Mothers continue to feed cereals despite advice that starches not be fed to younger infants because of pancreatic and salivary amylase immaturity. Because intestinal glucoamylase digests starch, we hypothesize that human MGA may serve as an alternate starch digestion pathway. Overlap exists between in vitro activities of the closely associated glucohydrolase enzyme complexes which has prevented direct enzymatic testing of this hypothesis. The objective of our study was the cloning of MGA cDNA to allow expression analysis of individual hydrolytic and binding sites for intestinal enzyme hydrolase activities. Methods: We sequenced 4 CNBr peptides and the N terminus from MGA isolated from human mucosa with HBB 2/143/17 mAb. RT used human intestinal RNA. PCR used degenerate primers with specific primers for extensions. Results: A 6515 bp cDNA coding a 1853 aa, 209 kDa protein was cloned. 92% identity was found between the sequence deduced from the cDNA and the purified peptide sequences. MGA peptide has 2 WIDMNE hydrolytic sites and the maltase and glucoamylase domains have additional internal homologies. We are now expressing recombinant maltase and glucoamylase in E. coli and COS-1 cells in order to determine maltose and starch specificities of the isolated hydrolytic domains. Conclusion: MGA is a member of the Glycosyl Hydrolase Family 31. The identical WIDMNE sequences suggests a common hydrolytic mechanism with differences in the binding regions responsible for substrate specificities. This work is supported by the USDA/ARS Children's Nutrition Research Center, Department of Pediatrics, Baylor College of Medicine, Houston, TX. This project has been funded in part by federal funds from the USDA/ARS under Cooperative Agreement No. 58-625
