E-type ATPases: Identification of a novel protein family and biochemical characterization of the potato apyrase and rat brain ecto-apyrase/CD3

Extracellular nucleotides and nucleosides play key functional roles in intercellular signaling for numerous physiological processes. Cells are capable of extruding adenosine triphosphate (ATP) and adenosine diphosphate (ADP) to elicit specific responses mediated through various nucleotide receptors. Cell surface ecto-apyrases capable of hydrolyzing extracellular ATP and ADP to the monophosphate form (AMP) are also present, and are speculated to be involved in the deactivation of the receptors. Subsequent hydrolysis of AMP to adenosine, by the 5$spprime$-nucleotidase, can either activate adenosine receptors and/or be recycled back into the cell through nucleoside transporters. In this thesis, we describe the identification and characterization of a novel protein family consisting of apyrases and related enzymes from various eukaryotic organisms.;There are four highly conserved regions in all the polypeptides that we termed Apyrase Conserved Regions (ACR) 1-4. Site-directed mutagenesis of completely conserved residues in the ACR sequences of the rat brain polypeptide resulted in a total loss of enzymatic activity, confirming our hypothesis that these regions were critical for nucleotide hydrolysis function.;Biochemical studies on the divalent cation dependency of the enzymatic activities for the rat brain ecto-apyrase/CD39 and potato apyrase, revealed that both enzymes possess two distinct calcium dependent activities associated with the monomeric polypeptides: a high affinity calcium dependent activity $rm(Ksb{M} lbrack Casp{2+}rbracksb{free}sim10sp{-7}$ M) that requires high concentrations of nucleotides $rm(Ksb{M} lbrack ATPrbracksb{free}sim10sp{-3}$ M) and a low affinity calcium dependent activity $rm(Ksb{M} lbrack Casp{2+}rbracksb{free}sim10sp{-5}$ M) that requires comparable levels of nucleotides $rm(Ksb{M} lbrack ATPrbracksb{free}sim10sp{-5}$ M).;Based on the biphasic nature of the nucleotide hydrolysis activity and the measurements of various kinetic properties, we have proposed a mechanistic model to describe the enzymatic activities. We believe the polypeptides contain a single active site that can interact with either free Ca$rmsp{2+} (Ksb{M}sim10sp{-7}$ M) and free ATP $rm(Ksb{M}sim10sp{-3}$ M) or free $rm Casp{2+} (Ksb{M}sim10sp{-7}$ M) and CaATP $rm(Ksb{M}sim10sp{-4}$ M) with separate rate constants for the hydrolysis of the terminal phosphates.