Purification and heterogeneity of inorganic pyrophosphatase of pig scapula cartilage.
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SUBMITTER: Felix R
PROVIDER: S-EPMC1165379 | biostudies-other | 1975 Apr
REPOSITORIES: biostudies-other
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Project description:The properties of a highly purified inorganic pyrophosphatase (pyrophosphate phosphohydrolase; EC 3.6.1.1) from pig scapula cartilage were studied. The enzyme had a molecular weight of 66 000 and a pH optimum of 7-8. It was markedly activated by magnesium, but not, or only to a much smaller degree, by other metal ions. PP1 was the only substrate found and had a Km value of 11 muM. The enzyme was not inhibited by phosphate and other inhibitors of alkaline phosphatase such as CN- minus, amino acids and theophylline; it was slightly inhibited by tartrate, formaldehyde and ammonium molybdate and strongly inhibited by F- minus, Ca2+ and other metal ions. The properties of the enzyme in the presence of concentrations of PP1 present in plasma (3.5 muM) were similar to those found at higher (2 mM) concentrations of PP1. The diphosphonates ethane-1-hydroxy-1,1-diphosphonate and dichloromethylenediphosphonate inhibited the enzyme in the presence of low PP1 concentrations. The characteristics of this enzyme are therefore similar to pyrophosphatases from other sources, such as from yeast and erythrocytes, and do not support a specific role of this enzyme in the calcification process.
| S-EPMC1165380 | biostudies-other
Project description:Inorganic pyrophosphatase (iPPase) is an enzyme that cleaves pyrophosphate into two phosphate molecules. This enzyme is an essential component of in vitro transcription (IVT) reactions for RNA preparation as it prevents pyrophosphate from precipitating with magnesium, ultimately increasing the rate of the IVT reaction. Large-scale RNA production is often required for biochemical and biophysical characterization studies of RNA, therefore requiring large amounts of IVT reagents. Commercially purchased iPPase is often the most expensive component of any IVT reaction. In this paper, we demonstrate that iPPase can be produced in large quantities and high quality using a reasonably generic laboratory facility and that laboratory-purified iPPase is as effective as commercially available iPPase. Furthermore, using size exclusion chromatography coupled with multi-angle light scattering and dynamic light scattering, analytical ultracentrifugation, and small-angle X-ray scattering, we demonstrate that yeast iPPase can form tetramers and hexamers in solution as well as the enzymatically active dimer. Our work provides a robust protocol for laboratories involved with RNA in vitro transcription to efficiently produce active iPPase, significantly reducing the financial strain of large-scale RNA production.
| S-EPMC10311840 | biostudies-literature
Project description:1. Dialysed extracts of rat costal cartilage were shown to possess an enzyme that hydrolyses inorganic pyrophosphate. 2. Inorganic pyrophosphatase activity assayed in the presence of 2mm substrate was maximal at pH6.8. 3. Mg(2+) was essential for activity, which was greatest with 10mm or higher concentrations of Mg(2+). 4. Extracts prepared from cartilage taken from suckling rats (<20g.) showed little or no hydrolytic activity, but as rat weight increased inorganic pyrophosphatase activity was detected, increased to a maximum in tissue from animals weighing about 40g., and then rapidly declined. 5. The increase in inorganic pyrophosphatase activity was associated with an increase in the uptake of (45)Ca by the cartilage in vivo. 6. Accumulation of calcium, inorganic phosphate and magnesium occurred when inorganic pyrophosphatase activity was at its maximum. 7. Alkaline phosphatase activity, measured in the same extracts used to determine pyrophosphatase activity, was highest in the tissues of the animals weighing <20g., and decreased as inorganic pyrophosphatase activity increased to its maximum. 8. There was no direct relationship between alkaline phosphatase activity and the onset of calcification.
| S-EPMC1187739 | biostudies-other
Project description:IntroductionThe physiologic selectivity of calcification in bone tissue reflects selective co-expression by osteoblasts of fibrillar collagen I and of tissue nonspecific alkaline phosphatase (TNAP), which hydrolyzes the calcification inhibitor pyrophosphate (PP(i)) and generates phosphate (P(i)). Humans and mice deficient in the PP(i)-generating ecto-enzyme NPP1 demonstrate soft tissue calcification, occurring at sites of extracellular matrix expansion. Significantly, the function in osteoblasts of cytosolic inorganic pyrophosphatase (abbreviated iPP(i)ase), which generates P(i) via PP(i) hydrolysis with neutral pH optimum, remains unknown. We assessed iPP(i)ase in Enpp1(-/-) and wild type (WT) mouse osteoblasts and we tested the hypothesis that iPP(i)ase regulates collagen I expression.MethodsWe treated mouse calvarial osteoblasts with ascorbate and beta-glycerol phosphate to promote calcification, and we assessed cytosolic P(i) and PP(i) levels, sodium-dependent P(i) uptake, Pit-1 P(i) co-transporter expression, and iPP(i)ase and TNAP activity and expression. We also assessed the function of transfected Ppa1 in osteoblasts.ResultsInorganic pyrophosphatase but not TNAP was elevated in Enpp1(-/-) calvariae in situ. Cultured primary Enpp1(-/-) calvarial osteoblasts demonstrated increased calcification despite flat TNAP activity rather than physiologic TNAP up-regulation seen in WT osteoblasts. Despite decreased cytosolic PP(i) in early culture, Enpp1(-/-) osteoblasts maintained cytosolic P(i) levels comparable to WT osteoblasts, in association with increased iPP(i)ase, enhanced sodium-dependent P(i) uptake and expression of Pit-1, and markedly increased collagen I synthesis. Suppression of collagen synthesis in Enpp1(-/-) osteoblasts using 3,4-dehydroproline markedly suppressed calcification. Last, transfection of Ppa1 in WT osteoblasts increased cytosolic P(i) and decreased cytosolic but not extracellular PP(i), and induced both collagen I synthesis and calcification.ConclusionsIncreased iPP(i)ase is associated with "P(i) hunger" and increased calcification by NPP1-deficient osteoblasts. Furthermore, iPP(i)ase induces collagen I at the levels of mRNA expression and synthesis and, unlike TNAP, stimulates calcification by osteoblasts without reducing the extracellular concentration of the hydroxyapatite crystal inhibitor PP(i).
| S-EPMC2818162 | biostudies-literature
Project description:Inorganic pyrophosphatase (PPase) is a ubiquitous enzyme that converts pyrophosphate (PPi) to phosphate and, in this way, controls numerous biosynthetic reactions that produce PPi as a byproduct. PPase activity is generally assayed by measuring the product of the hydrolysis reaction, phosphate. This reaction is reversible, allowing PPi synthesis measurements and making PPase an excellent model enzyme for the study of phosphoanhydride bond formation. Here we summarize our long-time experience in measuring PPase activity and overview three types of the assay that are found most useful for (a) low-substrate continuous monitoring of PPi hydrolysis, (b) continuous and fixed-time measurements of PPi synthesis, and (c) high-throughput procedure for screening purposes. The assays are based on the color reactions between phosphomolybdic acid and triphenylmethane dyes or use a coupled ATP sulfurylase/luciferase enzyme assay. We also provide procedures to estimate initial velocity from the product formation curve and calculate the assay medium's composition, whose components are involved in multiple equilibria.
| S-EPMC8073611 | biostudies-literature
Project description:cDNA corresponding to two type-I vacuolar H(+)-inorganic pyrophosphatases (V-PPases) (SlVP1, SlVP2) and one type-II V-PPase (SlVP3) was isolated from tomato fruit to investigate their role in fruit development. Southern analysis revealed that type-I V-PPase genes form a multigene family, whereas there is only one type-II V-PPase gene in the tomato genome. Although SlVP1 and SlVP2 were differentially expressed in leaves and mature fruit, the highest levels of both SlVP1 and SlVP2 mRNA were observed in fruit at 2-4 days after anthesis. The expression pattern of type-II SlVP3 was similar to that of SlVP2, and the highest levels of SlVP3 mRNA were also observed in fruit at 2-4 days after anthesis, thus suggesting that SlVP3 plays a role in early fruit development. Because SlVP1 and SlVP2 mRNA was more abundant than SlVP3 mRNA, expression of type-I V-PPases was analysed further. Type-I V-PPase mRNA was localized in ovules and their vicinities and in vascular tissue at an early stage of fruit development. Tomato RNAi lines in which the expression of type-I V-PPase genes was repressed using the fruit-specific promoter TPRP-F1 exhibited fruit growth retardation at an early stage of development. Although the major function of V-PPases in fruit has been believed to be the accumulation of materials such as sugars and organic acids in the vacuole during cell expansion and ripening, these results show that specific localization of V-PPase mRNA induced by pollination has a novel role in the cell division stage.
| S-EPMC3444275 | biostudies-literature
Project description:The present work is focused on testing enzyme-based agents for the partial dissolution of calcium pyrophosphate (CaPPi) deposits in the cartilages and synovial fluid of patients with pyrophosphate arthropathy (CPPD disease). Previously, we suggested that inorganic pyrophosphatases (PPases) immobilized on nanodiamonds of detonation synthesis (NDs) could be appropriate for this purpose. We synthesized and characterized conjugates of NDs and PPases from Escherichia coli and Mycobacterium tuberculosis. The conjugates showed high enzymatic activity and resistance to inhibition by calcium and fluoride. Here, we tested the effectiveness of pyrophosphate (PPi) hydrolysis by the conjugates in an in vitro model system simulating the ionic composition of the synovial fluid and in the samples of synovial fluid of patients with CPPD via NMR spectroscopy. The conjugates of both PPases efficiently hydrolyzed triclinic crystalline calcium pyrophosphate (t-CPPD) in the model system. We evaluated the number of phosphorus-containing compounds in the synovial fluid, showed the possibility of PPi detection in it, and estimated the hydrolytic activity of the PPase conjugates. The soluble and immobilized PPases were able to hydrolyze a significant amount of PPi (1 mM) in the synovial fluid in short periods of time (24 h). The maximum activity was demonstrated for Mt-PPase immobilized on ND-NH-(CH2)6-NH2 (2.24 U mg-1).
| S-EPMC7178341 | biostudies-literature
Project description:Pyrophosphate arthropathy is the mineralization defect in humans caused by the deposition of microcrystals of calcium pyrophosphate dihydrate in joint tissues. As a potential therapeutic strategy for the treatment of pyrophosphate arthropathy, delivery of exogenous pyrophosphate-hydrolyzing enzymes, inorganic pyrophosphatases (PPases), to the synovial fluid has been suggested. Previously, we synthesized the conjugates of Escherichia coli PPase (Ec-PPase) with detonation synthesis nanodiamonds (NDs) as a delivery platform, obtaining the hybrid biomaterial retaining high pyrophosphate-hydrolyzing activity in vitro. However, most known PPases including Ec-PPase in the soluble form are strongly inhibited by Ca2+ ions. Because synovial fluid contains up to millimolar concentrations of soluble calcium, this inhibition might limit the in vivo application of Ec-PPase-based material in joint tissues. In this work, we proposed other bacterial PPases from Mycobacterium tuberculosis (Mt-PPase), which are resistant to the inhibition by Ca2+ ions, as an active PPi-hydrolyzing agent. We synthesized conjugates of Mt-PPase with NDs and tested their activity under various conditions. Unexpectedly, conjugates of both Ec-PPase and Mt-PPase with aminated NDs retained significant hydrolytic activity in the presence of well-known mechanism-based PPase inhibitors, fluoride or calcium. The incomplete inhibition of PPases by fluoride or calcium was found for the first time.
| S-EPMC7114608 | biostudies-literature
Project description:1. Protein-polysaccharides from pig laryngeal cartilage extracted by two procedures described in the preceding paper (Tsiganos & Muir, 1969) were shown to consist of macromolecules of various sizes as assessed by gel filtration in 4% and 6% agarose. 2. A larger proportion of the smaller molecules was present in the preparation obtained by brief extraction in iso-osmotic sodium acetate (procedure I) than in that obtained by more prolonged extraction in 10% (w/v) calcium chloride (procedure II). 3. Two fractions were separated by gel filtration in 6% agarose and by electrophoresis in compressed glass fibre. These fractions differed in chemical composition and in antigenic determinants. The gel-retarded fraction R and that of higher electrophoretic mobility possessed the same single antigen, whereas the gel-excluded fraction E and the slower electrophoretic fraction contained all the antigens of the starting material including that of fraction R. 4. Five N-terminal amino acid residues were identified in preparation I and fraction E, only two of which were present in fraction R. 5. The relative proportions of gel-excluded and gel-retarded fractions did not change when solutions of high ionic strength, urea or guanidine hydrochloride were used for elution. 6. The differences in chemical and amino acid composition between fractions R and E showed that the latter was not a simple aggregate of the former. Fraction E contained more basic and aromatic amino acids, and some methionine and cystine; the last two were absent from fraction R. Hydroxyproline was not detected in either fraction. 7. The number of glycosidic linkages in both fractions was estimated by alkaline beta-elimination. Appreciable amounts of threonine as well as serine were destroyed in both fractions. An average chain length for chondroitin sulphate was calculated from the galactosamine content of both fractions and the amounts of hydroxy amino acid destroyed. Average chain lengths were also calculated from the xylose and galactosamine content of each fraction. Each independent method gave a value of approximately 28 disaccharide units for the chain length in both fractions and hence their difference in size could not be explained by differences in the length of carbohydrate chains. 8. All fractions contained glucosamine, which was attributed to keratan sulphate. Content of both protein and keratan sulphate increased with the size of the macromolecules. 9. It is suggested, from these results, that chondroitin sulphate-protein complexes normally exist as a heterogeneous population of macromolecules in cartilage, and that keratan sulphate is involved in the formation of larger molecules.
| S-EPMC1184780 | biostudies-other
Project description:Inorganic pyrophosphatase (IPPase) from the archaeon Thermococcus thioreducens was cloned, overexpressed in Escherichia coli, purified and crystallized in restricted geometry, resulting in large crystal volumes exceeding 5?mm3. IPPase is thermally stable and is able to resist denaturation at temperatures above 348?K. Owing to the high temperature tolerance of the enzyme, the protein was amenable to room-temperature manipulation at the level of protein preparation, crystallization and X-ray and neutron diffraction analyses. A complete synchrotron X-ray diffraction data set to 1.85?Å resolution was collected at room temperature from a single crystal of IPPase (monoclinic space group C2, unit-cell parameters a=106.11, b=95.46, c=113.68?Å, ?=?=90.0, ?=98.12°). As large-volume crystals of IPPase can be obtained, preliminary neutron diffraction tests were undertaken. Consequently, Laue diffraction images were obtained, with reflections observed to 2.1?Å resolution with I/?(I) greater than 2.5. The preliminary crystallographic results reported here set in place future structure-function and mechanism studies of IPPase.
| S-EPMC3509969 | biostudies-literature