This revised application describes studies designed to continue to elucidate the biosynthesis, intracellular processing, and molecular biology of the microvillus membrane (MVM) enzyme lactase-phlorizin hydrolase (EC 3.2.1.23-EC 3.2.1.62). Using organ culture and immunochemical techniques, the labeling patterns of newly synthesized intracellular and MVM lactase will be examined in rat intestine at various times during development and maturation. Subunit composition of synthetic precursors and MVM lactase will be analyzed using SDS-PAGE and HPLC. The role of glycosylation in the intracellular processing of lactase will be studied using tunicamycin B2 and castanospermine. These inhibition experiments will also yield information on the type of oligosaccharide chains which make up the lactase glycoprotein. To examine the molecular biology of lactase, experiments will continue using cDNA clones encoding the enzyme isolated from a fetal rat intestinal cDNA library prepared in the expression vector lambda gt11. Positive identification of 7 cDNA clones has been made with a panel of monoclonal antibodies to lactase using an alkaline phosphatase ELISA to detect positive plaques. Amino acid sequencing will be performed on proteolytic fragments generated from purified lactase subunits, and this information utilized to synthesize oligonucleotide probes for verification of the authenticity of the immunopositive cDNA clones or for screening the lambda gt11 library directly. The cDNA clones identified in this fashion will be sequenced and used to obtain and sequence, from a genomic library, the full-length gene encoding lactase. In order to determine whether developmentally regulated changes in levels of enzyme activity are associated with changes in specific lactase mRNA levels or synthesis, intestinal mRNA will be quantified by northern blot analysis and hybridization, and changes in mRNA function will be measured by nuclear run-off assay. Taken together, the studies described should elucidate the development and molecular biology of lactase. They will provide new information regarding the microvillus form of this enzyme, its synthesis and intracellular processing,and its genetic control. These data should provide the basis for further study of the lactase gene and for analysis of the genetic control of human lactase deficiency.
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