DEVELOPMENT AND CONTROL OF SYNTHESIS AND SECRETION

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The central focus of the research described in this proposal is an understanding of the mechanisms which regulate the expression of lactase- phlorizin hydrolase (L-Ph) during the development of the mammalian small intestine. This enzyme is solely responsible for the hydrolysis of lactose, the primary carbohydrate in milk, and is essential for survival of mammalian neonates; adult animals and most human adults are considered to be lactase-deficient. The laboratory of the principal investigator has recently isolated and partially sequenced a 2.3 kb cDNA encoding a portion of rat L-Ph. On the basis of recent data obtained using probes derived from this clone, it now seems clear that, in addition to the expected high levels of L-Ph mRNA in suckling animals, significant quantities also persist in adult enterocytes. In combination with data obtained during the previous grant period demonstrating hydrolysis of glycolipids by L-Ph and the continued presence of total L-Ph activity in adult small intestine, these observations suggest a more complex function of the L-Ph subsequent to the suckling period than previously understood. These findings alter the interpretation of all previous studies of the development of the L-Ph which relied exclusively on measurements of specific activity. In order to elucidate the mechanisms regulating developmental L-Ph expression studies have been designed to: 1) determine the temporal pattern of L-Ph gene expression during development in the rat total small intestine by measuring concurrently L-Ph mRNA, L-Ph enzyme activity and L-Ph protein content; 2) characterize the cellular basis for alteration sin L-Ph activity and content using in situ hybridization and immunohistochemistry; 3) define regional differences in L-Ph enzyme activity and protein content in relation to L-Ph mRNA levels along the length of the small intestine, and examine the mechanisms by which L-Ph gene expression is influenced by hormonal status; 4) isolate a full-length cDNA encoding L-Ph; 5) elucidate the structural requirements for correct processing and intracellular targeting of L-Ph using transfection of the full length cDNA and specific constructs or deletions; and 6) identify the active sites in L-Ph and their structural features by peptide mapping and use these data to guide deletional analysis using expression cDNAs. Taken together, these patterns, derived in rats which are considered to be lactase deficient in adulthood, should be representative of the developmental changes occurring in other mammals and in the intestine of lactase-deficient humans.