The amkinooligopeptidase, a brush border integral membrane enzyme of the small intestine that is essential for the final hydrolysis of peptide nutrients, has been isolated in pure form and a monospecific antiserum has been raised in rabbits. Initial studies show that this protein is synthesized with a transient phase in the cytosol compartment followed by a prolonged association with endoplasmic reticulum and Golgi membranes prior to its final brush border insertion. Studies have been designed to determine the functional relationship of the cytosol AOP to the overall intracellular synthesis and assembly process and to analyze the oligomeric structure of the functional AOP molecule in its native state in the brush border membrane by the use of bifunctional cross-linking reagents such as suberimidate and dihydrazides. Highly purified subcellular organelles including endoplasmic reticulum, Golgi membranes, laterobasal membrane and brush border membranes will be isolated by differential centrifugation, equilibrium centrifugation and finally by sequential separation between phases of dextran T-500 and polyethylene glycol 6000. The structural and functional relationship of the nascent aminooligopeptidase in endoplasmic reticulum and Golgi will be studied and the nascent AOP peptide will be translated in a cell-free rabbit reticulocyte system and the final AOP protein product compared structurally with that in the cytosol, endoplasmic reticulum and Golgi. After the details of the normal intracellular synthesis, assembly, transport and insertion into the brush border have been determined structurally and functionally, the regulation of the overall synthesis process and maintenance of the brush border aminooligopeptidase enzyme protein by nutrients such as peptides, proteins and amino acids will be thoroughly analyzed.
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