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Three anti-peptide antisera were raised against three distinct amino acid sequences of ecto-nucleoside triphosphate diphosphohydrolase 3 (NTPDase3), characterized by Western blot analyses, and used to determine the distribution of NTPDase3 protein in adult rat brain. The three antisera all yielded similar immunolocalization data, leading to increased(More)
An extracellular ATPase (E-type ATPase) clone was isolated from a human brain cDNA library and sequenced. The transcript shows similarity to the previously published chicken smooth muscle and rat brain ecto-ATPase cDNAs, human CD39L1 cDNA (putative human ecto-ATPase), and mammalian CD39 (lymphoid cell activation antigen, ecto-apyrase, ATPDase,(More)
Cell-surface nucleotidases (NTPDases) contain 10 invariant cysteine residues in their extracellular regions. To investigate disulfide structure in human NTPDase3, we made single and double mutants of these 10 cysteines, and analyzed their enzymatic activity, glycosylation pattern, trafficking to the cell membrane, and sensitivity to reduction. The mutants(More)
The study and therapeutic modulation of purinergic signaling is hindered by a lack of specific inhibitors for NTP diphosphohydrolases (NTPDases),which are the terminating enzymes for these processes. In addition, little is known of the NTPDase protein structural elements that affect enzymatic activity and which could be used as targets for inhibitor design.(More)
Triadin has been characterized as an abundant protein co-localized with the calcium release channel on the terminal cisternae of the sarcoplasmic reticulum of the skeletal muscle. Its localization to terminal cisternae of the sarcoplasmic reticulum and functional studies suggest that it has an important role in excitation-contraction coupling. In this study(More)
Procedures which allow rapid, quantitative, and selective fluorescent labeling of protein cyst(e)ine residues prior to electrophoresis by reaction with 4-(aminosulfonyl)-7-fluoro-2,1,3-benzoxadiazole (ABD-F) under mild conditions are described. After labeling, the protein(s) of interest is easily monitored throughout electrophoresis and subsequent(More)
On the basis of sequence homologies observed between members of the E-type ATPases and the phosphate binding motifs of the actin/heat shock protein 70/sugar kinase superfamily, a human ecto-apyrase was analyzed by site-directed mutagenesis of conserved amino acids in apyrase conserved regions (ACR) I and IV. The expressed proteins were analyzed to assess(More)
Several "specific" inhibitors of P2 purinergic receptors (purinoceptors) were evaluated for their ability to inhibit purified chicken gizzard cell membrane ecto-ATPase. The P2 purinoceptor antagonists tested included suramin, triazine based reactive textile dyes, and non-specific total protein dyes. All inhibited the purified ecto-ATPase, with half maximal(More)
For the first time, a soluble, dedicated E-type ecto-ATPase has been identified and purified. This fully soluble ecto-ATPase is released into the growth media of the single-celled eukaryote, Tetrahymena, at a constant rate over time (independent of the growth phase of the cells) and it has characteristics similar to those previously described for the(More)
The salivary apyrases of blood-feeding arthropods are nucleotide-hydrolyzing enzymes implicated in the inhibition of host platelet aggregation through the hydrolysis of extracellular adenosine diphosphate. A human cDNA homologous to the apyrase cDNA of the blood-feeding bed bug was identified, revealing an open reading frame encoding a 371-amino acid(More)