence for the presence and physiological roles of PP(i)-PFK and ATP-PFK isoenzymes in a bacterium. The novel ATP-PFK enzyme was purified to homogeneity and characterized in detail at the biochemical and molecular levels. The A. methanolica ATP-PFK and PP(i)-PFK proteins possess a low level of amino acid sequence similarity (24%), clearly showing that the two proteins are not the result of a gene duplication event. PP(i)-PFK is closely related to other (putative) actinomycete PFK enzymes. Surprisingly, the A. methanolica ATP-PFK is most similar to ATP-PFK from the protozoon Trypanosoma brucei and PP(i)-PFK proteins from the bacteria Borrelia burgdorferi and Treponema pallidum, both spirochetes, very distinct from actinomycetes. The data thus suggest that A. methanolica obtained the ATP-PFK-encoding gene via a lateral gene transfer event.     

The Crystal Structure of ATP-bound Phosphofructokinase from Trypanosoma brucei Reveals Conformational Transitions Different from those of Other Phosphofructokinases

The crystal structure of the ATP-bound form of the tetrameric phosphofructokinase (PFK) from Trypanosoma brucei enables detailed comparisons to be made with the structures of the apoenzyme form of the same enzyme, as well as with those of bacterial ATP-dependent and PP_i-dependent PFKs. The active site of T. brucei PFK (which is strictly ATP-dependent but belongs to the PP_i-dependent family by sequence similarities) is a chimera of the two types of PFK. In particular, the active site of T. brucei PFK possesses amino acid residues and structural features characteristic of both types of PFK. Conformational changes upon ATP binding are observed that include the opening of the active site to accommodate the two substrates, MgATP and fructose 6-phosphate, and a dramatic ordering of the C-terminal helices, which act like reaching arms to hold the tetramer together. These conformational transitions are fundamentally different from those of other ATP-dependent PFKs. The substantial differences in structure and mechanism of T. brucei PFK compared with bacterial and mammalian PFKs give optimism for the discovery of species-specific drugs for the treatment of diseases caused by protist parasites of the trypanosomatid family.     

Investigation of the Secretory Pathway in Trichomonas vaginalis Argues against a Moonlighting Function of Hydrogenosomal Enzymes

The enzymes pyruvate ferredoxin oxidoreductase (PFO), malic enzyme (ME), and the α‐ and β‐subunits of succinyl‐CoA synthetase (SCS) catalyze key steps of energy metabolism in Trichomonas vaginalis hydrogenosomes. These proteins have also been characterized as the adhesins AP120 (PFO), AP65 (ME), AP33, and AP51 (α‐ and β‐SCS), which are localized on the cell surface and mediate the T. vaginalis cytoadherence. However, the mechanisms that facilitate the targeting of these proteins to the cell surface via the secretory pathway and/or to hydrogenosomes are not known. Here we adapted an in vivo biotinylation system to perform highly sensitive tracing of protein trafficking in T. vaginalis. We showed that α‐ and β‐SCS are biotinylated in the cytosol and imported exclusively into the hydrogenosomes. Neither α‐ nor β‐SCS is biotinylated in the endoplasmic reticulum and delivered to the cell surface via the secretory pathway. In contrast, two surface proteins, tetratricopeptide domain‐containing membrane‐associated protein and tetraspanin family surface protein, as well as soluble‐secreted β‐amylase‐1 are biotinylated in the endoplasmic reticulum and delivered through the secretory pathway to their final destinations. Taken together, these results demonstrate that the α‐ and β‐SCS subunits are targeted only to the hydrogenosomes, which argues against their putative moonlighting function.     

Discovery of PPi-type Phosphoenolpyruvate Carboxykinase Genes in Eukaryotes and Bacteria

Phosphoenolpyruvate carboxykinase (PEPCK) is one of the pivotal enzymes that regulates the carbon flow of the central metabolism by fixing CO₂ to phosphoenolpyruvate (PEP) to produce oxaloacetate or vice versa. Whereas ATP- and GTP-type PEPCKs have been well studied, and their protein identities are established, inorganic pyrophosphate (PP_i)-type PEPCK (PP_i-PEPCK) is poorly characterized. Despite extensive enzymological studies, its protein identity and encoding gene remain unknown. In this study, PP_i-PEPCK has been identified for the first time from a eukaryotic human parasite, Entamoeba histolytica, by conventional purification and mass spectrometric identification of the native enzyme, followed by demonstration of its enzymatic activity. A homolog of the amebic PP_i-PEPCK from an anaerobic bacterium Propionibacterium freudenreichii subsp. shermanii also exhibited PP_i-PEPCK activity. The primary structure of PP_i-PEPCK has no similarity to the functional homologs ATP/GTP-PEPCKs and PEP carboxylase, strongly suggesting that PP_i-PEPCK arose independently from the other functional homologues and very likely has unique catalytic sites. PP_i-PEPCK homologs were found in a variety of bacteria and some eukaryotes but not in archaea. The molecular identification of this long forgotten enzyme shows us the diversity and functional redundancy of enzymes involved in the central metabolism and can help us to understand the central metabolism more deeply.     

Functional and fluorescence analyses of tryptophan residues in H+-pyrophosphatase of Clostridium tetani

Homodimeric proton-translocating pyrophosphatase (H⁺-PPase; EC 3.6.1.1) maintains the cytoplasmic pH homeostasis of many bacteria and higher plants by coupling pyrophosphate (PP_i) hydrolysis and proton translocation. H⁺-PPase accommodates several essential motifs involved in the catalytic mechanism, including the PP_i binding motif and Acidic I and II motifs. In this study, 3 intrinsic tryptophan residues, Trp-75, Trp-365, and Trp-602, in H⁺-PPase from Clostridium tetani were used as internal probes to monitor the local conformational state of the periplasm domain, transmembrane region, and cytoplasmic domain, respectively. Upon binding of the substrate analog Mg-imidodiphosphate (Mg-IDP), local structural changes prevented the modification of tryptophan residues by N-bromosuccinimide (NBS), especially at Trp-602. Following Mg-P_i binding, Trp-75 and Trp-365, but not Trp-602, were slightly protected from structural modifications by NBS. These results reveal the conformation of H⁺-PPase is distinct in the presence of different ligands. Moreover, analyses of the Stern-Volmer relationship and steady-state fluorescence anisotropy also indicate that the local structure around Trp-602 is more exposed to solvent and varied under different environments. In addition, Trp-602 was identified to be a crucial residue in the H⁺-PPase that may potentially be involved in stabilizing the structure of the catalytic region by site-directed mutagenesis analysis.     

A method for the concentration and for the colorimetric determination of nanomoles of inorganic pyrophosphate

A generally applicable, inexpensive, and sensitive method for the determination of inorganic pyrophosphate (PP_i) was developed. PP_i was quantitatively separable from solution even in nanomolar concentrations by filtration through a membrane filter in the presence of CaCl₂ and KF. The separated PP_i was dissolved by immersing the filter in 0.5 n H₂SO₄. Inorganic phosphate (P_i) was removed by precipitating it as a phosphomolybdate-triethylamine complex and the PP_i was measured as a green pyrophosphomolybdate in the presence of 2-mercaptoethanol. Nucleotides and phosphate esters do not react. PP_i can be accurately assayed even when there is a 10⁴-fold excess of P_i. Trimetaphosphate, tripolyphosphate, and tetrapolyphosphate also give this green color, but the rate of the color formation is 50 times slower than that with PP_i. Thus this interference of the polyphosphates can be eliminated or the polyphosphates can be assayed simultaneously with the PP_i in the same sample.     

Utilization of PPi as an Energy Source by a Clostridium sp

The growth of an anaerobic, spore-forming rod we have isolated from the cockroach gut after enrichment on media containing PP_i was stimulated by the presence of PP_i. The doubling time decreased and cell yield increased proportionately to PP_i concentrations of up to 0.35%. A similar stimulation of the growth of Desulfotomaculum sp. by PP_i has been reported. The PP_i-stimulated Clostridium sp. fermented a number of sugars with the production of hydrogen, acetate, and butyrate, with smaller amounts of ethanol and butanol being produced from some substrates. The fermentation products were not qualitatively changed by the presence of PP_i, but significantly more hydrogen was produced. The organism contained several of the enzymes previously reported from Entamoeba sp. and Propionibacterium sp., in which PP_i serves as a source of a high-energy bond in place of ATP. These include significant amounts of pyruvate-phosphate dikinase and phosphoenolpyruvate carboxytransphosphorylase. The activities of many of the catabolic enzymes of the organism, as well as of its phosphatases and pyrophosphatase, were similar whether it was grown in the presence or absence of PP_i. The organism did not accumulate intracellular polyphosphate granules but stored large amounts of glycogen.     

A sensitive method for measuring pyrophosphate in the presence of a 10,000-fold excess of orthophosphate using inorganic pyrophosphatase

A simple method for measuring PP_i at concentrations down to 2 μm has been devised using the ability of inorganic pyrophosphatase to be inactivated by fluoride in the presence of PP_i. Orthophosphate (20 mm) and a number of other compounds did not interfere with the assay. The applicability of the method for direct measurement of PP_i in urine is demonstrated.     

Targeting of tail‐anchored proteins to Trichomonas vaginalis hydrogenosomes

Tail‐anchored (TA) proteins are membrane proteins that are found in all domains of life. They consist of an N‐terminal domain that performs various functions and a single transmembrane domain (TMD) near the C‐terminus. In eukaryotes, TA proteins are targeted to the membranes of mitochondria, the endoplasmic reticulum (ER), peroxisomes and in plants, chloroplasts. The targeting of these proteins to their specific destinations correlates with the properties of the C‐terminal domain, mainly the TMD hydrophobicity and the net charge of the flanking regions. Trichomonas vaginalis is a human parasite that has adapted to oxygen‐poor environment. This adaptation is reflected by the presence of highly modified mitochondria (hydrogenosomes) and the absence of peroxisomes. The proteome of hydrogenosomes is considerably reduced; however, our bioinformatic analysis predicted 120 putative hydrogenosomal TA proteins. Seven proteins were selected to prove their localization. The elimination of the net positive charge in the C‐tail of the hydrogenosomal TA4 protein resulted in its dual localization to hydrogenosomes and the ER, causing changes in ER morphology. Domain mutation and swap experiments with hydrogenosomal (TA4) and ER (TAPDI) proteins indicated that the general principles for specific targeting are conserved across eukaryotic lineages, including T. vaginalis; however, there are also significant lineage‐specific differences.     

A new, convenient method for the rapid analysis of inorganic pyrophosphate.

A new method for the rapid analysis of inorganic pyrophosphate (PP_i) which utilizes the enzyme ATP sulfurylase is described. All components of the assay system are commercially available and inexpensive. The assay is linear over the range of 0.5–50.0 nmol of PP_i and is not affected by inorganic phosphate. ATP and PP_i can both be analyzed using this method.     

Catalysis by isolated β-subunits of the ATP Synthase/ATPase from Thermophilic bacillus PS3. Hydrolysis of Pyrophosphate

Although the capacity of isolated β-subunits of the ATP synthase/ATPase to perform catalysis has been extensively studied, the results have not conclusively shown that the subunits are catalytically active. Since soluble F₁ of mitochondrial H⁺-ATPase can bind inorganic pyrophosphate (PP_i) and synthesize PP_i from medium phosphate, we examined if purified His-tagged β-subunits from Thermophilic bacillus PS3 can hydrolyze PP_i. The difference spectra in the near UV CD of β-subunits with and without PP_i show that PP_i binds to the subunits. Other studies show that β-subunits hydrolyze [³²P] PP_i through a Mg²⁺-dependent process with an optimal pH of 8.3. Free Mg²⁺ is required for maximal hydrolytic rates. The Km for PP_i is 75 μM and the Vmax is 800 pmol/min/mg. ATP is a weak inhibitor of the reaction, it diminishes the Vmax and increases the Km for PP_i. Thus, isolated β-subunits are catalytically competent with PP_i as substrate; apparently, the assembly of β-subunits into the ATPase complex changes substrate specificity, and leads to an increase in catalytic rates.