The long term goal of the proposed research is to understand the structures and the functions of ecto-ATPases and ecto-apyrases in extracellular nucleotide metabolism in both normal and pathophysiological states. These enzymes, along with the related soluble apyrases, constitute the class of enzymes known as the """"""""E-type ATPases"""""""". The E-type ATPases are important for the termination of purinergic receptor-mediated responses, including termination of responses to nucleotides used as neurotransmitters. They are also involved in cell adhesion processes, and are important for maintenance of hemostasis in the cardiovasculature. The cDNAs encoding the three human E-type ATPases will be expressed. Site directed mutagenesis will be performed to identify amino acid residues which are important for ATP and ADP hydrolysis and binding. The site-directed mutagenesis experiments will be designed based on: (1) the newly discovered homologies to the actin/heat shock protein/sugar kinase superfamily of enzymes; (2) careful analysis of amino acid residues that are conserved in all the E-type ATPases; (3) analysis of residues that are different between the ecto-ATPases and ecto-apyrases, yet conserved among the sub-types. Thus, amino acid residues important for nucleotide hydrolysis will be identified, the hypothesis that the E-type ATPases are members of the actin superfamily of proteins will be tested, and the residues important for hydrolysis of ADP in addition to ATP (i.e., residues distinguishing biochemically the ecto-apyrases from ecto-ATPases) will be identified. In addition, in situ nucleotide hybridization and immunolocalization using specific anti-peptide antibodies will be performed to learn which areas of the brain and muscle express which E-type ATPase(s), thereby establishing putative functional roles for the enzymes in extracellular nucleotide metabolism. If site-directed mutagenesis experiments continue to support the hypothesis that the E-type ATPases are members of the actin superfamily of proteins, then three dimensional computer modeling of the mutational results will be done by using the known crystal structures of members of the actin/heat shock protein/sugar kinase superfamily of proteins as templates for small segments of wild-type and mutated E-type ATPase sequences.
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