Identification of the inorganic pyrophosphate metabolizing, ATP substituting pathway in mammalian spermatozoa

Inorganic pyrophosphate (PPi) is generated by ATP hydrolysis in the cells and also present in extracellular matrix, cartilage and bodily fluids. Fueling an alternative pathway for energy production in cells, PPi is hydrolyzed by inorganic pyrophosphatase (PPA1) in a highly exergonic reaction that can under certain conditions substitute for ATP-derived energy. Recombinant PPA1 is used for energy-regeneration in the cell-free systems used to study the zymology of ATP-dependent ubiquitin-proteasome system, including the role of sperm-borne proteasomes in mammalian fertilization. Inspired by an observation of reduced in vitro fertilization (IVF) rates in the presence of external, recombinant PPA1, this study reveals, for the first time, the presence of PPi, PPA1 and PPi transporter, progressive ankylosis protein ANKH in mammalian spermatozoa. Addition of PPi during porcine IVF increased fertilization rates significantly and in a dose-dependent manner. Fluorometric assay detected high levels of PPi in porcine seminal plasma, oviductal fluid and spermatozoa. Immunofluorescence detected PPA1 in the postacrosomal sheath (PAS) and connecting piece of boar spermatozoa; ANKH was present in the sperm head PAS and equatorial segment. Both ANKH and PPA1 were also detected in human and mouse spermatozoa, and in porcine spermatids. Higher proteasomal-proteolytic activity, indispensable for fertilization, was measured in spermatozoa preserved with PPi. The identification of an alternative, PPi dependent pathway for ATP production in spermatozoa elevates our understanding of sperm physiology and sets the stage for the improvement of semen extenders, storage media and IVF media for animal biotechnology and human assisted reproductive therapies.     

Microcytosis in ank/ank mice and the role of ANKH in promoting erythroid differentiation

Progressive ankylosis (Ank and the human homolog, ANKH) is a transmembrane protein which regulates transport of inorganic pyrophosphate (PPi). ank/ank mice with a mutated ank gene, have calcification and bone ankylosis of the affected joints. In the course of studying these mutant mice, we found that they have microcytosis. These mutant mice have lower mean red blood cell volume (MCV) and lower hemoglobin content in red cells (mean corpuscular hemoglobin, MCH) than normal mice. Using quantitative real-time PCR analysis, we showed that Ank was expressed in the E/Meg bipotent precursor, BFU-E, CFU-E, but there was no Ank expression in the hemoglobinizing erythroblasts. Stable ANKH transfectants in K562 cells highly expressed two immature erythroid cell markers, E-cadherin and endoglin. Enhanced Erythropoietin (Epo) expression and downregulation of SHP-1 were detected in these transfectants. Consequently, the autocrine Epo-EpoR signaling pathway was activated, as evidenced by higher p-Tyr JAK2, p-Tyr EpoR and p-Tyr STAT5B in the ANKH transfectants. Our results revealed a novel function of ANKH in the promotion of early erythroid differentiation in K562 cells. We also showed that ank/ank mice have lower serum levels of Epo than the normal littermates, and this is the likely cause of microcytosis in these mutant mice.     

Phosphoribosyl pyrophosphate and the measurement on inorganic pyrophosphate in plant tissues

This work provides further evidence that plants contain appreciable amounts of inorganic pyrophosphate (PPi), and that breakdown of phosphoribosyl pyrophosphate (PPRibP) does not contribute significantly to the PPi detected in plant extracts. Inorganic pyrophosphate in extracts of the roots of Pisum sativum L., clubs of the spadices of Arum maculatum L., and the developing endosperm of Zea mays L. was assayed with pyrophosphate fructose 6-phosphate 1-phosphotransferase (EC 2.7.1.90), and with sulphate adenyltransferase (EC 2.7.7.4). The two different assays gave the same value for PPi content, and for recovery of added PPi. It was shown that PPRibP is converted to PPi during the extraction of PPi. However, the amounts of PPRibP in clubs of A. maculatum and the developing endosperm of Z. mays were negligible in comparison with the contents of PPi.Abbreviations EDTA ethylenediaminetetraacetic acid - PFK(PPi) pyrophosphate fructose 6-phosphate 1-phosphotransferase - PPi inorganic pyrophosphate - PPRibP phosphoribosyl pyrophosphate     

Effects of 2,4-dinitrophenol and anoxia on the inorganic-pyrophosphate content of the spadix of Arum maculatum and the root apices of Pisum sativum

This work was done to determine whether the inorganic-pyrophosphate (PPi) content of plant tissues changes when the rate of glycolysis is altered. Treatment of excised clubs of the spadix of Arum maculatum L. and root apices of Pisum sativum L. with 2,4-dinitrophenol increased the rates of respiration but had no detectable effects on PPi contents. When the two tissues were subjected to up to 60 min anoxia, no changes in PPi were detected. Anoxia was shown to lead to a fall in ATP and concomitant rises in ADP and AMP in pea roots. It is argued (i) that variation in the rate of glycolysis was not accompanied by detectable changes in PPi content, (ii) that this observation does not favour the view that pyrophosphate fructose 6-phosphate 1-phosphotransferase mediates appreciable entry into glycolysis, and (iii) that PPi content can be maintained when respiratory-chain phosphorylation is inhibited.Abbreviations FW fresh weight - PFK(PPi) pyrophosphate fructose 6-phosphate 1-phosphotransferase - PPi inorganic pyrophosphate     

Molecular dynamics simulation studies of the effect of phosphocitrate on crystal-induced membranolysis

In this study, following our earlier work on calcium pyrophosphate dihydrate (CPPD) crystal-induced membranolysis, we demonstrate, using the CHARMM method of molecular dynamics simulation, the protective role of phosphocitrate (PC) against solvated dimyristoyl phosphatidylcholine phospholipid bilayer disintegration on contact with the CPPD crystal. Our molecular dynamics simulations studies show that coverage of the CPPD crystal with a layer of phosphocitrate molecules results in the conservation of phospholipid bilayer integrity. We show that the rupture of the lipid bilayer in presence of CPPD and the protective effect of PC are primarily due to electrostatic interactions. The protective role of PC, which may also play an important and potentially therapeutic function against crystal-induced membranolysis is also discussed.     

Augmentation of inorganic pyrophosphate elaboration in cartilage by serum factors

The disordered production of inorganic pyrophosphate (PPi) by articular cartilage is thought to have an important role in the pathogenesis of calcium pyrophosphate dihydrate deposition disease and perhaps osteoarthritis. We have previously shown that fetal calf serum added to the culture media of porcine articular cartilage explants increases the elaboration of PPi into the ambient media. We have examined this PPi stimulatory activity by studying the effects of adult human serum (HS), serum derived from adult human plasma (HP), and an acid-alcohol extract of human platelets (PE) on PPi production in cartilage organ culture. Ten percent HS produces a 1.4-fold increase in PPi production after 48 h of culture, while cartilage incubated in media containing 10% HP produces no more PPi than that incubated in media alone. PE stimulates a mean 2-fold increase in PPi production at 48 h in the presence of low concentrations of HP, and has no effect alone. It does not appear to up-regulate the activity of the ectoenzyme nucleoside triphosphate pyrophosphohydrolase (NTPPPH), nor does it promote the release of enzyme substrate into the extracellular space. Cartilage exposed to 0.5% HP and PE has 1.51 +/- 0.36 units of NTPPPH activity whereas cartilage exposed to 0.5% HP alone has 1.52 +/- 0.41 units of enzyme activity. PE does not increase the release of [14C]adenine-labeled compounds into the media. Approximately 13% of soluble 14C counts was found in the media of chondrocytes treated with PE while 18% of counts was released in the presence of HP alone. We have demonstrated a factor or factors present in FCS, HS, and an acid-ethanol extract of human platelets which represent(s) the first known physiologic modulators of PPi production in articular cartilage and may increase PPi production without affecting NTPPPH activity.     

Thermodynamics, kinetics, and mechanism in yeast inorganic pyrophosphatase catalysis of inorganic pyrophosphate: inorganic phosphate equilibration

We have developed two methods for quantitatively measuring inorganic pyrophosphate (PPi) in the presence of 10(3)--10(4) molar excesses of inorganic phosphate (Pi) and used them to measure the extent of enzyme-bound pyrophosphate (EPPi) formation in solutions of yeast inorganic pyrophosphatase and Pi. We have also measured the rate of enzyme-catalyzed H2O--phosphate oxygen exchange. We find both processes to have essentially identical dependence on Mg2+ and Pi concentrations, thus providing important confirmation for the recent proposal by Janson et al. (1979) that oxygen exchange proceeds via EPPi formation. Our results are consistent with a model in which three Mg2+ per active site are required for EPPi formation but inconsistent with a model requiring only two Mg2+ per active site and permit the formulation of an overall scheme for inorganic pyrophosphatase catalysis of PPi--Pi equilibration as well as the evaluation of equilibrium and rate constants in this scheme. The major results and conclusions of our work are the following: (a) the equilibrium constant for PPi (enzyme-bound) in equilibrium with 2Pi (enzyme-bound) is 4.8; (b) following PPi hydrolysis, the first released Pi contains an oxygen from solvent water; (c) the steps for PPi hydrolysis on the enzyme and for release of both product Pi's are all partially rate determining in overall enzyme-catalyzed PPi hydrolysis; (d) PPi formation on the enzyme is rate determining for H2O--Pi oxygen exchange; (e) PPi dissociation from the enzyme is very slow and is the rate-determining step in Pi--PPi exchange (Cohn, 1958; Janson et al., 1979). This also accounts for the observation that the calculated dissociation constant for MgPPi complex binding to enzyme is considerably lower than the measured Km for enzyme-catalyzed MgPPi hydrolysis.     

Osteoarthritis, calcium pyrophosphate deposition disease, and osseous infection in Old World primates.

Uncertainties as to the nature and implications of osteoarthritis and calcium pyrophosphate deposition disease (CPPD) in primates were subject to critical review through examination of 153 prosimians and 1,250 Old World non-prosimian primates. Osteoarthritis, calcium pyrophosphate deposition disease, and infectious arthritis/osteomyelitis were relatively rare phenomena, affecting only 1.7% and 2.5%, respectively, of free-ranging prosimians and other Old World non-prosimian primates. Frequency of infection in Indri and Presbytis appears to reflect a unique susceptibility or exposure. Papio may have a unique predisposition to CPPD. The dichotomy (frequency and joint distribution) between free-ranging and artificially restrained animals suggests caution in interpretation of osteoarthritis or CPPD in non-free-ranging animals.     

Calcium pyrophosphate dihydrate crystal associated induction of neutrophil activation and repression of TNF-alpha-induced apoptosis is mediated by the p38 MAP kinase

The role of p38 mitogen-activated protein (MAP) kinase in the activation of human neutrophils and repression of TNF-alpha-induced apoptosis in response to plasma opsonized crystals of calcium pyrophosphate dihydrate (CPPD) was investigated. We monitored the endogenous phosphotransferase activity of p38 kinase in neutrophils stimulated with CPPD crystals (25 mg/ml) alone or in the presence of TNF-alpha (10 ng/ml), and with TNF-alpha alone. CPPD crystals induced a 2-fold activation of p38 kinase activity over the basal activity that was observed in untreated neutrophils. Furthermore, CPPD crystals repressed the TNF-alpha associated 6-fold induction of p38 kinase phosphotransferase activity to levels associated with CPPD crystal incubation alone in a PD98059 (20 ng/ml) and Wortmannin (100 nM) sensitive manner. Inhibition of CPPD crystal-induced activation of the neutrophil inflammatory response as measured by chemiluminescence, superoxide anion generation and degranulation as determined by myeloperoxidase and lysozyme release was observed in the presence of the specific p38 MAP kinase inhibitor SB203580 (5 microM). CPPD crystal associated repression of TNF-alpha-induced activation of neutrophil apoptosis as determined by DNA fragmentation correlated with the CPPD crystal mediated inhibition of p38 kinase activity, probably through crystal inhibition of caspase 3. Together, our results indicate that the CPPD crystal associated inflammatory response is regulated through the activation of p38 kinase to sub-apoptotic levels, and that the repression of the TNF-alpha-induced apoptosis program in neutrophils is mediated via the repression of caspase 3 mediated apoptosis-associated p38 kinase activity.     

Pyrophosphate:fructose 6-phosphate 1-phosphotransferase and glycolysis in non-photosynthetic tissues of higher plants.

The activity of pyrophosphate:fructose-6-phosphate 1-phosphotransferase [PFK (PPi); EC 2.7.1.90] in extracts of the storage tissues of leek (Allium porrum), beetroot (Beta vulgaris) and roots of darnel (Lolium temulentum) exceeded 0.15 mumol/min per g fresh wt. As net flux from fructose 1,6-bisphosphate to fructose 6-phosphate in these tissues is unlikely, it is suggested that PFK (PPi) does not contribute to gluconeogenesis or starch synthesis. The maximum catalytic activities of PFK (PPi) in apex, stele and cortex of the root of pea (Pisum sativum) and in the developing and the thermogenic club of the spadix of cuckoo-pint (Arum maculatum) were measured and compared with those of phosphofructokinase, and to estimates of the rates of carbohydrate oxidation. PPi and fructose 2,6-bisphosphate in Arum clubs were measured. The above measurements are consistent with a glycolytic role for PFK (PPi) in tissues where there is marked biosynthesis, but not in the thermogenic club of Arum. The possibility that PFK (PPi) is a means of synthesizing pyrophosphate is discussed.     

Promotion of neutrophil adherence to human osteoblasts by microcrystals and f-Met-Leu-Phe

Human osteoblast-like cells (hOB) stimulated by monosodium urate monohydrate (MSUM) or calcium pyrophosphate dihydrate (CPPD) microcrystals produce the neutrophil chemoattractant IL-8. We investigated whether human neutrophils can adhere to hOB and respond to hOB preactivated by MSUM, CPPD, or by f-Met-Leu-Phe (fMLP). Confluent hOB were coincubated with human blood neutrophils in the presence of MSUM, CPPD or fMLP. MSUM, CPPD, and fMLP stimulated a significant adherence of neutrophils to hOB after a 1h incubation. This effect was not abrogated by pretreating the cells with an anti-CD18 mAb. MSUM stimulated more efficiently the adherence of neutrophils to non-preactivated hOB while CPPD were more efficient when hOB were preactivated. Crystal-free conditioned media from MSUM- or CPPD-stimulated hOB mobilized intracellular free calcium in human neutrophils. Thus, microcrystals were powerful promoters of neutrophil adherence to hOB via a CD18-independent mechanism. The bacterial peptide fMLP also stimulated the adherence of neutrophils to hOB. Functional neutrophil-hOB interactions could be important in bone pathophysiology of crystal- or infection-associated arthritis.     

Stabilization of stannous pyrophosphate kits with gentisic acid

We evaluated the ability of gentisic acid, an antioxidant, to stabilize stannous pyrophosphate (Sn:PPi) kits and extend the shelf-life of the kit after reconstitution. In vitro studies showed that gentisic acid (0.5 mg/mL) stabilized the stannous ion against oxidation by various levels of exogenous hydrogen peroxide. In patients who received stabilized Sn:PPi for in vivo red blood cell labelling, the left ventricle-to-background activity ratio was significantly higher than that in patients who received a standard formulation of Sn:PPi. Gentisic acid is now used routinely in the Sn:PPi kit formulation in this institution.     

The interaction of adenyl-5'-yl imidodiphosphate and PPi with actomyosin

We previously studied the equilibrium binding of ADP, adenyl-5'-yl imidodiphosphate (AMP-PNP), and inorganic pyrophosphate (PPi) to actomyosin-subfragment 1 (acto.S-1) and found that AMP-PNP and PPi bind considerably more weakly to acto.S-1 than does ADP. In this study, we investigated the pre-steady-state kinetics of the binding of AMP-PNP and PPi to acto.S-1 and of S-1.AMP-PNP and S-1.PPi to actin to determine if the pre-steady-state kinetic data are consistent with our previous equilibrium data. We find that the kinetic data are consistent with the equilibrium data and agree with a model in which acto.S-1 forms a collision intermediate with the ATP analog, followed by a slower conformational change to a ternary complex that rapidly dissociates into actin and the S-1.ATP analog. Although this scheme fits the AMP-PNP as well as the PPi data, we find that the isomerization of the collision intermediate to the ternary complex is approximately 10 times faster in the presence of PPi than in the presence of AMP-PNP, which is consistent with previous physiological studies (Schoenberg, M., and Eisenberg, E. (1985) Biophys. J. 48, 863-872).     

Characterization of pyrophosphate exchange by the reconstituted adenine nucleotide translocator from mitochondria

The transport of inorganic pyrophosphate (PPi) by the adenine nucleotide translocator from beef heart mitochondria was studied in a reconstituted system. The transport of PPi is dependent on appropriate transmembrane substrates. The activity of PPi exchange is about one tenth as compared to the ADP/ATP exchange, whereas the transport affinity for PPi is very low (2-5 mM). The adenine nucleotide carrier catalyzes a strict counterexchange of PPi and nucleotides with an exchange stoichiometry close to 1. The inhibitor specificity of PPi exchange is comparable to that of ADP/ATP exchange.     

Osteopontin promotes pathologic mineralization in articular cartilage.

Calcium pyrophosphate dihydrate (CPPD) crystals are commonly found in osteoarthritic joint tissues, where they predict severe disease. Unlike other types of calcium phosphate crystals, CPPD crystals form almost exclusively in the pericellular matrix of damaged articular cartilage, suggesting a key role for the extracellular matrix milieu in their development. Osteopontin is a matricellular protein found in increased quantities in the pericellular matrix of osteoarthritic cartilage. Osteopontin modulates the formation of calcium-containing crystals in many settings. We show here that osteopontin stimulates ATP-induced CPPD crystal formation by chondrocytes in vitro. This effect is augmented by osteopontin's incorporation into extracellular matrix by transglutaminase enzymes, is only modestly affected by its phosphorylation state, and is inhibited by integrin blockers. Surprisingly, osteopontin stimulates transglutaminase activity in cultured chondrocytes in a dose-responsive manner. As elevated levels of transglutaminase activity promote extracellular matrix changes that permit CPPD crystal formation, this is one possible mechanism of action. We demonstrate the presence of osteopontin in the pericellular matrix of chondrocytes adjacent to CPPD deposits and near active transglutaminases. Thus, osteopontin may play an important role in facilitating CPPD crystal formation in articular cartilage.     

焦磷酸显色法检测PCR产物及酶活性

聚合酶链反应（polymerase chain reaction, PCR）是分子生物学领域的一项具有划时代意义的技术,但定量PCR产物或测定能生成焦磷酸的酶活性仍需要新技术的发展。本文提供了一种PCR产物定性及定量检测方法(Color PCR kit)及其用途;该方法通过焦磷酸(pyrophosphate,PPi)显色测定PCR的副产物PPi。利用试剂盒中的试剂与PPi反应,最终生成物为甲暨（formazan）,呈红色。根据显色现象(红色)判断PCR的阳性结果,由目测实现PCR的定性检测;或通过测定490 nm 处的吸光度值定量检测PCR过程中生成的副产物PPi的含量,定量检测PCR 产物的生成量;目测情况下Color PCR kit可检测到2.5 ng水平的PCR产物,用紫外分光光度计可检测到低限为1 pg;Color PCR kit法比琼脂糖凝胶电泳检测法（低限为4 ng）灵敏。Color PCR kit还可用于连接酶和转移酶活性的测定。     

A multienzyme bioluminescent time-resolved pyrophosphate assay

We have developed a high-sensitivity assay for measurement of inorganic pyrophosphate (PPi) in adenosine 5'-triphosphate (ATP)-contaminated samples. The assay is based on time-resolved measurements of the luminescence kinetics and implements multiple enzymes to convert PPi to ATP that is, in turn, utilized to produce light and to hydrolyze PPi for measurement of the steady state background luminescence. A theoretical model for describing luminescence kinetics and optimizing composition of the assay detection mixture is presented. We found that the model is in excellent agreement with the experimental results. We have developed and evaluated two algorithms for PPi measurement from luminescence kinetics acquired from ATP-contaminated samples. The first algorithm is considered to be the method of choice for analysis of long, i.e., 3-5 min, kinetics. The activity of enzymes is controlled during the experiment; the sensitivity of PPi detection is about 7 pg/ml or 15 pM of PPi in ATP-contaminated samples. The second algorithm is designed for analysis of short, i.e., less than 1-min, luminescence kinetics. It has about 20 pM PPi detection sensitivity and may be the better choice for assays in microplate format, where a short measurement time is required. The PPi assay is primarily developed for RNA expression analysis, but it also can be used in various applications that require high-sensitivity PPi detection in ATP-contaminated samples.     

Plant proton pumping pyrophosphatase: the potential for its pyrophosphate synthesis activity to modulate plant growth

Cellular pyrophosphate (PPi) homeostasis is vital for normal plant growth and development. Plant proton‐pumping pyrophosphatases (H⁺‐PPases) are enzymes with different tissue‐specific functions related to the regulation of PPi homeostasis. Enhanced expression of plant H⁺‐PPases increases biomass and yield in different crop species. Here, we emphasise emerging studies utilising heterologous expression in yeast and plant vacuole electrophysiology approaches, as well as phylogenetic relationships and structural analysis, to showcase that the H⁺‐PPases possess a PPi synthesis function. We postulate this synthase activity contributes to modulating and promoting plant growth both in H⁺‐PPase‐engineered crops and in wild‐type plants. We propose a model where the PPi synthase activity of H⁺‐PPases maintains the PPi pool when cells adopt PPi‐dependent glycolysis during high energy demands and/or low oxygen environments. We conclude by proposing experiments to further investigate the H⁺‐PPase‐mediated PPi synthase role in plant growth.     

Inhibition of TNF-alpha-induced neutrophil apoptosis by crystals of calcium pyrophosphate dihydrate is mediated by the extracellular signal-regulated kinase and phosphatidylinositol 3-kinase/Akt pathways up-stream of caspase 3

The role of protein kinases in the inhibition of TNF-alpha associated apoptosis of human neutrophils by crystals of calcium pyrophosphate dihydrate (CPPD) (25 mg/ml) was investigated. We monitored the activities of the p44 extracellular signal-regulated kinase 1 (ERK1) and p42 ERK2 mitogen-activated protein (MAP) kinases and phosphatidylinositol 3-kinase (PI3-K)-regulated protein kinase B (Akt) in neutrophils incubated with TNF-alpha and CPPD crystals, separately and in combination, in parallel with the endogenous caspase 3 activity and DNA fragmentation. CPPD crystals were observed to induce a robust and transient activation of ERK1, ERK2, and Akt, whereas TNF-alpha produced only a modest and delayed activation of Akt. In the presence of TNF-alpha, Akt activity was enhanced, and CPPD crystal-induced activation of ERK1 and ERK2 was more sustained than with CPPD crystals alone, but TNF-alpha itself reduced the basal phosphotransferase activities of these MAP kinases. Preincubation with the MAP kinase kinase (MEK1) inhibitors PD98059 (20 ng/ml) and U0126 (250 nM), or the PI3-K inhibitors wortmannin (100 nM) and LY294002 (50 microM) repressed the activation of ERK1, ERK2, and Akt in association with CPPD crystal incubation, in the absence or presence of TNF-alpha. Furthermore, the inhibition of the Mek1/Mek2-->ERK1/ERK2 or PI3-K/Akt pathways reversed CPPD crystal-associated suppression of TNF-alpha-induced caspase 3 activation and neutrophil apoptosis. Together, these results indicate that CPPD crystals function to induce acute inflammatory responses through ERK1/ERK2 and PI3-K/Akt-mediated stimulation of neutrophil activation and repression of apoptosis.     

Pyrophosphate-caused inhibition of the aminoacylation of tRNA by the leucyl-tRNA synthetase from Neurospora crassa

Inorganic pyrophosphate inhibits the aminoacylation of tRNALeu by the leucyl-tRNA synthetase from Neurospora crassa giving very low Kapp.i, PPi values of 3-20 microM. The inhibition by pyrophosphate, together with earlier kinetic data, suggest a reaction mechanism where leucine, ATP and tRNA are bound to the enzyme in almost random order, and pyrophosphate is dissociated before the rate-limiting step. A kinetic analysis of this mechanism shows that the measured Kapp.i values do not give the real dissociation constant but it is about 0.4 mM. Other dissociation constants are 90 microM for leucine, 2.2 mM for ATP and 1 microM for tRNALeu. At the approximate conditions of the living cell (2 mM ATP, 100 microM leucine and 150 microM PPi) the leucyl-tRNA synthetase is about 85% inhibited by pyrophosphate.