Bovine intestinal mucosal membranes are a rich source of alkaline phosphatase (ALPase) and 5'-nucleotide phosphodiesterase (PDase). These enzymes catalyze different reactions (the product of PDase is a substrate for ALPase) but they are remarkably similar in several catalytic and physicochemical characteristics, and they are immunologically cross-reactive. We propose to establish the chemical basis for these similarities by identifying certain common structures in these molecules, and sequencing the amino acids in these segments. The structures to be examined in both enzymes include: a) the peptide which contains the site of attachment of substrate in the covalent enzyme-substrate intermediate, b) peptide around the site oxidized by ferrate, c) peptides cross-reactive to monoclonal antibodies, d) peptides around the sites of oligosaccharide attachment (and the structures of the oligosaccharides themselves), and 5) (tentatively) the membrane binding sites. The identification of sequence homology between ALPase and PDase will serve to emphasize the role of particular amino acids in functional characteristics, and will support our hypothesis that these enzymes descended from a common ancestor. The cross-reacting antibodies will be used to identify other proteins from the same tissue which share a common antigenic determinant, and to examine the relatedness of intestinal ALPase and PDase to the corresponding enzymes of other tissues and unrelated organisms. Other points to be examined include the question of whether intestinal ALPase and PDase are part of a phosphate uptake system, whether venom PDase has a catalytic mechanism which includes a covalent nucleotidyl-enzyme intermediate like intestinal PDase, and the chemical basis for anomalous kinetics and specificity of intestinal PDase.