Presence and Regulation of α-Ketoglutarate Dehydrogenase Complex in a Glutamate-Producing Bacterium,Brevibacterium flavum

The presence of α-ketoglutarate (α-KG) dehydrogenase complex in the glutamate-producing bacteria was demonstrated for the first time with Brevibacterium flavum. The partially purified enzyme, which was specific to KG and NAD⁺ with the usual requirements for other co-factors, was labile and stabilized by glycerol, Mg²⁺, and thiamine pyrophosphate. The enzyme showed an optimum pH of 7.6 and K_ms of 80, 86, and 61 μm for KG, NAD⁺, and CoA, respectively, cis-Aconitate, succinyl-CoA, NADPH, NADH, pyruvate, and oxalacetate strongly inhibited the activity, while it was activated by acetyl-CoA, but not by AMP. Various inorganic and organic salts also inhibited the activity. When cells were cultured in glucose and acetate media, the specific activity of the cell extracts increased markedly and reached to a maximum at the late-logarithmic phase. Then, it decreased to the basal level. The addition of glutamate stimulated the synthesis of the enzyme.     

A rapid,enzymatic assay for the measurement of inorganic pyrophosphate in animal tissues

A simple, rapid enzymatic assay for the determination of inorganic pyrophosphate in tissue and plasma has been developed using the enzyme pyrophosphate-fructose-6-phosphate 1-phosphotransferase (EC 2.7.1.90) which was purified from extracts of Propionibacterium shermanii. The enzyme phosphorylates fructose-6-phosphate to produce fructose-1,6-bisphosphate using inorganic pyrophosphate as the phosphate donor. The utilization of inorganic pyrophosphate is measured by coupling the production of fructose-1,6-bisphosphate with the oxidation of NADH using fructose-bisphosphate aldolase (EC 4.1.2.13), triosephosphate isomerase (EC 5.3.1.1), and glycerol-3-phosphate dehydrogenase (NAD⁺)(EC 1.1.1.8). The assay is completed in less than 5 min and is not affected by any of the components of tissue or plasma extracts. The recovery of pyrophosphate added to frozen tissue powder was 97 ± 1% (n = 4). In this assay the change in absorbance is linearly related to the concentration of inorganic pyrophosphate over the cuvette concentration range of 0.1 μm to 0.1 mm.     

Purification and properties of pyrophosphatase of Acinetobacter johnsonii 210A and its involvement in the degradation of polyphosphate

Inorganic pyrophosphatase (E.C. 3.6.1.1) of Acinetobacter johnsonii210A was purified 200-fold to apparent homogeneity. The enzyme catalyzedthe hydrolysis of inorganic pyrophosphate and triphosphate to orthophosphate.No activity was observed with other polyphosphates and a wide variety oforganic phosphate esters. The molecular mass of the enzyme was estimatedto be 141 kDa by gelfiltration. Sodium dodecyl sulfate-polyacrylamide gelelectrophoresis indicated a subunit composition of six identical polypeptideswith a molecular mass of 23 kDa. The cation Mg² was required foractivity, the activity with Mn², Co² and Zn² was 48, 48 and 182% of the activity observed with Mg², respectively. The enzyme was heat-stable and inhibited by fluoride and iodoacetamide. The analysis of the kinetic properties of the enzyme revealed an apparent K_m for pyrophosphate of 0.26 mM. In A. johnsonii 210A, pyrophosphatase may be involved in the degradation of high-molecular polyphosphates under anaerobic conditions: (i) it catalyses the further hydrolysis of pyrophosphate and triphosphate formed from high-molecular weight polyphosphates by the action of exopolyphosphatase, and (ii) it abolishes the inhibition of polyphosphate: AMP phosphotransferase-mediated degradation by pyrophosphate and triphosphate.     

Cardiotoxin of the Indian cobra(Naja naja) is a pyrophosphatase

An inorganic pyrophosphatase has been purified to apparent homogeniety from Indian cobra(Naja naja) venom, with a ten-fold increase in specific activity. The enzyme activity is intrinsic to a protein fraction in the venom which is normally termed cardiotoxin, cobramine, cytotoxin and so on. The enzyme shows a lowK _m (70 μI) and high heat stability. The enzyme was active against sodium pyrophosphate; it also hydrolyses a few mononucletides and sugar phosphates at much lower rates. The physiological significance of inorganic pyrophosphatase in venom is discussed.     

Substrate and metal specificity in the enzymic synthesis of cyclic monoterpenes from geranyl and neryl pyrophosphate

A partially purified enzyme (carbocyclase) from the flavedo of Citrus limonum formed α-pinene, β-pinene, limonene, and γ-terpinene from geranyl pyrophosphate (GPP) and neryl pyrophosphate. The maximum specific activities obtained were 7.0 and 3.6 nmol/ min/mg, respectively. Cross-inhibition by the two substrates were observed and the ability to utilize neryl pyrophosphate was almost completely lost with aging. Citronellyl pyrophosphate and dimethylallyl pyrophosphate were the most effective inhibitors of carbocyclase. Isopentenyl pyrophosphate, the monophosphate esters of nerol and geraniol, as well as inorganic pyrophosphate were much less effective inhibitors. The enzyme had an absolute requirement for Mn²⁺. It could be replaced with about 2% effectiveness by Mg²⁺ and Co²⁺. Kinetic studies showed that the observed reaction rate correlates with the calculated concentration of the GPP (Mn²⁺)₂ species. Previous evidence with nonenzymatic reactions and the results presented support the view that the mechanism of carbocyclase may be the intramolecular analog of prenyltransferase.     

Measurement of the inorganic pyrophosphate in tissues of Pisum sativum L.

Purified pyrophosphate: fructose 6-phosphate 1-phosphotransferase (EC 2.7.1.90) was used to measure the inorganic pyrophosphate in unfractionated extracts of tissues of Pisum sativum L. The fructose 1,6-bisphosphate produced by the above enzyme was measured by coupling to NADH oxidation via aldolase (EC 4.1.2.13), triosephosphate isomerase (EC 5.3.1.1) and glycerol-3-phosphate dehydrogenase (EC 1.1.1.8). Amounts of pyrophosphate as low as 1 nmol could be measured. The contents of pyrophosphate in the developing embryo of pea, and in the apical 2 cm of the roots, were appreciable; 9.4 and 8.9 nmol g^-1 fresh weight, respectively. The possibility that pyrophosphate acts in vivo as an energy source for pyrophosphate: fructose 6-phosphate 1-phosphotransferase and for UDPglucose pyrophosphorylase (EC 2.7.7.9) is considered.     

A fluoride-insensitive inorganic pyrophosphatase isolated from Methanothrix soehngenii

An inorganic pyrophosphatase [E.C. 3.6.1.1] was isolated from Methanothrix soehngenii. In three steps the enzyme was purified 400-fold to apparent homogeneity. The molecular mass estimated by gelfiltration was 139±7 kDa. Sodium dodecyl sulfate/polyacrylamide gel electrophoresis indicated that the enzyme is composed of subunits with molecular masses of 35 and 33 kDa in an _{2 2} oligomeric structure. The enzyme catalyzed the hydrolysis of inorganic pyrophosphate, tri-and tetrapolyphosphate, but no activity was observed with a variety of other phosphate esters. The cation Mg²⁺ was required for activity. The pH optimum was 8 at 1 mM PP _i and 5 mM Mg²⁺. The enzyme was heat-stable, insensitive to molecular oxygen and not inhibited by fluoride. Analysis of the kinetic properties revealed an apparent K _m for PP _i of 0.1 mM in the presence of 5 mM Mg²⁺. The V _max was 590 mol of pyrophosphate hydrolyzed per min per mg protein, which corresponds to a K _cat of 1400 per second.The enzyme was found in the soluble enzyme fraction after ultracentrifugation, when cells were disrupted by French Press. Upto 5% of the pyrophosphatase was associated with the membrane fraction, when gentle lysis procedyre were applied.Abbreviation PMSF phenylmethylsulfonyl fluoride     

The Identification of Fucosterol in the Marine Brown Algae Hizikia fusiformis

The enzyme uridine diphosphate N-acetylglucosamine pyrophosphorylase was purified about 330-fold from an extract of baker’s yeast by the treatment with protamine sulfate and column chromatographies on DEAE-cellulose, hydroxylapatite and Sephadex G–150. The purified enzyme was proved to be homogeneous by disc gel electrophoresis. The molecular weight was determined to be approximately 37,000 by gel filtration. The enzyme had an optimum reactivity in the pH range of 7.5-8.5 and was stable at 4°C in potassium phosphate buffer, pH 7.5, containing 0.1 mm dithiothreitol, but was unstable when stored at ?20°C. The addition of dithiothreitol also increased the thermal stability of enzyme. The enzyme was specific for UDP-N-acetylglucosamine as substrate, and none of the other sugar nucleotides could serve as nucleotide substrate. The estimated values of Km were 6.1 × 10^?3 m for UDP-N-acetylglucosamine and 5.0 × 10^?3 m for inorganic pyrophosphate. The enzyme required some divalent cations for activity. Magnesium ion was the most effective among the cations tested. The enzyme activity was highly stimulated by the addition of dithiothreitol or dithioerythritol.     

A membrane-bound alkaline inorganic pyrophosphatase in isolated spinach chloroplasts

An alkaline inorganic pyrophosphatase is found in association with isolated spinach chloroplast membranes. The enzyme is not removed from chloroplasts by repeated washings in an iso-osmotic medium. Suspension of the chloroplasts in hyper- or hypo-osmotic medium, however, results in the loss of pyrophosphatase activity in the chloroplasts. Fractionation of an isolated chloroplast suspension by differential centrifugation yields chloroplast fractions possessing high levels of alkaline pyrophosphatase activity but practically devoid of cytoplasmic acid pyrophosphatase.The alkaline pyrophosphatase exhibits a pH optimum of 8.2–8.5. In addition, there is an absolute requirement for Mg²⁺, with maximal rates of pyrophosphate hydrolysis occurring at $\text{Mg}{}^{\mathrm{2+}}\text{PP}{}_{\mathrm{i}}$ ratios greater than 2. From these findings the actual substrate for the enzyme is evidently Mg₂P₂O₇⁰ with pyrophosphate (P₂O₇^4?) acting as a potent inhibitor.The enzyme is inhibited by high concentrations of ATP (>3 mm), but increasing the concentration of Mg²⁺ effectively relieves this inhibition. At lower ATP concentrations, however, there is a stimulation of pyrophosphatase activity.The rate of hydrolysis of pyrophosphate by isolated chloroplasts is not affected by methylamine, 4′-deoxyphlorizin, and light. The possible role of this enzyme in photophosphorylation is discussed.     

Studies on 5′-Phosphodiesterases in Microorganisms

5′-Phosphodiesterase, which degrades RNA into nucleoside-5′-monophosphates but does not attack DNA, is present not only in mycelium but also in culture filtrate of Penicillium citrinum Thorn 1131. For the formation of this enzyme pH of the culture medium must be kept below 7.0 during culture, as this enzyme is inactivated rapidly in alkaline solution. The pH optimum of this enzyme is in the region of pH 5. Cysteine, Mg⁺⁺, sodium fluoride, and inorganic ortho- or pyrophosphate are without appreciable effect on this enzyme. Nucleoside-5′-monophosphates, which have been regarded as new chemical seasonings, can be produced economically in a large scale by using the microbial 5′-phosphodiesterase.     

The partial purification and properties of a phytoene synthesizing enzyme system.

An enzyme system catalyzing the synthesis of phytoene from isopentenyl pyrophosphate has been isolated from tomato fruit plastids and purified approximately 350-fold in specific activity. This enzyme system has a molecular weight of approximately 200,000. The rate of phytoene formation is maximal at pH 7.0 and 23 °C and the apparent K_m for isopentenyl pyrophosphate is 10 μm The rates of phytoene synthesis when geranylgeranyl pyrophosphate and isopentenyl pyrophosphate were used as substrates were 0.08 and 0.17 nmol of phytoene/mg of protein/h, respectively. The enzyme complex showed an absolute requirement for Mn²⁺, but not for NADP⁺. At a concentration of 2 mm, NADP⁺ produced only a 1.5- to 3-fold stimulation, and this effect varied from preparation to preparation. The addition of NADPH to the incubation mixture produced inhibition of phytoene synthesis and there was no evidence for the concomitant accumulation of lycopersene. The acid labiles produced on acid treatment of the incubation mixture indicated that geranylgeranyl pyrophosphate was formed by the enzyme complex. The enzyme system is stabilized in the presence of 30% glycerol and 10 mm dithiothreitol and it can be stored at ?20 °C for over 1 month without significant loss of activity. However, the enzyme activity for phytoene formation is heat labile, and it is not stable when attempts are made to purify it further by ion-exchange chromatography.     

Inorganic pyrophosphate: D-fructose-6-phosphate 1-phosphotransferase in mung beans and its activation by D-fructose 1,6-bisphosphate and D-glucose 1, 6-bisphosphate

Inorganic pyrophosphate: D-fructose-6-phosphate 1-phosphotransferase was detected in extracts of mung bean sprouts, the first such detection in C₃ plants. The enzyme had an absolute requirement for a divalent metal (Mg⁺⁺) as well as for D-fructose 6-phosphate and inorganic pyrophosphate. An examination of anomalous kinetics revealed that the enzyme was activated by a product of the reaction, D-fructose 1,6-bisphosphate; micromolar concentrations of this effector increased the activity of the enzyme about 20-fold. D-Glucose 1,6-bisphosphate at higher concentrations could substitute for D-fructose 1,6-bisphosphate as an activator, but not as a substrate in the reverse reaction. The enzyme was fully active under conditions wherein ATP: D-fructose-6-phosphate 1-phosphotransferase from the same source was inhibited >99% (e.g., in the presence of 10 μM phosphoenolpyruvate).     

Evidence for the role of vacuolar soluble pyrophosphatase and inorganic polyphosphate in Trypanosoma cruzi persistence

Trypanosoma cruzi infection leads to development of a chronic disease but the mechanisms that the parasite utilizes to establish a persistent infection despite activation of a potent immune response by the host are currently unknown. Unusual characteristics of T. cruzi are that it possesses cellular levels of pyrophosphate (PP_i) at least 10 times higher than those of ATP and molar levels of inorganic polyphosphate (polyP) within acidocalcisomes. We characterized an inorganic soluble EF‐hand containing pyrophosphatase from T. cruzi (TcVSP) that, depending on the pH and cofactors, can hydrolyse either pyrophosphate (PP_i) or polyphosphate (polyP). The enzyme is localized to both acidocalcisomes and cytosol. Overexpression of TcVSP (TcVSP‐OE) resulted in a significant decrease in cytosolic PP_i, and short and long‐chain polyP levels. Additionally, the TcVSP‐OE parasites showed a significant growth defect in fibroblasts, less responsiveness to hyperosmotic stress, and reduced persistence in tissues of mice, suggesting that PP_i and polyP are essential for the parasite to resist the stressful conditions in the host and to maintain a persistent infection.     

Phosphoenol-pyruvate-carboxylase activity in cotton and Sorghum seeds and its relation to seedling development

Cotton (Gossypium hirsutum) seeds and Sorghum vulgare caryopses are able to incorporate CO₂ through a PEP-carboxylating enzyme (EC 4.1.1.38). The enzyme activity is optimal at pH 8.2 and is unaffected by ATP, GDP or acetyl CoA. The partially purified cotton enzyme is stimulated by inorganic phosphate with an apparent Km of 0.3 mM. The enzymes from both cultivars are inhibited by pyrophosphate, malate, and aspartate but not by succinate. Kinetic studies for Sorghum and cotton seed enzymes show apparent Km values for carbonate of 5 mM and 1.2 mM and for PEP of 36 M and 5 mM, respectively. The V_max values are 90 and 3.3 nmol min^-1 mg protein^-1, respectively.A two-fold increase in the enzyme activity from cotton seeds occurs after 2 h under laboratory germination conditions after which the activity drops sharply to 1/3 of the original activity after 5 h imbibition. No such change was observed in Sorghum caryopses enzyme. A correlation between PEP-carboxylase activity and seed vigor in both cultivars was demonstrated.Abbreviations GOT glutamicoxaloacetic-transaminase - MDH malic dehydrogenase-NADH₂ - RH relative humidity     

Biosynthesis of monoterpenes: partial purification and characterization of 1,8-cineole synthetase from Salvia officinalis.

The heterocyclic monoterpene 1,8-cineole is one of the major components of the volatile oil produced by sage (Salvia officinalis), and soluble enzyme extracts prepared from young sage leaves catalyzed the anaerobic conversion of the acyclic precursor neryl pyrophosphate to 1,8-cineole. This enzymatic activity was partially purified by a combination of ammonium sulfate precipitation and chromatography on hydroxylapatite, and the bulk of the competing activities, including phosphatases, were removed from the preparation. Cineole synthetase activity had a pH optimum at 6.1. The rate of 1,8-cineole formation was linear up to 1 h, and up to a protein concentration of 450 μg/ml. A divalent cation was required for catalysis, and maximum activity was obtained with MnCl₂ (1 mm). ZnCl₂ was nearly as effective as MnCl₂, and MgCl₂ could substitute for MnCl₂ only at tenfold higher concentrations. The apparent K_m and V of the enzyme were 10^?5m and 5.6 nmol/h-mg-ml, respectively. Inhibition of activity was observed at neryl pyrophosphate concentrations above 2 × 10^?4m. Nerol, neryl phosphate, 6,7-dihydroneryl pyrophosphate, citronellyl pyrophosphate, and 3,7-dimethyloctyl pyrophosphate were inactive as substrates for 1,8-cineole biosynthesis, indicating that the pyrophosphate and both double bonds of neryl pyrophosphate were required for catalysis. Geranyl pyrophosphate and linaloyl pyrophosphate were converted to 1,8-cineole at only 9 and 15%, respectively, of the rate of neryl pyrophosphate. Thus, the enzyme was highly specific for neryl pyrophosphate. α-Terpineol and its phosphorylated derivatives were not converted to 1,8-cineole, and this observation, coupled with the resolution of cineole synthetase activity from α-terpineol synthetase activity, proved conclusively that α-terpineol was not an intermediate in 1,8-cineole biosynthesis. p-Hydroxymercuribenzoate strongly inhibited the conversion of neryl pyrophosphate to 1,8-cineole (90% inhibition at 4 × 10^?5m); however, other thiol-directed reagents such as N-ethylmaleimide were much less effective. The enzyme was insensitive to NaF and to several other metabolic inhibitors. This is the first report on the properties of cineole synthetase, a novel enzyme which catalyzes both a carbocyclization and a heterocyclization.     

Lactobacillus plantarum undecaprenyl pyrophosphate synthetase: purification and reaction requirements.

Undecaprenyl pyrophosphate synthetase was partially purified from Lactobacillus plantarum by DEAE-cellulose, hydroxyapatite, and Sephadex G-100 chromatography in Triton X-100. The enzyme has a molecular weight between 53,000 and 60,000. The enzyme demonstrated a fivefold preference for farnesyl pyrophosphate rather than geranyl pyrophosphate as the allylic cosubstrate, whereas dimethylallyl pyrophosphate was not effective as a substrate. Polyprenyl pyrophosphates obtained using either farnesyl or geranyl pyrophosphate as cosubstrate were chromatographically identical. Hydrolysis of these polyprenyl pyrophosphates with either a yeast or liver phosphatase preparation yielded undecaprenol as the major product. Incorporation of radioactive label from mixtures of Δ³-[1-¹⁴C]isopentenyl pyrophosphate and Δ³-2R-[2-³H]isopentenyl pyrophosphate into enzymic product indicated that each isoprene unit added to the allylic pyrophosphate substrate has a cis configuration about the newly formed double bond. The removal of detergent from enzyme solutions resulted in a parallel loss in enzyme activity when analyzed with either farnesyl or geranyl pyrophosphate as cosubstrates. Enzymic activity was restored on addition of Triton X-100 or deoxycholate. The enzyme exhibited a pH-activity profile with optima at pH 7.5 and 10.2. It also demonstrated a divalent cation requirement, with Mg²⁺, Mn²⁺, Zn²⁺, and Co²⁺ exhibiting comparable activities.     

P5L mutation in Ank results in an increase in extracellular inorganic pyrophosphate during proliferation and nonmineralizing hypertrophy in stably transduced ATDC5 cells

Ank is a multipass transmembrane protein that regulates the cellular transport of inorganic pyrophosphate. In the progressive ankylosis (ank) mouse, a premature termination mutation at glutamic acid 440 results in a phenotype characterized by inappropriate deposition of basic calcium phosphate crystals in skeletal tissues. Mutations in the amino terminus of ANKH, the human homolog of Ank, result in familial calcium pyrophosphate dihydrate deposition disease. It has been hypothesized that these mutations result in a gain-of-function with respect to the elaboration of extracellular inorganic pyrophosphate. To explore this issue in a mineralization-competent system, we stably transduced ATDC5 cells with wild-type Ank as well as with familial chondrocalcinosis-causing Ank mutations. We evaluated the elaboration of inorganic pyrophosphate, the activity of pyrophosphate-modulating enzymes, and the mineralization in the transduced cells. Expression of transduced protein was confirmed by quantitative real-time PCR and by ELISA. Levels of inorganic pyrophosphate were measured, as were the activities of nucleotide pyrophosphatase phosphodiesterase and alkaline phosphatase. We also evaluated the expression of markers of chondrocyte maturation and the nature of the mineralization phase elaborated by transduced cells. The cell line expressing the proline to leucine mutation at position 5 (P5L) consistently displayed higher levels of extracellular inorganic pyrophosphate and higher phosphodiesterase activity than the other transduced lines. During hypertrophy, however, extracellular inorganic pyrophosphate levels were modulated by alkaline phosphatase activity in this cell system, resulting in the deposition of basic calcium phosphate crystals only in all transduced cell lines. Cells overexpressing wild-type Ank displayed a higher level of expression of type X collagen than cells transduced with mutant Ank. Other markers of hypertrophy and terminal differentiation, such as alkaline phosphatase, osteopontin, and runx2, were not significantly different in cells expressing wild-type or mutant Ank in comparison with cells transduced with an empty vector or with untransduced cells. These results suggest that the P5L Ank mutant is capable of demonstrating a gain-of-function with respect to extracellular inorganic pyrophosphate elaboration, but this effect is modified by high levels of expression of alkaline phosphatase in ATDC5 cells during hypertrophy and terminal differentiation, resulting in the deposition of basic calcium phosphate crystals.     

Cytoplasmic factors that affect the intensity and stability of bioluminescence from firefly luciferase in living mammalian cells

In order to improve calibration of firefly luciferase signals obtained by injecting the enzyme into single, isolated heart and liver cells we have investigated why the luminescence from cells is greatly depressed compared with in vitro (in mammalian ionic milieu) and why the decay of the intracellular signal is remarkably slow. We have shown that inorganic pyrophosphate greatly depresses the signal in vitro and that micromolar concentrations of inoragnic pyrophosphate, comparable with that in cytoplasm, reverse this inhibition and stabilize the signal, eliminating its decay. Higher concentrations of pyrophosphate depress the signal by inhibiting ATP-binding to luciferase. Luciferse-injected cells exposed to extracellular luciferin concentrations above about 100 μmol/1 (corresponding to a cytoplasmic level of c. 5–10 μmol/1 because of a transplasmalemmal gradient) show a gradual, irreversible loss of signal. We attribute this phenomenon (which is not seen in vitro) to the gradual accumlation of a luminescently inactive, irreversible, luciferase-oxyluciferin complex. At low luciferin levels this complex is prevented from forming by cytoplasmic pyrophosphate. Above c. 100μmol/1 extracellular luciferin, the pyrophosphate level in the cytoplasm fails to fully prevent the complex forming. In vitro this phenomenon does not occur because the luciferase concentrations and hence oxyluciferin levels are orders of magnitude lower than in cells injected with concentrated luciferase solutions, which have a cytoplasmic luciferase concentration of approximately 2-4 μmol/1.     

Small-angle X-ray scattering studies on yeast inorganic pyrophosphatase and its interactions with divalent metal ions, inorganic phosphate, and hydroxymethane bisphosphonate

Small-angle x-ray scattering studies have been carried out on the enzyme yeast inorganic pyrophosphatase (PPase), and its overall conformational changes on interaction with divalent metal ions (Mg²⁺ and Mn²⁺) and with phosphoryl ligands [inorganic phosphate (P_i) and hydroxymethane bisphosphonate (PCHOHP), a nonhydrolyzable inorganic pyrophosphate analog] were assessed. The enzyme undergoes an apparent reduction in size on simultaneous addition of Mg²⁺ and high P_i concentration, although neithough neither Mg²⁺ nor P_i added separately induced any measurable conformational changes. By contrast, simultaneous addition of Mn²⁺ and P_i to PPase does not result in an observable conformational change. However, the overall structure of the enzyme appears to enlarge in the simultaneous presence of Mn²⁺ ions and PCHOHP. The significance of the structural changes seen in PPase under various conditions is discussed.     

The ank gene story

The underlying molecular defect resulting in the abnormal calcification observed in ank/ank mice has been identified. The responsible nonsense mutation affects the protein product of ank, resulting in diminished production of extracellular inorganic pyrophosphate, an important inhibitor of nucleation and of the growth of apatite crystals. The ank gene product is one of several cell membrane proteins, including ectonucleoside triphosphate pyrophosphohydrolase enzymes and alkaline phosphatase, that regulate extracellular inorganic pyrophosphate levels and thereby regulate mineralization.