Multigene enzyme families are a common feature of the mammalian genome. To study this general phenomenon, detailed immunologic and structural studies will be carried out on alkaline phosphatases (ALPs). In humans at least three gene loci encode the protein moieties of the various forms of these enzyme glycoproteins; one for the placental form, at least one for the intestinal forms (adult and fetal) and at least one for the liver, bone and kidney forms. Panels of monoclonal antibodies will be produced against the enzyme products of the loci. Since monoclonal antibodies each recognize distinct antigenic determinants on a protein surface, these antibody panels will allow the characterization and discrimination of the various locus and allelic products on a determinant by determinant basis. By pairwise antibody competition experiments, topologic maps of the relative positions of the various determinant sites on the surfaces of the different enzymes will be obtained. They will be correlated with characteristics revealed by biochemical methods; electrophoretic phenotyping, peptide mapping, sensitivity to specific inhibitors, etc. The various monoclonal antibodies will also be used to prepare immunoaffinity columns for direct enzyme purifications. Peptide analysis of the purified enzymes after cyanogen bromide and also trypsin digestion will be carried out by HPLC. In collaboration with others, N-terminal aminoacid sequencing of the enzymes and of selected peptides of special interest will be carried out. From these and the related immunologic studies much detailed information, about the similarities and differences of the various ALP locus products, will be obtained. Similar immunologic and structural studies of the ALPs from various tissues over an extensive range of primate and other mammalian species will also be carried out. Such studies will be informative about the occurrence in evolution, of gene duplications and of mutations affecting differential tissue expression in this multigene system. Genetic heterogeneity in hypophosphatasia will be studied by identifying immunologically reactive but enzymatically inactive ALP protein using fibroblast cultures from patients with different clinical types. Such CRM+ cases will be characterized and differentiated using an appropriate panel of monoclonal antibodies. Monoclonal antibodies will also be used to develop a routine method for serum ALP analysis, which will be useful clinically; for immunocytochemical studies of normal tissues and of malignancies; and in somatic cell hybridization studies for gene assignment.
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