The intestinal oligosaccharides sucrase-a-dextrinase (S-D) and lactase (L), both synthesized as precursors of >200 kDa which are proteolytically processed either intracellularly or after insertion into the brush border membrane (BB), undergo maturational activity changes (decrease of L; increase of S-D) correlating with specific mRNA levels, but structural subunit changes also appear to be important for regulation. S-D is present in intact BB as 330 kDa and 260 kDa oligomers composed of alpha (140 kDa), beta (125 kDa) and gamma (110 kDa) subunits in varying combinations; L exists as a 225 kDa precursor which is cleaved to a 130 kDa unit in neonates but extensively replaced by a 100 kDa inactive unit during maturation and loss of activity. Post-insertional oligomeric subunit reassociation will be examined by reconstitution experiments of binding of metabolically labeled (35-S-methionine) to solubilized BB's (and purified fractions) renatured after SDS electrophoresis by blotting to nitrocellulose sheets. Synthetic peptide probes from N-terminal and active site amino acid sequences will be used directly as inhibitors of reassembly and catalytic activity and also to raise antibodies as additional probes. The mechanism of change in catalytic activity of S-D in response to diet (decrease with carbohydrate-free; increase with high carbohydrate) and L decline in adult rats will be examined by correlating catalytic activity, immunological activity (for total hydrolase protein), mRNA levels (with cDNA probes from PCR) and transcription rates (by nuclear run-on assays) with the amount of carbohydrate in the diet, both acutely and at steady state. Since adult rats produce relatively large amounts of a 100 kDa inactive subunit replacing much of the 130 kDa """"""""active"""""""" unit, the 100 kDa unit will be isolated, purified and the amino acid sequence of its N- terminus and peptide fragments will be determined to allow structural assignment to either the N-terminus of the 225 kDa precursor or to the 130 subunit (the C-terminal half of the precursor). The oligomers required for full activity will be examined in intact BB by differential exposure to 1% SDS at 4o->55oC and analyzing SDS gel nitrocellulose blots for in situ immunoreactivity and catalytic activity. Neonatal and adult rat intestinal cDNA and genomic libraries will be screened with 32P-labeled PCR probes and full length clones and 5' upstream regulatory regions will be isolated and used for transfection experiments. The regulatory elements of S-D and L gene expression will be studied by transfecting full length cDNA's into the COS cell expression system and appropriate 5' upstream genomic fragments into the CAT and hGH expression systems. These studies of the regulation of essential digestive enzymes have implications for our understanding of the changes in intestinal surface digestion occuring with maturation and in intestinal diseases.
Showing the most recent 10 out of 14 publications
