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The importance of the dimeric state for enzymatic activity in human calcium-activated nucleotidase (CAN) is investigated. The results demonstrate that a single mutation in the center of the dimmer interface disrupts the calcium-dependent dimerization in soluble CAN and affects enzyme activity.

Results reveal that residues glutamate182 and serine224 in apyrase conserved region 3 and 4, respectively are essential for the activity of ecto-nucleoside triphosphate diphosphohydrolases. Data further indicate that arginine67, 146 and asparagine191 and glutamine226 are involved in substrate specificity.

Mutagenesis was performed on a human brain E-type ATPase to evaluate the role of two highly conserved tryptophan residues (Trp187 and Trp459), the only two tryptophans conserved in almost all E-type ATPases. Mutation of tryptophan 187 to alanine resulted in a poorly expressed ecto-apyrase completely lacking nucleotidase activity. Findings indicated a modification of the tertiary structure, causing the enzyme to be inappropriately folded and retained within the cell.

Site-directed mutagenesis of conserved amino acids in apyrase conserved regions (ACR) I and IV was used in studying the human brain ecto-apyrase HB6. These are glycosylated enzymes with nucleotide binding and hydrolysis active sites on the exterior of the cell. These enzymes are both soluble and membrane-associated proteins. Results reveal HB6 has the consensus ATP phosphate binding motifs found in the actin family.

Monoclonal antibodies and newly generated polyclonal antibodies, both raised against the chicken gizzard ecto-ATPase, were tested for their capacity to cross-react with mammalian ecto-ATPases and were utilized as specific immunochemical probes to identify non-cross-linked and cross-linked ecto-ATPase. Unlike previous findings with the rabbit skeletal muscle ecto-ATPase enzyme, cross-linking the chicken gizzard smooth muscle ecto-ATPase with 3,3'-dithiobis-(sulfosuccinimidylpropionate) and dithiobis(succinimidylpropionate) increased the activity of the enzyme.

The studies related to the structural and functional roles of the conserved polar residues in the N- and C-terminal transmembrane helices of human nucleoside triphosphate diphosphohydrolase 3 (NTPDase3) are described. The data obtained proposed a model for putative hydrogen bond interactions of the conserved polar residues in the transmembrane domain of native, oligomeric NTPDase3 which are critical for proper protein expression, full enzymatic activity, and susceptibility to membrane perturbations.

The human ecto-nucleoside triphosphate diphosphohydrolases (NTPDase3) in COS-1 cells was expressed to block the secretory transport with monensin or brefeldin A, or by targeting to endoplasmic reticulum (ER) with signal peptide to study the subcellular location and the mechanism of ecto-nucleotidase activation. The findings from ER-targeted NTPDase3 could be applied as an experimental tool to study the effects of ER ATP depletion on ER function under normal and stress conditions.

The disulfide structure in human Cell-surface nucleotidases (NTPDase)3 that contain 10 invariant cysteine residues in their extracellular regions is investigated by making single and double mutants of these 10 cysteines, and their enzymatic, activity, glycosylation pattern, trafficking to the cell membrane and sensitivity to reduction is analyzed. It is suggested that the structural basis for the importance of several apyrase conserved regions for the nucleotidase activities of the NTPDases.

The amino acids used for catalytic activity and enzyme specificity is examined. The seven point mutations constructed for this purpose are explained and the raminifications of the results on the interpretations of a published crystal structure are discussed.

NTPDase soluble human nucleotidase has been expressed in bacteria and refolded to a very high specific activity. A structural model for NTPDase6, incorporating these and other findings obtained with other NTPDases, is proposed.

Research describes the expression of a bed bug apyrase, a human soluble calcium-activated nucleotidase-1 in bacteria and its characterization. Results demonstrate that it is a calcium-binding protein and calcium triggers a conformational change rendering an enzymatically inactive enzyme to the active form.