The role of nucleoside triphosphate pyrophosphohydrolase in in vitro nucleoside triphosphate-dependent matrix vesicle calcification

Nucleoside triphosphate pyrophosphohydrolase (EC 3.6.1.8) activity is associated with matrix vesicles purified from collagenase digests of fetal calf epiphyseal cartilage. This enzyme hydrolyzes nucleoside triphosphates to nucleotides and PPi, the latter inducing precipitation in the presence of Ca2+ and Pi. An assay for matrix vesicle nucleoside triphosphate pyrophosphohydrolase is developed using beta, gamma-methylene ATP as substrate. The assay is effective in the presence of matrix vesicle-associated ATPase, pyrophosphatase, and alkaline phosphatase activities. A soluble nucleoside triphosphate pyrophosphohydrolase is obtained from matrix vesicles by treatment with 5 mM sodium deoxycholate. The solubilized enzyme induced the precipitation of calcium phosphate in the presence of ATP, Ca2+, and Pi. Extraction of deoxycholate-solubilized enzymes from matrix vesicles with 1-butanol destroys nucleoside triphosphate pyrophosphohydrolase activity while enhancing the specific activities of ATPase, pyrophosphatase, and alkaline phosphatase. In solutions devoid of ATP and matrix vesicles, concentrations of PPi between 10 and 100 microM induce calcification in mixtures containing initial Ca2+ X P ion products of 3.5 to 7.9 mM2. This finding plus the discovery of nucleoside triphosphate pyrophosphohydrolase in matrix vesicles supports the view that these extracellular organelles induce calcium precipitation by the enzymatic production of PPi. Nucleoside triphosphate pyrophosphohydrolase is more active against pyrimidine nucleoside triphosphates than the corresponding purine derivatives. The pH optimum is 10.0 and the enzyme is neither activated nor inhibited by Mg2+ or Ca2+ ions or mixtures of the two. Vmax at pH 7.5 for beta, gamma-methylene ATP is 0.012 mumol of substrate hydrolyzed per min per mg of protein and Km is below 10 microM. The enzyme is irreversibly destroyed at pH 4 and is stable at pH 10.5.     

Distribution, cloning, and characterization of porcine nucleoside triphosphate diphosphohydrolase-1.

In this study, we have investigated the distribution of the enzyme nucleoside triphosphate diphosphohydrolase-1 (NTPDase1; EC 3.6.1.5) in a subset of pig tissues by biochemical activity and Western blotting with antibodies against porcine NTPDase1. The highest expression of this enzyme was found in vascular endothelium, smooth muscle, spleen and lung. The complete cDNA of NTPDase1 from aorta endothelial cells was sequenced using primer walking. The protein consists of 510 amino acids, with a calculated molecular mass of 57 756 Da. The amino-acid sequence indicated seven putative N-glycosylation sites and one potential intracellular cGMP- and cAMP-dependent protein kinase phosphorylation site. As expected, the protein has a very high homology to other known mammalian ATPDases and CD39 molecules, and includes all five apyrase conserved regions. Expression of the complete cDNA in COS-7 cells confirmed that NTPDase1 codes for a transmembrane glycoprotein with ecto-ATPase and ecto-ADPase activities. Two proteolytic products of NTPDase1, with molecular mass of 54 and 27 kDa, respectively, were consistently present in proteins from transfected COS-7 cells and in particulate fractions from different tissues. A trypsin cleavage site, giving rise to these two cleavage products, was identified. In order to remain enzymatically active, the two cleavage products have to interact by non-covalent interactions.     

Cloning and characterization of mouse nucleoside triphosphate diphosphohydrolase-8

A novel mammalian plasma membrane bound nucleoside triphosphate diphosphohydrolase (NTPDase), named NTPDase8, has been cloned and characterized. Analysis of cDNA reveals an open reading frame of 1491 base pairs encoding a protein of 497 amino acid residues with an estimated molecular mass of 54650 Da and a predicted isoelectric point of 5.94. In a mouse, the genomic sequence is located on chromosome 2A3 and is comprised of 10 exons. The deduced amino acid sequence reveals eight putative N-glycosylation sites, two transmembrane domains, five apyrase-conserved regions, and 20-50% amino acid identity with other mammalian NTPDases. mRNA expression was detected in liver, jejunum, and kidney. Both intact cells and crude cell lysates from COS-7 cells expressing NTPDase8 hydrolyzed P2 receptor agonists, namely, ATP, ADP, UTP, and UDP, but did not hydrolyze AMP. There was an absolute requirement for divalent cations for the catalytic activity (Ca(2+) > Mg(2+)) with an optimal pH between 5.5 and 8.0 for ATP and 6.4 for ADP hydrolysis. Kinetic parameters derived from analysis of crude cell lysates showed that the enzyme had lower apparent K(m) values for adenine nucleotides and for triphosphonucleosides (K(m,app) of 13 microM for ATP, 41 microM for ADP, 47 microM for UTP, and 171 microM for UDP). Hydrolysis of triphosphonucleosides resulted in a transient accumulation of the corresponding diphosphonucleoside, as expected from the apparent K(m) values. Enzymatic properties of NTPDase8 differ from those of other NTPDases suggesting an alternative way to modulate nucleotide levels and consequently P2 receptor activation.     

Identification of a dithiol-dependent nucleoside triphosphate hydrolase in Sarcocystis neurona

A putative nucleoside triphosphate hydrolase (NTPase) gene was identified in a database of expressed sequence tags (ESTs) from the apicomplexan parasite Sarcocystis neurona. Analysis of culture-derived S. neurona merozoites demonstrated a dithiol-dependent NTPase activity, consistent with the presence of a homologue to the TgNTPases of Toxoplasma gondii. A complete cDNA was obtained for the S. neurona gene and the predicted amino acid sequence shared 38% identity with the two TgNTPase isoforms from T. gondii. Based on the obvious homology, the S. neurona protein was designated SnNTP1. The SnNTP1 cDNA encodes a polypeptide of 714 amino acids with a predicted 22-residue signal peptide and an estimated mature molecular mass of 70kDa. Southern blot analysis of the SnNTP1 locus revealed that the gene exists as a single copy in the S. neurona genome, unlike the multiple gene copies that have been observed in T. gondii and Neospora caninum. Analyses of the SnNTP1 protein demonstrated that it is soluble and secreted into the culture medium by extracellular merozoites. Surprisingly, indirect immunofluorescence analysis of intracellular S. neurona revealed apical localisation of SnNTP1 and temporal expression characteristics that are comparable with the microneme protein SnMIC10. The absence of SnNTP1 during much of endopolygeny implies that this protein does not serve a function during intracellular growth and development of S. neurona schizonts. Instead, SnNTP1 may play a role in events that occur during or proximal to merozoite egress from and/or invasion into cells.     

Maleimide-mediated protein conjugates of a nucleoside triphosphate gamma-S and an internucleotide phosphorothioate diester.

The purpose of this study was to determine whether the gamma-S of nucleoside thiotriphosphates and the non-bridging sulfur of internucleotide phosphorothioate diesters possess sufficient thiol character to form adducts with maleimides. Adenosine triphosphate gamma-S (ATPS) and thymidyl-PS-thymidine (TPST) were each reacted with the reporter molecule N-1 pyrene maleimide (PM) and the fluorescence intensity was recorded. The observed reactivity of the phosphorothioate nucleotides towards maleimide was used as a basis for preparing covalent protein-nucleotide conjugates of ATPS and of the internucleotide phosphorothioate diester, deoxyadenylyl-PS-deoxy-adenylyl-PS-deoxyadenosine (dA3(PS)2). The absorbance spectra of bovine serum albumin (BSA) conjugates of ATPS and of dA3(PS)2 showed the formation of protein-nucleotide conjugates, with absorbance maxima near 260 nm. The degree of conjugation was 1.69 nucleotides (nt)/BSA molecule for ATPS and 0.44 nt/BSA molecule for dA3(PS)2. The extent of conjugation of the gamma-S of the nucleoside thiotriphosphate and of the non-bridging sulfur of the internucleotide phosphorothioate diester with maleimide-derivatized protein agreed with their relative reactivity towards PM. Both the gamma-S of the nucleoside thiotriphosphate and the internucleotide phosphorothioate diester were found to possess sufficient thiol character to permit formation of maleimide-mediated protein conjugates.     

Protein P4 of the bacteriophage phi 6 procapsid has a nucleoside triphosphate-binding site with associated nucleoside triphosphate phosphohydrolase activity.

Bacteriophage phi 6 contains three segments of double-stranded RNA. The procapsid consists of proteins P1, P2, P4, and P7, which are encoded by the viral L segment. cDNA copies of this segment have been cloned into plasmids that direct the production of these proteins, which assemble into polyhedral procapsids. These procapsids are capable of packaging plus-sense phi 6 RNA in the presence of nucleoside triphosphate and synthesizing the complementary minus strand to form double-stranded RNA. In this article, we report the presence of a nucleotide-binding site in protein P4. The viral procapsid and nucleocapsid exhibit a nucleoside triphosphate phosphohydrolase activity that converts nucleoside triphosphates into nucleoside diphosphates.     

An RNA-dependent nucleoside triphosphate hydrolase from Krebs-II ascites tumor cells. Detection and preliminary characterization

A novel enzymatic activity, RNA-dependent, NTPase, was isolated from Krebs-II ascites tumor cells. This activity is associated with ribosomes and can be detached from them by washing in KCl solutions of a higher than 0.3 M concentration. The enzyme hydrolyzes all the four nucleoside triphosphates to the corresponding nucleoside diphosphates and orthophosphate. The rate of NTP hydrolysis increases about 10-fold in the presence of natural RNAs and synthetic polyribonucleotides [except poly(G)]. Natural DNAs, both double and single-stranded, are poor cofactors, although pol(dA) and poly(dT) stimulate, to a certain extent, the rate of ATP hydrolysis. Possible involvement of RNA-dependent NTPase in protein biosynthesis is discussed.     

Stimulation of adenine phosphoribosyltransferase by adenosine triphosphate and other nucleoside triphosphates

1. Adenine phosphoribosyltransferase was protected from inactivation on heating at 55° by the presence of 5-phosphoribosyl pyrophosphate. ATP, adenine, AMP or GMP had no protective effect on the activity of this enzyme. The presence of either 5-phosphoribosyl pyrophosphate or ATP did not protect adenine phosphoribosyltransferase against the loss of ATP stimulation obtained by heating at 55°. 2. At pH5·3 and 6·0 adenine phosphoribosyltransferase was stimulated by a narrow range of ATP concentration (15–25μm). At pH6·5 and 7·0 maximum stimulation was obtained with 25–30μm-ATP, and at pH7·4, 8·2 and 8·85 maximum stimulation was obtained over a wide range of ATP concentrations (60–200μm). With extracts that had been heated for 30min. at 55° no stimulation was observed at either pH5·3 or 7·4 with ATP concentrations up to 100μm. 3. Short periods of heating at 55° (1, 2 or 5min.) increased the stimulation of adenine phosphoribosyltransferase obtained with various concentrations of ATP. 4. The addition of CTP, GTP, deoxy-GTP, deoxy-TTP or XTP to assay mixtures resulted in weak stimulation of adenine-phosphoribosyltransferase activity. 5. It is suggested that there are at least three different forms of adenine phosphoribosyltransferase, each with a different affinity for ATP.     

Thermolability characteristics and inheritance of human red cell nucleoside triphosphate pyrophosphohydrolase

Biochemical and genetic data which serve to define further the variation of nucleoside triphosphate pyrophosphohydrolase (NTPH) activity in red cells are presented [see also Verhoef, V.L., Fuller, S.A., and Morris, A.J. (1980). Biochem. Genet. 18:235; Soder, C., Henderson, J.F., Zombor, G., McCoy, E. E., Verhoef, V., and Morris, A. J. (1976). Can. J. Biochem. 54:843]. Examination of the in vivo stability of red cell NTPH via separation of cells by density (age) reveals that loss of NTPH activity during the lifetime of the erythrocytes is the same in individuals with high and low NTPH specific activity. However, when the thermolability of lysate NTPH was measured, three phenotypes could be distinguished. In order of decreasing thermolability, these correspond to NTPH specific activities of 0-5, 12-25, and greater than 25. These data provide the first evidence of physical differences in the molecules of NTPH associated with the various specific activities of NTPH present in the human population. Family data are also presented which show that the mode of inheritance of differences in NTPH specific activity cannot be ascribed to a simple autosomal one gene-two allele system. We propose alternatively that the family data conform well to an hypothesis of three alleles at one NTPH locus controlling NTPH activity.     

Novel nucleoside triphosphate analogs for the enzymatic labeling of nucleic acids

We have evaluated several novel nucleotide analogs suitable for enzymatic labeling of nucleic acid targets for a variety of array-based assays. Two new reagents in particular, a C4-labeled 1-(2',3'-dideoxy-beta-D-ribofuranosyl) imidazole-4-carboxamide 5'-triphosphate 5 and an N1-labeled 5-(beta-D-ribofuranosyl)-2,4(1H,3H)-pyrimidinedione 5'-triphosphate 3, were found to be excellent substrates for labeling with terminal deoxynucleotidyl transferase and T7 RNA polymerase, respectively.