Biochemical characterization of an ectonucleotide pyrophosphatase/phosphodiesterase (E-NPP, E.C. 3.1.4.1) from rat cardiac soluble and microsomal fractions

In this study, we have reported the kinetic and biochemical characterization of ectonucleotide pyrophosphatase/phosphodiesterase (E-NPP) activity in rat cardiac fractions, one soluble and the other enriched in vesicles derived from sarcoplasmic reticulum. Both fractions demonstrated E-NPP activities, which could be observed by extracellular hydrolysis of p-nitrophenyl-5'-thymidine monophosphate (p-Nph-5'-TMP) and other biochemical characteristics. The K(M) values for the hydrolysis of p-Nph-5'-TMP in soluble and microsomal fractions were 118.53?±?27.28 and 91.92?±?12.49 μM, respectively. The V(max) values calculated were 2.56?±?0.15 and 113.87?±?21.09 nmol p-nitrophenol/min/mg of protein in soluble and microsomal fractions, respectively. Among the compounds tested to evaluate the possible activity of other enzymes on p-Nph-5'-TMP hydrolysis, only suramin (0.25?mM) produced a significant inhibition of substrate hydrolysis. Thus, our results strongly suggest the presence of E-NPP enzymes in subcellular fractions of rat heart, which could be involved in nucleotide signalling in the cardiac tissue.     

Biochemical characterization of soluble nucleotide pyrophosphatase/phosphodiesterase activity in rat serum

Biochemical properties of nucleotide pyrophosphatase/phosphodiesterase (NPP) in rat serum have been described by assessing its nucleotide phosphodiesterase activity, using p-nitrophenyl-5′-thymidine monophosphate (p-Nph-5′-TMP) as a substrate. It was demonstrated that NPP activity shares some typical characteristics described for other soluble NPP, such as divalent cation dependence, strong alkaline pH optimum (pH 10.5), inhibition by glycosaminoglycans, and K _m for p-Nph-5′-TMP hydrolysis of 61.8 ± 5.2 μM. In order to characterize the relation between phosphodiesterase and pyrophosphatase activities of NPP, we have analyzed the effects of different natural nucleotides and nucleotide analogs. ATP, ADP, and AMP competitively inhibited p-Nph-5′-TMP hydrolysis with K _i values ranging 13–43 μM. Nucleotide analogs, α,β-metATP, BzATP, 2-MeSATP, and dialATP behaved as competitive inhibitors, whereas α,β-metADP induced mixed inhibition, with K _i ranging from 2 to 20 μM. Chromatographic analysis revealed that α,β-metATP, BzATP, and 2-MeSATP were catalytically degraded in the serum, whereas dialATP and α,β-metADP resisted hydrolysis, implying that the former act as substrates and the latter as true competitive inhibitors of serum NPP activity. Since NPP activity is involved in generation, breakdown, and recycling of extracellular adenine nucleotides in the vascular compartment, the results suggest that both hydrolyzable and non-hydrolyzable nucleotide analogs could alter the amplitude and direction of ATP actions and could have potential therapeutic application.     

Assessment of human left ventricle flow using statistical shape modelling and computational fluid dynamics

Blood flow patterns in the human left ventricle (LV) have shown relation to cardiac health. However, most studies in the literature are limited to a few patients and results are hard to generalize. This study aims to provide a new framework to generate more generalized insights into LV blood flow as a function of changes in anatomy and wall motion. In this framework, we studied the four-dimensional blood flow in LV via computational fluid dynamics (CFD) in conjunction with a statistical shape model (SSM), built from segmented LV shapes of 150 subjects. We validated results in an in-vitro dynamic phantom via time-resolved optical particle image velocimetry (PIV) measurements. This combination of CFD and the SSM may be useful for systematically assessing blood flow patterns in the LV as a function of varying anatomy and has the potential to provide valuable data for diagnosis of LV functionality.     

Tomographic PIV in a model of the left ventricle: 3D flow past biological and mechanical heart valves

Left ventricular flow is intrinsically complex, three-dimensional and unsteady. Its features are susceptible to cardiovascular pathology and treatment, in particular to surgical interventions involving the valves (mitral valve replacement). To improve our understanding of intraventricular fluid mechanics and the impact of various types of prosthetic valves thereon, we have developed a custom-designed versatile left ventricular phantom with anatomically realistic moving left ventricular membrane. A biological, a tilting disc and a bileaflet valve (in two different orientations) were mounted in the mitral position and tested under the same settings. To investigate 3D flow within the phantom, a four-view tomographic particle image velocimetry setup has been implemented. The results compare side-by-side the evolution of the 3D flow topology, vortical structures and kinetic energy in the left ventricle domain during the cardiac cycle. Except for the tilting disc valve, all tested prosthetic valves induced a crossed flow path, where the outflow crosses the inflow path, passing under the mitral valve. The biological valve shows a strong jet with a peak velocity about twice as high compared to all mechanical heart valves, which makes it easier to penetrate deeply into the cavity. Accordingly, the peak kinetic energy in the left ventricle in case of the biological valve is about four times higher than the mechanical heart valves. We conclude that the tomographic particle imaging velocimetry setup provides a useful ground truth measurement of flow features and allows a comparison of the effects of different valve types on left ventricular flow patterns.     

Nucleotide pyrophosphatase/phosphodiesterase from Euphorbia characias latex: Purification and characterization

An authentic soluble metallo-protein nucleotide pyrophosphatase/phosphodiesterase (ELNPP) was purified to homogeneity from Euphorbia characias latex. The native protein had a molecular mass of 80 ± 5 kDa and was shown to be formed by two apparently identical subunits, each containing 1 Ca²⁺ and 1 Mg²⁺ ion. Whereas Mg²⁺ was shown to be strongly bound to the enzyme, Ca²⁺ was easily removed by treatment with EDTA. Ca²⁺-demetalated enzyme was shown to be almost totally inactive and the activity was fully restored incubating the demetalated ELNPP with Ca²⁺ ions. ELNPP exhibited hydrolytic activities toward pyrophosphate/phosphodiester bonds of a broad range of substrates and very efficiently hydrolyzed the artificial substrate thymidine 5′-monophosphate 4-nitrophenyl ester generating 4-nitrophenolate as a final product, and it has been used for enzyme kinetic experiments. ELNPP represents the first example of a nucleotide pyrophosphatase/phosphodiesterase enzyme purified from the latex of a plant belonging to the large genus Euphorbia.     

The non-classical N-glycan processing pathway of bovine brain ecto-nucleotide phosphodiesterase/pyrophosphatase 6 (eNPP6) is brain specific and not due to mannose-6-phosphorylation

Ecto-nucleotide phosphodiesterase/pyrophosphatase 6 (eNPP6) is a glycosylphosphatidylinositol (GPI)-anchored alkaline lysophospholipase C which is predominantly expressed in brain myelin and kidney. Due to shedding of the GPI-anchor eNPP6 occurs also as a soluble isoform (s-eNPP6). eNPP 6 consists of two identical monomers of 55 kDa joined by a disulfide bridge, and possesses four N-glycans in each monomer. In brain s-eNPP6 the N-glycans are mainly hybrid and high mannose type structures, reminiscent of processed mannose-6-phosphorylated glycans. Here we completed characterization of the site-specific glycan structures of bovine brain s-eNPP6, and determined the endo H-sensitivity glycan profiles of s-eNPP6 from bovine liver and kidney. Whereas in brain s-eNPP6 all of the N-glycans were endo H-sensitive, in liver and kidney only one of the glycosylation sites was occupied by an endo H-sensitive glycan, likely N406, which is located within the cleft formed by the dimer interface. Thus, the non-classical glycan processing pathway of brain eNPP 6 is not due to mannose-6-phosphorylation, suggesting that there is an alternative Golgi glycan-processing pathway of eNPP6 in brain. The resulting brain-specific expression of accessible hybrid and oligomannosidic glycans may be physiologically important within the cell–cell communication system of the brain.     

Purification and characterization of an endonuclease (E.C. 3.1.30.1) from Streptomyces tendae

A single-strand-specific endonuclease which converted negatively supercoiled DNA to open-circular and linear DNA was purified to homogeneity with Hb-Sepharose 4B, DEAE Trisacryl M, HA-Ultrogel and PBE-94 chromatofocusing from extracts of Streptomyces tendae ATCC 31160. Bio-Gel P-200 chromatography and electrophoresis in SDS-PAGE indicated the native protein was a monomer with a molecular weight of approximately 40-kDa. This enzyme did not hydrolyze double-stranded linear DNA but digested RNA and circular single-strand DNA. Sequence specificity for nicking of negatively supercoiled DNA was not detected.     

Synthesis,biological evaluation,and molecular docking study of sulfonate derivatives as nucleotide pyrophosphatase/phosphodiesterase (NPP) inhibitors

A new series of sulfonate derivatives 1a–zk were synthesized and evaluated as inhibitors of nucleotide pyrophosphatases. Most of the compounds exhibited good to moderate inhibition towards NPP1, NPP2, and NPP3 isozymes. Compound 1m was a potent and selective inhibitor of NPP1 with an IC₅₀ value of 0.387 ± 0.007 µM. However, the most potent inhibitor of NPP3 was found as 1x with an IC₅₀ value of 0.214 ± 0.012 µM. In addition, compound 1e was the most active inhibitor of NPP2 with an IC₅₀ value of 0.659 ± 0.007 µM. Docking studies of the most potent compounds were carried out, and the computational results supported the in vitro results.     

Torsional motion of the left ventricle does not affect ventricular fluid dynamics of both foetal and adult hearts

Left ventricular torsion is caused by shortening and relaxation of the helical fibres in the myocardium, and is thought to be an optimal configuration for minimizing myocardial tissue strains. Characteristics of torsional motion has also been proposed to be markers for cardiac dysfunction. However, its effects on fluid and energy dynamics in the left ventricle have not been comprehensively investigated. To investigate this, we performed image-based flow simulations on five healthy adult porcine and two healthy human foetal left ventricles (representing two different length scales) at different degrees of torsional motions. In the adult porcine ventricles, cardiac features such as papillary muscles and mitral valves, and cardiac conditions such as myocardial infarctions, were also included to investigate the effect of twist. The results showed that, for all conditions investigated, ventricular torsional motion caused minimal changes to flow patterns, and consistently accounted for less than 2% of the energy losses, wall shear stresses, and ejection momentum energy. In contrast, physiological characteristics such as chamber size, stroke volume and heart rate had a much greater influence on flow patterns and energy dynamics. The results thus suggested that it might not be necessary to model the torsional motion to study the flow and energy dynamics in left ventricles.     

Cyclic nucleotide phosphodiesterase from a particulate fraction of rat heart. Solubilization and characterization of a single enzymatic form

Approximatively 2–8% of the cyclic nucleotide phosphodiesterase activity of a crude 1000 g supernatant from rat heart was associated with the washed 105,000 g pellet fraction. This activity exhibited biphasic Lineweaver-Burk plots over a large range of cyclic nucleotides concentrations. Concave-Bownward plots were obtained with cyclic AMP as the assay substrate, while cyclic GMP gave rise to concave-upward plots. Treatment of this particulate fraction by freezing and thawing and then with 2% Lubrol PX released the major part of phosphodiesterase activity into the supernatant (70 and 90% for cyclic AMP and cyclic GMP phosphodiesterase activities respectively). Isoelectric focusing of the solubilized enzyme revealed a single peak of phosphodiesterase activity. While the Lineweaver-Burk plots of cyclic AMP phosphodiesterase activity were not markedly modified by detergent treatment kinetic plots of cyclic GMP phosphodiesterase activity underwent a drastic transformation during the overall solubilization procedure. The substantial increase in the cyclic GMP rate of hydrolysis observed at low substrate level might explain the difference in the apparent yield of solubilization between cyclic AMP and cyclic GMP phosphodiesterase activities.     

Cloning, molecular characterization and chromosome localization of the inorganic pyrophosphatase (PPA) gene from S. cerevisiae.

The gene for Saccharomyces cerevisiae inorganic pyrophosphatase, PPA, has been cloned by hybridization of "long" oligonucleotide probes with both cDNA and genomic S. cerevisiae libraries. The nucleotide sequence of 1612 bp from a genomic subclone that includes the entire coding region gives a deduced amino acid sequence that has nine differences (out of a total of 286 residues) from the previously published amino acid sequence that was determined directly. The codon usage in PPA is as expected for a "highly expressed" yeast gene. The upstream region contains a poly dA/dT sequence that might comprise a constitutive promoter. The PPA gene appears to be present in a single copy within the S. cerevisiae genome and has been localized to chromosome II.     

Electron microscopy description of cardiomyocytes from the left ventricle of rat heart after apoptosis induction by isoproterenol

Changes in cardiomyocytes from the left ventricle of rat heart were studied by light and electron microscopic and morphometric methods in the myocardial regions neighboring necrotic foci formed after the injection of 80 mg/kg β adrenomimetic isoproterenol. TUNEL assay was used to detect apoptotic cardiomyocytes. Three types of cardiomyocytes (A, B, and C) differing by the ultrastructure of the nucleus and the degree of mitochondrial changes were identified at all studied stages of necrotic focus development (4–48 h). B and C type cardiomyocytes could represent cells at different stages of apoptosis. The apoptotic changes in cardiomyocytes proved to prevail in early lesion foci (4–18 h), while cardiomyocytes at later stages were prone to necrosis; cardiomyocytes can exhibit signs of apoptosis and necrosis at the same time.     

Biochemical characterization of membrane-associated septin from rat brain

Within the cell membrane there exist various microdomains (lipid rafts) in which specific lipids and proteins are assembled and these microdomains are recovered in the detergent-resistant low-density membrane fraction (DRM). Septin is a novel GTP-binding, cytoskeletal protein having various isoforms that assemble into homo- and heterooligomers and filaments. As the localization of septin 3 in DRM was found through a proteomics analysis of brain-derived DRM, the presence of other septin isoforms in DRM was studied. Western blotting analysis showed maturation-dependent enrichment of several septin isoforms in DRM prepared from synaptic plasma membrane (SPM). These isoforms were solubilized with high MgCl₂ solution and recovered as the precipitate after dialysis to low ionic solution. Three times cycling of the extraction-dialysis process resulted in the partial purification of septin complex and electron microscopic observation of this fraction revealed rod-like structures in which building units were observed. The presence of heterooligomers was shown with western blotting after the separation of the MgCl₂ extract with blue-native polyacrylamide gel electrophoresis. Immunoprecipitation assay using monoclonal anti-septin11 antibody also showed the presence of heterooligomers. These results show that septin localizes in the membrane microdomains of the SPM in adult brain and may have important roles in the membrane dynamics of neurons.     

Extracellular phytase (E.C. 3.1.3.8) from Aspergillus ficuum NRRL 3135: purification and characterization

Extracellular phytase from Aspergillus ficuum, a glycoprotein, was purified to homogeneity in 3 column chromatographic steps using ion exchange and chromatofocusing. Results of gel filtration chromatography and SDS-polyacrylamide gel electrophoresis indicated the approximate molecular weight of the native protein to be 85-100-KDa. On the basis of a molecular weight of 85-KDa, the molar extinction coefficient of the enzyme at 280 nm was estimated to be 1.2 X 10(4) M-1 cm-1. The isoelectric point of the enzyme, as deduced by chromatofocusing, was about 4.5. The purified enzyme is remarkably stable at 0 degree C. Thermal inactivation studies have shown that the enzyme retained 40% of its activity after being subjected to 68 degrees C for 10 minutes, and the enzyme exhibited a broad temperature optimum with maximum catalytic activity at 58 degrees C. The Km of the enzyme for phytate and p-nitrophenylphosphate is about 40 uM and 265 uM, respectively, with an estimated turnover number of the enzyme for phytate of 220 per sec. Enzymatic deglycosylation of phytase by Endoglycosidase H lowered the molecular weight of native enzyme from 85-100-KDa to about 76-KDa; the digested phytase still retained some carbohydrate as judged by positive periodic acid-Schiff reagent staining of the electrophoresed protein. Immunoblotting of the phytase with monoclonal antibody 7H10 raised against purified native enzyme recognized not only native but also partially deglycosylated protein.     

Extracellular alpha galactosidase (E.C. 3.2.1.22) from Aspergillus ficuum NRRL 3135 purification and characterization

Extracellular alpha-galactosidase, a glycoprotein from the extracellular culture fluid of Aspergillus ficuum grown on glucose and raffinose in a batch culture system, was purified to homogeneity in five steps by ion exchange and hydrophobic interaction chromatography. The molecular mass of the enzyme was 70.8 Kd by SDS polyacrylamide gel electrophoresis and 74.1 Kd by gel permeation HPLC. On the basis of a molecular mass of 70.7 Kd, the molar extinction coefficient of the enzyme at 279 nm was estimated to be 6.1 X10(4) M-1 cm-1. The purified enzyme was remarkably stable at 0 degrees C. It had a broad temperature optimum and maximum catalytic activity was at 60 degrees C. It retained 33% of its activity after 10 min. at 65 degrees C. It had a pH optimum of 6.0. It retained 62% of its activity after 12 hours at pH 2.3. The Kms for p-nitrophenyl-alpha-D-galactopyranoside, o-nitrophenyl-alpha-D-galactopyranoside and m-nitrophenyl-alpha-D-galactopyranoside are: 1462, 839 and 718 microM. The enzyme was competitively inhibited by mercury (19.8 microM), silver (21.5 microM), copper (0.48 mM), zinc (0.11 mM), galactose (64.0 mM) and fructose (60.3 mM). It was inhibited non-competitively by glucose (83.2 mM) and uncompetitively by mannose (6.7 mM).     

Sequence, structure and functional diversity of PD-(D/E)XK phosphodiesterase superfamily

Proteins belonging to PD-(D/E)XK phosphodiesterases constitute a functionally diverse superfamily with representatives involved in replication, restriction, DNA repair and tRNA–intron splicing. Their malfunction in humans triggers severe diseases, such as Fanconi anemia and Xeroderma pigmentosum. To date there have been several attempts to identify and classify new PD-(D/E)KK phosphodiesterases using remote homology detection methods. Such efforts are complicated, because the superfamily exhibits extreme sequence and structural divergence. Using advanced homology detection methods supported with superfamily-wide domain architecture and horizontal gene transfer analyses, we provide a comprehensive reclassification of proteins containing a PD-(D/E)XK domain. The PD-(D/E)XK phosphodiesterases span over 21 900 proteins, which can be classified into 121 groups of various families. Eleven of them, including DUF4420, DUF3883, DUF4263, COG5482, COG1395, Tsp45I, HaeII, Eco47II, ScaI, HpaII and Replic_Relax, are newly assigned to the PD-(D/E)XK superfamily. Some groups of PD-(D/E)XK proteins are present in all domains of life, whereas others occur within small numbers of organisms. We observed multiple horizontal gene transfers even between human pathogenic bacteria or from Prokaryota to Eukaryota. Uncommon domain arrangements greatly elaborate the PD-(D/E)XK world. These include domain architectures suggesting regulatory roles in Eukaryotes, like stress sensing and cell-cycle regulation. Our results may inspire further experimental studies aimed at identification of exact biological functions, specific substrates and molecular mechanisms of reactions performed by these highly diverse proteins.     

Biochemical and structural characterization of alanine racemase from Bacillus anthracis (Ames)

Background  

Bacillus anthracis is the causative agent of anthrax and a potential bioterrorism threat. Here we report the biochemical and structural characterization of B. anthracis (Ames) alanine racemase (Alr _Bax ), an essential enzyme in prokaryotes and a target for antimicrobial drug development. We also compare the native Alr _Bax structure to a recently reported structure of the same enzyme obtained through reductive lysine methylation.